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The present invention relates to formulations comprising a solid dispersion product of an active agent having at least one of a hydrogen bond donor moiety (e.g. ibuprofen, fenofibric acid or naproxen) and a proton donor moiety and a pharmaceutically acceptable polyvinyllactam polyvinylacetate poly(alkylene glycol) graft copolymer, and to methods for preparing such formulations.

1. A formulation comprising a solid dispersion product comprising (a) an active agent having at least one of a hydrogen bond donor moiety and a proton donor moiety, (b)a pharmaceutically acceptable polyvinyllactam polyvinylacetate poly(alkylene glycol) graft copolymer, and (c) a pharmaceutically acceptable pH modifier; wherein the active agent is selected from pharmaceutically active agents, cosmetically active agents and nutritional supplements, and is a compound with a solubility in water at 25° C. and pH 7.0 of 0.01 g/100 ml or less. 2. The formulation of claim 1, wherein the active agent has at least one hydrogen bond donor moiety. 3. The formulation of claim 2, wherein the hydrogen bond donor moiety is selected from primary amino, secondary amino, hydroxy, carbamoyl, thiocarbamoyl, sulfamoyl, sulfinamoyl and ureido. 4. The formulation of claim 1, wherein the active agent has at least one proton donor moiety. 5. The formulation of claim 4, wherein the proton donor moiety is selected from organic acid moieties and CH-acidic moieties. 6. The formulation of claim 1, wherein the active agent is non-ionic. 7. (canceled) 8. The formulation of claim 1, wherein the active agent is selected from (RS)-2-(4-(2-methylpropyl)phenyl)propanoic acid, 2-{4-[(4-chlorophenyl)carbonyl]phenoxy}-2-methylpropanoic acid and (2S)-2-(6-methoxynaphthalen-2-yl)propanoic acid. 9. The formulation of claim 1, wherein the graft copolymer comprises (i) 10 to 50 wt % poly(alkylene glycol) moieties, (ii) 30 to 80 wt % N-vinyllactam moieties, and (iii) 10 to 50 wt % vinyl acetate moieties. 10. The formulation of claim 9, wherein the N-vinyllactam moieties are N-vinylcaprolactam moieties. 11. The formulation of claim 1, wherein the pH modifier is an alkaline pH modifier. 12. The formulation of claim 11, wherein the alkaline pH modifier is selected from basic salts of organic acids and inorganic acids, basic amino acids, metal oxides and metal hydroxides. 13. The formulation of claim 1, wherein the pH modifier is an acidic pH modifier. 14. The formulation of claim 13, wherein the acidic pH modifier is selected from mono-, di- and polybasic carboxylic acids, mono-, di- and poly-sulfonic acids, and acidic salts thereof. 15. The formulation of claim 1, comprising 0.5 to 20 wt % of the pH modifier relative to the weight of the solid dispersion product. 16. A method for producing the formulation of claim 1, wherein the active agent having at least one hydrogen bond donor moiety or proton donor moiety, the pharmaceutically acceptable pH modifier and the pharmaceutically acceptable polyvinyllactam polyvinylacetate poly(alkylene glycol) graft copolymer are intimately blended. 17. The method of claim 16, comprising the steps of (a) blending the active agent, the pH modifier and the graft copolymer; (b) heating the blend to obtain a homogeneous melt; (c) forcing the thus obtained melt through one or more nozzles; and (d) allowing the melt to solidify to obtain a solid dispersion product. 18. The method of claim 17, wherein step (b) is carried out in an extruder and the blend is subjected to a mixing action in a mixing section of the extruder. 19. The formulation of claim 1, wherein the sum of components (a), (b) and (c) makes up at least 80% of the solid dispersion product. 20. A pharmaceutically acceptable pH modifier as defined in claim 1 for increasing the dispersion rate of a solid dispersion product in an aqueous medium relative to the dispersion rate of a comparative product in the aqueous medium and for increasing the release rate of an active agent from the solid dispersion product into the aqueous medium relative to the release rate of the active agent from the comparative product into the aqueous medium; wherein the active agent has at least one of a hydrogen bond donor moiety and a proton donor moiety, is selected from pharmaceutically active agents, cosmetically active agents and nutritional supplements, and is a compound with a solubility in water at 25° C. and pH 7.0 of 0.01 g/100 ml or less; the solid dispersion product comprises (a) the active agent, (b) a pharmaceutically acceptable polyvinyllactam polyvinylacetate poly(alkylene glycol) graft copolymer, and (c) the pharmaceutically acceptable pH modifier; and the comparative product differs from the solid dispersion product in lacking the pharmaceutically acceptable pH modifier.

The present invention relates to formulations comprising a solid dispersion product of an active agent having at least one of a hydrogen bond donor moiety (e.g. ibuprofen, fenofibric acid or naproxen) and a proton donor moiety and a pharmaceutically acceptable polyvinyllactam polyvinylacetate poly(alkylene glycol) graft copolymer, and to methods for preparing such formulations.1. A formulation comprising a solid dispersion product comprising (a) an active agent having at least one of a hydrogen bond donor moiety and a proton donor moiety, (b)a pharmaceutically acceptable polyvinyllactam polyvinylacetate poly(alkylene glycol) graft copolymer, and (c) a pharmaceutically acceptable pH modifier; wherein the active agent is selected from pharmaceutically active agents, cosmetically active agents and nutritional supplements, and is a compound with a solubility in water at 25° C. and pH 7.0 of 0.01 g/100 ml or less. 2. The formulation of claim 1, wherein the active agent has at least one hydrogen bond donor moiety. 3. The formulation of claim 2, wherein the hydrogen bond donor moiety is selected from primary amino, secondary amino, hydroxy, carbamoyl, thiocarbamoyl, sulfamoyl, sulfinamoyl and ureido. 4. The formulation of claim 1, wherein the active agent has at least one proton donor moiety. 5. The formulation of claim 4, wherein the proton donor moiety is selected from organic acid moieties and CH-acidic moieties. 6. The formulation of claim 1, wherein the active agent is non-ionic. 7. (canceled) 8. The formulation of claim 1, wherein the active agent is selected from (RS)-2-(4-(2-methylpropyl)phenyl)propanoic acid, 2-{4-[(4-chlorophenyl)carbonyl]phenoxy}-2-methylpropanoic acid and (2S)-2-(6-methoxynaphthalen-2-yl)propanoic acid. 9. The formulation of claim 1, wherein the graft copolymer comprises (i) 10 to 50 wt % poly(alkylene glycol) moieties, (ii) 30 to 80 wt % N-vinyllactam moieties, and (iii) 10 to 50 wt % vinyl acetate moieties. 10. The formulation of claim 9, wherein the N-vinyllactam moieties are N-vinylcaprolactam moieties. 11. The formulation of claim 1, wherein the pH modifier is an alkaline pH modifier. 12. The formulation of claim 11, wherein the alkaline pH modifier is selected from basic salts of organic acids and inorganic acids, basic amino acids, metal oxides and metal hydroxides. 13. The formulation of claim 1, wherein the pH modifier is an acidic pH modifier. 14. The formulation of claim 13, wherein the acidic pH modifier is selected from mono-, di- and polybasic carboxylic acids, mono-, di- and poly-sulfonic acids, and acidic salts thereof. 15. The formulation of claim 1, comprising 0.5 to 20 wt % of the pH modifier relative to the weight of the solid dispersion product. 16. A method for producing the formulation of claim 1, wherein the active agent having at least one hydrogen bond donor moiety or proton donor moiety, the pharmaceutically acceptable pH modifier and the pharmaceutically acceptable polyvinyllactam polyvinylacetate poly(alkylene glycol) graft copolymer are intimately blended. 17. The method of claim 16, comprising the steps of (a) blending the active agent, the pH modifier and the graft copolymer; (b) heating the blend to obtain a homogeneous melt; (c) forcing the thus obtained melt through one or more nozzles; and (d) allowing the melt to solidify to obtain a solid dispersion product. 18. The method of claim 17, wherein step (b) is carried out in an extruder and the blend is subjected to a mixing action in a mixing section of the extruder. 19. The formulation of claim 1, wherein the sum of components (a), (b) and (c) makes up at least 80% of the solid dispersion product. 20. A pharmaceutically acceptable pH modifier as defined in claim 1 for increasing the dispersion rate of a solid dispersion product in an aqueous medium relative to the dispersion rate of a comparative product in the aqueous medium and for increasing the release rate of an active agent from the solid dispersion product into the aqueous medium relative to the release rate of the active agent from the comparative product into the aqueous medium; wherein the active agent has at least one of a hydrogen bond donor moiety and a proton donor moiety, is selected from pharmaceutically active agents, cosmetically active agents and nutritional supplements, and is a compound with a solubility in water at 25° C. and pH 7.0 of 0.01 g/100 ml or less; the solid dispersion product comprises (a) the active agent, (b) a pharmaceutically acceptable polyvinyllactam polyvinylacetate poly(alkylene glycol) graft copolymer, and (c) the pharmaceutically acceptable pH modifier; and the comparative product differs from the solid dispersion product in lacking the pharmaceutically acceptable pH modifier.

A phosphoprotein detection reagent that selectively binds phosphoamino acids. Methods of generating and employing the reagent are also provided, as are methods of detecting modulation of protein phosphorylation are disclosed. Methods of detecting a change in state of a cell are also disclosed. Additionally, a kit for the detection of phosphoproteins is also disclosed.

1. A phosphoprotein detection reagent (PPDR) comprising: (a) a polydentate chelator that coordinates a Zn2+ ion; and (b) a detectable moiety conjugated to the polydentate chelator, wherein when the Zn2+ ion is coordinated to the chelator, a chelator-metal ion moiety is formed that can selectively bind to a phosphorylated amino acid residue in a phosphoprotein, if present, to create a chelator-metal-ion-phosphoprotein (CMPP) complex, and the detectable moiety allows the CMPP complex to be detected, if present. 2. The phosphoprotein detection reagent (PPDR) of claim 1, wherein the PPDR is soluble in an aqueous medium. 3. The phosphoprotein detection reagent (PPDR) of claim 1, wherein the chelator is nitriloacetic acid or iminodiacetic acid. 4. The phosphoprotein detection reagent (PPDR) of claim 1, wherein the detectable moiety is biotin, a fluorescent molecule, or an enzyme. 5. The phosphoprotein detection reagent (PPDR) of claim 1, wherein the polydentate chelator is bidentate, tridentate, tetradentate, or pentadentate. 6. The reagent of claim 1, further comprising a spacer between the polydentate chelator and the detectable moiety. 7. A method for synthesizing a phosphoprotein detection reagent (PPDR), the method comprising: (a) reacting a polydentate chelator donor molecule with a detectable moiety donor under conditions wherein a detectable moiety is transferred to a polydentate chelator to form a chelator-detectable moiety complex; and (b) mixing the chelator-detectable moiety complex and a Zn2+ ion-containing solution under conditions wherein the chelator-detectable moiety complex coordinates the Zn2+ ion, forming a PPDR that is soluble in aqueous medium. 8. The method of claim 7, wherein the polydentate chelator is bidentate, tridentate, tetradentate, or pentadentate. 9. The method of claim 7, wherein the detectable moiety is selected from the group consisting of biotin, a fluorescent molecule, and an enzyme. 10. A method for detecting a phosphoprotein, the method comprising: (a) obtaining a protein-containing solution; (b) contacting the protein-containing solution with a reagent under conditions wherein the reagent selectively binds to a phosphorylated amino acid residue present in one or more of the proteins to form a reagent/amino acid complex, wherein: (i) the reagent comprises a polydentate chelator and an detectable moiety conjugated to the polydentate chelator; (ii) the polydentate chelator is coordinated to a Zn2+ ion to form a polydentate chelator-Zn2+ ion moiety: (iii) the polydentate chelator-Zn2+ ion moiety selectively binds to a phosphorylated amino acid residue; and (iv) the detectable moiety allows the reagent/amino acid complex to be detected if present; and (c) detecting the reagent/amino acid complex, wherein the detection of the reagent/amino acid complex detects a phosphoprotein. 11. The method of claim 10, further comprising separating the proteins present in solution from each other by electrophoresis. 12. The method of claim 10, further comprising immobilizing the separated proteins on a solid support. 13. The method of claim 10, further comprising contacting the reagent and immobilized proteins at a pH between about 5.0 and 7.0, and washing at a pH between about 6.9 and 9.5. 14. A method for detecting a change in phosphorylation status of a protein present within a target tissue in response to a change in state, the method comprising: (a) obtaining a protein lysate from a cell from the target tissue prior to the change in state; (b) separating the proteins present in the lysate from each other; (c) contacting the proteins with a reagent under conditions wherein the reagent selectively binds to a phosphorylated amino acid residue present in at least one of the proteins to form a reagent/amino acid complex, wherein: (i) the reagent comprises a polydentate chelator and an detectable moiety conjugated to the polydentate chelator; (ii) the polydentate chelator is coordinated to a Zn2+ ion to form a polydentate chelator-Zn2+ ion moiety: (iii) the polydentate chelator-Zn2+ ion moiety selectively binds to a phosphorylated amino acid residue; and (iv) the detectable moiety allows the reagent/amino acid complex to be detected if present; (d) detecting the reagent/amino acid complex, wherein the reagent/amino acid complex is indicative of a phosphoprotein in the cell lysate; (e) creating a profile indicative of the detected phosphoproteins of the lysate; (f) obtaining a protein lysate from a cell from the target tissue after the change in state; (g) repeating steps b) through e) for the lysate from a cell from the target tissue after the change in state; and (h) comparing the profile from the lysate from the cell from the target tissue prior to the change in state to the profile from the lysate from the cell from the target tissue after the change in state, wherein a difference between the two profiles is indicative of a change in the phosphorylation status of a protein present within the target tissue in response to the change in state of the target tissue. 15. A kit comprising: (a) a phosphoprotein detection reagent (PPDR) comprising: (i) a polydentate chelator that coordinates a Zn2+ ion; and (ii) a detectable moiety conjugated to the polydentate chelator, wherein when the Zn2+ ion is coordinated to the chelator, a chelator-metal ion moiety is formed that can selectively bind to a phosphorylated amino acid residue in a phosphoprotein, if present, to create a chelator-metal-ion-phosphoprotein (CMPP) complex, and the detectable moiety allows the CMPP complex to be detected, if present; and (b) instructions for using the PPDR. 16. The kit of claim 15, wherein the phosphoprotein detection reagent (PPDR) is soluble in aqueous medium. 17. The kit of claim 15, wherein the detectable moiety is biotin, a fluorescent molecule, or an enzyme. 18. The kit of claim 15, wherein the polydentate chelator is bidentate, tridentate, tetradentate, or pentadentate. 19. A composition comprising: (a) a Zn2+ ion; and (b) a phosphoprotein detection reagent (PPDR) comprising a chelator and a detectable moiety, wherein: (i) the detectable moiety is conjugated to the chelator; (ii) the chelator comprises a polydentate chelator coordinated to the Zn2+ ion to form a chelator-metal ion moiety that can selectively bind to a phosphorylated amino acid residue in a phosphoprotein, if present, to create a chelator-metal ion-phosphoprotein (CMPP) complex; and (iii) the detectable moiety allows the CMPP complex to be detected, if present.

A phosphoprotein detection reagent that selectively binds phosphoamino acids. Methods of generating and employing the reagent are also provided, as are methods of detecting modulation of protein phosphorylation are disclosed. Methods of detecting a change in state of a cell are also disclosed. Additionally, a kit for the detection of phosphoproteins is also disclosed.1. A phosphoprotein detection reagent (PPDR) comprising: (a) a polydentate chelator that coordinates a Zn2+ ion; and (b) a detectable moiety conjugated to the polydentate chelator, wherein when the Zn2+ ion is coordinated to the chelator, a chelator-metal ion moiety is formed that can selectively bind to a phosphorylated amino acid residue in a phosphoprotein, if present, to create a chelator-metal-ion-phosphoprotein (CMPP) complex, and the detectable moiety allows the CMPP complex to be detected, if present. 2. The phosphoprotein detection reagent (PPDR) of claim 1, wherein the PPDR is soluble in an aqueous medium. 3. The phosphoprotein detection reagent (PPDR) of claim 1, wherein the chelator is nitriloacetic acid or iminodiacetic acid. 4. The phosphoprotein detection reagent (PPDR) of claim 1, wherein the detectable moiety is biotin, a fluorescent molecule, or an enzyme. 5. The phosphoprotein detection reagent (PPDR) of claim 1, wherein the polydentate chelator is bidentate, tridentate, tetradentate, or pentadentate. 6. The reagent of claim 1, further comprising a spacer between the polydentate chelator and the detectable moiety. 7. A method for synthesizing a phosphoprotein detection reagent (PPDR), the method comprising: (a) reacting a polydentate chelator donor molecule with a detectable moiety donor under conditions wherein a detectable moiety is transferred to a polydentate chelator to form a chelator-detectable moiety complex; and (b) mixing the chelator-detectable moiety complex and a Zn2+ ion-containing solution under conditions wherein the chelator-detectable moiety complex coordinates the Zn2+ ion, forming a PPDR that is soluble in aqueous medium. 8. The method of claim 7, wherein the polydentate chelator is bidentate, tridentate, tetradentate, or pentadentate. 9. The method of claim 7, wherein the detectable moiety is selected from the group consisting of biotin, a fluorescent molecule, and an enzyme. 10. A method for detecting a phosphoprotein, the method comprising: (a) obtaining a protein-containing solution; (b) contacting the protein-containing solution with a reagent under conditions wherein the reagent selectively binds to a phosphorylated amino acid residue present in one or more of the proteins to form a reagent/amino acid complex, wherein: (i) the reagent comprises a polydentate chelator and an detectable moiety conjugated to the polydentate chelator; (ii) the polydentate chelator is coordinated to a Zn2+ ion to form a polydentate chelator-Zn2+ ion moiety: (iii) the polydentate chelator-Zn2+ ion moiety selectively binds to a phosphorylated amino acid residue; and (iv) the detectable moiety allows the reagent/amino acid complex to be detected if present; and (c) detecting the reagent/amino acid complex, wherein the detection of the reagent/amino acid complex detects a phosphoprotein. 11. The method of claim 10, further comprising separating the proteins present in solution from each other by electrophoresis. 12. The method of claim 10, further comprising immobilizing the separated proteins on a solid support. 13. The method of claim 10, further comprising contacting the reagent and immobilized proteins at a pH between about 5.0 and 7.0, and washing at a pH between about 6.9 and 9.5. 14. A method for detecting a change in phosphorylation status of a protein present within a target tissue in response to a change in state, the method comprising: (a) obtaining a protein lysate from a cell from the target tissue prior to the change in state; (b) separating the proteins present in the lysate from each other; (c) contacting the proteins with a reagent under conditions wherein the reagent selectively binds to a phosphorylated amino acid residue present in at least one of the proteins to form a reagent/amino acid complex, wherein: (i) the reagent comprises a polydentate chelator and an detectable moiety conjugated to the polydentate chelator; (ii) the polydentate chelator is coordinated to a Zn2+ ion to form a polydentate chelator-Zn2+ ion moiety: (iii) the polydentate chelator-Zn2+ ion moiety selectively binds to a phosphorylated amino acid residue; and (iv) the detectable moiety allows the reagent/amino acid complex to be detected if present; (d) detecting the reagent/amino acid complex, wherein the reagent/amino acid complex is indicative of a phosphoprotein in the cell lysate; (e) creating a profile indicative of the detected phosphoproteins of the lysate; (f) obtaining a protein lysate from a cell from the target tissue after the change in state; (g) repeating steps b) through e) for the lysate from a cell from the target tissue after the change in state; and (h) comparing the profile from the lysate from the cell from the target tissue prior to the change in state to the profile from the lysate from the cell from the target tissue after the change in state, wherein a difference between the two profiles is indicative of a change in the phosphorylation status of a protein present within the target tissue in response to the change in state of the target tissue. 15. A kit comprising: (a) a phosphoprotein detection reagent (PPDR) comprising: (i) a polydentate chelator that coordinates a Zn2+ ion; and (ii) a detectable moiety conjugated to the polydentate chelator, wherein when the Zn2+ ion is coordinated to the chelator, a chelator-metal ion moiety is formed that can selectively bind to a phosphorylated amino acid residue in a phosphoprotein, if present, to create a chelator-metal-ion-phosphoprotein (CMPP) complex, and the detectable moiety allows the CMPP complex to be detected, if present; and (b) instructions for using the PPDR. 16. The kit of claim 15, wherein the phosphoprotein detection reagent (PPDR) is soluble in aqueous medium. 17. The kit of claim 15, wherein the detectable moiety is biotin, a fluorescent molecule, or an enzyme. 18. The kit of claim 15, wherein the polydentate chelator is bidentate, tridentate, tetradentate, or pentadentate. 19. A composition comprising: (a) a Zn2+ ion; and (b) a phosphoprotein detection reagent (PPDR) comprising a chelator and a detectable moiety, wherein: (i) the detectable moiety is conjugated to the chelator; (ii) the chelator comprises a polydentate chelator coordinated to the Zn2+ ion to form a chelator-metal ion moiety that can selectively bind to a phosphorylated amino acid residue in a phosphoprotein, if present, to create a chelator-metal ion-phosphoprotein (CMPP) complex; and (iii) the detectable moiety allows the CMPP complex to be detected, if present.

The present invention relates to a method for identification of specific target proteins in a protein sample following a detection procedure, such as a Western blotting procedure, wherein the membrane is probed with at least two primary antibodies directed against the same and/or different epitopes of the same target protein, and wherein specific binding to the target protein in a sample is differentiated from unspecific binding to the target protein by comparing the resulting sample patterns, such as bands or spot patterns, with each other. In a further step signals from the true target proteins are enhanced while signals resulting from unspecific binding are diminished.

1. A method for identification of specific target biomolecules in a sample following a detection procedure, wherein a gel or membrane is probed with at least two probes directed against the same and/or different sites of the same target biomolecule, and wherein specific binding to the target biomolecule in a sample is differentiated from unspecific binding to the target biomolecule by comparing the resulting sample patterns, such as bands or spot patterns on said gel or membrane, with each other. 2. The method of claim 1, wherein the sample patterns are compared by overlapping. 3. The method of claim 1, wherein the detection procedure is a Western blotting procedure and the method comprises enhancing overlapping band features and sorting out non-overlapping features to produce image data with a pattern more specifically representing the target protein. 4. The method of claim 1, wherein the target biomolecule is a protein, peptide or aggregated biomolecule, such as a fusion protein, or a modified protein, such as a phosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylated protein. 5. The method of claim 1, wherein an algorithm is used to enhance overlapping band features, such as by pixel-wise multiplication. 6. The method of claim 5, wherein the resulting image data is scaled after the enhancement. 7. The method of claim 5, wherein resulting image data is scaled by using the square root function. 8. The method of claim 5, wherein resulting image data is scaled by using an A1/n function where A is the resulting pixel value after enhancement and n is the number of primary antibodies. 9. The method of claim 5, wherein resulting image data is scaled by using a function involving image data from the original images. 10. The method of claim 3, wherein image data is represented in the form of one or more intensity profiles or 3D surfaces. 11. The method of claim 3, wherein the final result is presented in the form of one or more images where optionally color coding has been used to highlight overlapping and/or non-overlapping features. 12. The method of claim 11, wherein the color coding represents the result of applying an algorithm to image data. 13. The method of claim 1, wherein at least two primary antibodies are used as probes. 14. The method of claim 13, wherein the primary antibodies are from different species. 15. The method of claim 13, wherein the primary antibodies are from the same species. 16. The method of claim 13, wherein the primary antibodies are differentially labelled. 17. The method of claim 13, wherein differentially labelled secondary antibodies are used against said primary antibodies. 18. The method of claim 1, wherein the primary and/or secondary antibodies are labelled with fluorescent dyes, preferably cyanine dyes. 19. The method of claim 13, wherein one or more of the antibodies are detected using Enhanced chemiluminescence (ECL) to provide multiplexing. 20. The method of claim 1, wherein at least two sample patterns represent different samples covalently labeled with different dyes and mixed together before electrophoresis is carried out. 21. The method of claim 1, wherein at least two images represent different pre-processing of sample aliquots covalently labeled with different dyes and mixed together before electrophoresis is carried out, wherein the pre-processing for example may involve affinity separation, for example using antibodies against the target protein. 22. The method of claim 1, wherein the resulting sample pattern is quantitatively analyzed. 23. The method of claim 1, implemented in a software package and/or in an instrument system for Western blotting analysis.

The present invention relates to a method for identification of specific target proteins in a protein sample following a detection procedure, such as a Western blotting procedure, wherein the membrane is probed with at least two primary antibodies directed against the same and/or different epitopes of the same target protein, and wherein specific binding to the target protein in a sample is differentiated from unspecific binding to the target protein by comparing the resulting sample patterns, such as bands or spot patterns, with each other. In a further step signals from the true target proteins are enhanced while signals resulting from unspecific binding are diminished.1. A method for identification of specific target biomolecules in a sample following a detection procedure, wherein a gel or membrane is probed with at least two probes directed against the same and/or different sites of the same target biomolecule, and wherein specific binding to the target biomolecule in a sample is differentiated from unspecific binding to the target biomolecule by comparing the resulting sample patterns, such as bands or spot patterns on said gel or membrane, with each other. 2. The method of claim 1, wherein the sample patterns are compared by overlapping. 3. The method of claim 1, wherein the detection procedure is a Western blotting procedure and the method comprises enhancing overlapping band features and sorting out non-overlapping features to produce image data with a pattern more specifically representing the target protein. 4. The method of claim 1, wherein the target biomolecule is a protein, peptide or aggregated biomolecule, such as a fusion protein, or a modified protein, such as a phosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylated protein. 5. The method of claim 1, wherein an algorithm is used to enhance overlapping band features, such as by pixel-wise multiplication. 6. The method of claim 5, wherein the resulting image data is scaled after the enhancement. 7. The method of claim 5, wherein resulting image data is scaled by using the square root function. 8. The method of claim 5, wherein resulting image data is scaled by using an A1/n function where A is the resulting pixel value after enhancement and n is the number of primary antibodies. 9. The method of claim 5, wherein resulting image data is scaled by using a function involving image data from the original images. 10. The method of claim 3, wherein image data is represented in the form of one or more intensity profiles or 3D surfaces. 11. The method of claim 3, wherein the final result is presented in the form of one or more images where optionally color coding has been used to highlight overlapping and/or non-overlapping features. 12. The method of claim 11, wherein the color coding represents the result of applying an algorithm to image data. 13. The method of claim 1, wherein at least two primary antibodies are used as probes. 14. The method of claim 13, wherein the primary antibodies are from different species. 15. The method of claim 13, wherein the primary antibodies are from the same species. 16. The method of claim 13, wherein the primary antibodies are differentially labelled. 17. The method of claim 13, wherein differentially labelled secondary antibodies are used against said primary antibodies. 18. The method of claim 1, wherein the primary and/or secondary antibodies are labelled with fluorescent dyes, preferably cyanine dyes. 19. The method of claim 13, wherein one or more of the antibodies are detected using Enhanced chemiluminescence (ECL) to provide multiplexing. 20. The method of claim 1, wherein at least two sample patterns represent different samples covalently labeled with different dyes and mixed together before electrophoresis is carried out. 21. The method of claim 1, wherein at least two images represent different pre-processing of sample aliquots covalently labeled with different dyes and mixed together before electrophoresis is carried out, wherein the pre-processing for example may involve affinity separation, for example using antibodies against the target protein. 22. The method of claim 1, wherein the resulting sample pattern is quantitatively analyzed. 23. The method of claim 1, implemented in a software package and/or in an instrument system for Western blotting analysis.

The invention relates to a composition comprising, in a physiologically acceptable aqueous medium, 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone and a solvent with solubility parameters in the Hansen solubility space such that 4.5<δ, <30 and 15<δ d <22. Use for caring for, making up and cleansing keratin materials.

1. Composition comprising, in a physiologically acceptable aqueous medium: 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone and an organic solvent with solubility parameters in the Hansen solubility space such that 14.5<δa<30 and 15<δd<22. 2. Composition according to the preceding claim, wherein the organic solvent is chosen from ethanol, 1,2-propylene glycol, 1,3-propanediol, PEG-8 (polyethylene glycol containing 8 ethylene glycol units), propylene carbonate, dipropylene glycol, 1,2-hexylene glycol and PEG-4 (polyethylene glycol containing 4 ethylene glycol units). 3. Composition according to claim 1, wherein the organic solvent is chosen from ethanol, 1,2-propylene glycol, 1,3-propanediol, PEG-8 and propylene carbonate. 4. Composition according to claim 1, wherein the organic solvent is present in a content ranging from 0.5% to 30% by weight relative to the total weight of the composition. 5. Composition according to claim 1, wherein the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone is present in a content ranging from 0.01% to 10% by weight, relative to the total weight of the composition. 6. Composition according to claim 1, wherein the said organic solvent and the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone are present in a solvent/4-(3-ethoxy-4-hydroxyphenyl)-2-butanone mass ratio of less than or equal to 10. 7. Composition according to claim 1, wherein it is in the form of a water-in-oil or oil-in-water emulsion, preferably an oil-in-water emulsion. 8. Non-therapeutic cosmetic treatment process for caring for and/or making up and/or cleansing keratin materials, comprising the application to the said keratin materials of a composition according to claim 1. 9. Composition according to claim 2, wherein the organic solvent is present in a content ranging from 0.05% to 30% by weight relative to the total weight of the composition. 10. Composition according to claim 3, wherein the organic solvent is present in a content ranging from 0.05% to 30% by weight relative to the total weight of the composition. 11. Composition according to claim 1, wherein the organic solvent is present in a content ranging from 0.05% to 10% by weight relative to the total weight of the composition. 12. Composition according to claim 2, wherein the organic solvent is present in a content ranging from 0.1% to 5% by weight relative to the total weight of the composition. 13. Composition according to claim 2, wherein the organic solvent is present in a content ranging from 0.1% to 2.5% by weight relative to the total weight of the composition. 14. Composition according to claim 2, wherein the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone is present in a content ranging from 0.01% to 10% by weight, relative to the total weight of the composition. 15. Composition according to claim 3, wherein the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone is present in a content ranging from 0.01% to 10% by weight, relative to the total weight of the composition. 16. Composition according to claim 4, wherein the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone is present in a content ranging from 0.01% to 10% by weight, relative to the total weight of the composition. 17. Composition according to claim 2, wherein the said organic solvent and the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone are present in a solvent/4-(3-ethoxy-4-hydroxyphenyl)-2-butanone mass ratio of less than or equal to 10. 18. Composition according to claim 3, wherein the said organic solvent and the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone are present in a solvent/4-(3-ethoxy-4-hydroxyphenyl)-2-butanone mass ratio of less than or equal to 10. 19. Composition according to claim 4, wherein the said organic solvent and the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone are present in a solvent/4-(3-ethoxy-4-hydroxyphenyl)-2-butanone mass ratio of less than or equal to 10. 20. Composition according to claim 5, wherein the said organic solvent and the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone are present in a solvent/4-(3-ethoxy-4-hydroxyphenyl)-2-butanone mass ratio of less than or equal to 10.

The invention relates to a composition comprising, in a physiologically acceptable aqueous medium, 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone and a solvent with solubility parameters in the Hansen solubility space such that 4.5<δ, <30 and 15<δ d <22. Use for caring for, making up and cleansing keratin materials.1. Composition comprising, in a physiologically acceptable aqueous medium: 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone and an organic solvent with solubility parameters in the Hansen solubility space such that 14.5<δa<30 and 15<δd<22. 2. Composition according to the preceding claim, wherein the organic solvent is chosen from ethanol, 1,2-propylene glycol, 1,3-propanediol, PEG-8 (polyethylene glycol containing 8 ethylene glycol units), propylene carbonate, dipropylene glycol, 1,2-hexylene glycol and PEG-4 (polyethylene glycol containing 4 ethylene glycol units). 3. Composition according to claim 1, wherein the organic solvent is chosen from ethanol, 1,2-propylene glycol, 1,3-propanediol, PEG-8 and propylene carbonate. 4. Composition according to claim 1, wherein the organic solvent is present in a content ranging from 0.5% to 30% by weight relative to the total weight of the composition. 5. Composition according to claim 1, wherein the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone is present in a content ranging from 0.01% to 10% by weight, relative to the total weight of the composition. 6. Composition according to claim 1, wherein the said organic solvent and the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone are present in a solvent/4-(3-ethoxy-4-hydroxyphenyl)-2-butanone mass ratio of less than or equal to 10. 7. Composition according to claim 1, wherein it is in the form of a water-in-oil or oil-in-water emulsion, preferably an oil-in-water emulsion. 8. Non-therapeutic cosmetic treatment process for caring for and/or making up and/or cleansing keratin materials, comprising the application to the said keratin materials of a composition according to claim 1. 9. Composition according to claim 2, wherein the organic solvent is present in a content ranging from 0.05% to 30% by weight relative to the total weight of the composition. 10. Composition according to claim 3, wherein the organic solvent is present in a content ranging from 0.05% to 30% by weight relative to the total weight of the composition. 11. Composition according to claim 1, wherein the organic solvent is present in a content ranging from 0.05% to 10% by weight relative to the total weight of the composition. 12. Composition according to claim 2, wherein the organic solvent is present in a content ranging from 0.1% to 5% by weight relative to the total weight of the composition. 13. Composition according to claim 2, wherein the organic solvent is present in a content ranging from 0.1% to 2.5% by weight relative to the total weight of the composition. 14. Composition according to claim 2, wherein the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone is present in a content ranging from 0.01% to 10% by weight, relative to the total weight of the composition. 15. Composition according to claim 3, wherein the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone is present in a content ranging from 0.01% to 10% by weight, relative to the total weight of the composition. 16. Composition according to claim 4, wherein the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone is present in a content ranging from 0.01% to 10% by weight, relative to the total weight of the composition. 17. Composition according to claim 2, wherein the said organic solvent and the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone are present in a solvent/4-(3-ethoxy-4-hydroxyphenyl)-2-butanone mass ratio of less than or equal to 10. 18. Composition according to claim 3, wherein the said organic solvent and the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone are present in a solvent/4-(3-ethoxy-4-hydroxyphenyl)-2-butanone mass ratio of less than or equal to 10. 19. Composition according to claim 4, wherein the said organic solvent and the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone are present in a solvent/4-(3-ethoxy-4-hydroxyphenyl)-2-butanone mass ratio of less than or equal to 10. 20. Composition according to claim 5, wherein the said organic solvent and the 4-(3-ethoxy-4-hydroxyphenyl)-2-butanone are present in a solvent/4-(3-ethoxy-4-hydroxyphenyl)-2-butanone mass ratio of less than or equal to 10.

The present invention relates to the use, in a cosmetic, dermatological or pharmaceutical composition, of at least one compound of formula (I): in which: R2 represents a hydrogen atom or a methyl or ethyl radical; R3 represents a linear C1-C12 alkyl radical, optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical, optionally substituted with a hydroxyl group; as a preserving agent. The invention also relates to certain novel compounds and to the cosmetic, dermatological or pharmaceutical compositions comprising them.

1. Use as a preserving agent, in particular in a cosmetic, dermatological, pharmaceutical, nutraceutical or oral cosmetic composition, of at least one compound of formula (I): in which: either R2 represents a hydrogen atom and R3 represents a linear C1-C6 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C6 alkenyl radical (C═C unsaturated), or alternatively a linear C2-C12 alkenyl radical optionally substituted with a hydroxyl group; or R2 represents a methyl or ethyl radical and R3 represents a linear C1-C12 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical (C═C unsaturated), optionally substituted with a hydroxyl group. 2. Use according to claim 1, in which the compounds correspond to the formula (I), in which: (i) R2 is H and R3 represents a methyl, ethyl, propyl, butyl or pentyl radical, optionally substituted with an OH and especially of structure —CH2-CH(OH)—R5 with R5 representing a linear C1-C4 alkyl radical; or a C2-C6 alkenyl radical, especially a radical —CH═CH—R4 with R4 representing a linear C1-C4 alkyl radical; or (ii) R2 represents CH3 and R3 represents (i) a C1-C10 alkyl radical, especially methyl, ethyl, propyl, butyl, pentyl or hexyl; (ii) a C2-C10 alkenyl radical, especially a radical —CH═CH—R4 with R4 representing a linear C1-C6 alkyl radical; or alternatively (iii) a hydroxyalkyl radical of structure —CH2-CH(OH)—R5 with R5 representing a linear C1-C10 and preferably C4-C10 alkyl radical. 3. Use according to claim 1, in which the compound of formula (I) is chosen from the following compounds: 4. Use according to claim 1, in which the compound of formula (I), alone or as a mixture, is present in a proportion of from 0.01% to 5% by weight and especially 0.1% to 2.5% by weight, relative to the weight of the composition. 5. Use according to Claim 1, in which the composition comprises a physiologically acceptable medium which comprises at least one ingredient chosen from silicone fatty substances such as silicone oils, gums and waxes; non-silicone fatty substances such as oils, pastes and waxes of plant, mineral, animal and/or synthetic origin; fatty acids having from 8 to 32 carbon atoms; synthetic esters and ethers, in particular of formula R1COOR2 and R1OR2 in which R1 represents the residue of a fatty acid comprising from 8 to 29 carbon atoms, and R2 represents a branched or unbranched hydrocarbon-based chain containing from 3 to 30 carbon atoms; linear or branched hydrocarbons of mineral or synthetic origin; fatty alcohols having from 8 to 26 carbon atoms; water; C2-C6 alcohols; glycols such as propylene glycol, ketones; thickeners, emulsifiers, surfactants, gelling agents, active cosmetic agents, fragrances, fillers, dyestuffs, moisturizers, vitamins and polymers. 6. Use according to claim 1, in which the composition is in the form of a product for making up the skin of the face, body or lips; an aftershave gel or lotion; a hair-removing cream; a body hygiene composition such as a shower gel or a shampoo; a pharmaceutical composition; a solid composition such as a soap or a cleansing bar; an aerosol composition also comprising a pressurized propellent; a hair-setting lotion, a hair-styling cream or gel, a dyeing composition, a hair-restructuring lotion, a permanent-wave composition, a lotion or a gel for combating hair loss; or a composition for oro dental use. 7. Process for preserving a cosmetic, dermatological, pharmaceutical, nutraceutical or oral cosmetic composition, wherein it consists in incorporating into the said composition at least one compound of formula (I) as defined in claim 1. 8. Compound of formula (I′): in which: either R2 represents a hydrogen atom and R3 represents a linear C2-C6 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C6 alkenyl radical (C═C unsaturated), or alternatively a linear C2-C12 alkenyl radical optionally substituted with a hydroxyl group; or R2 represents a methyl or ethyl radical and R3 represents a linear C1-C12 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical (C═C unsaturated), optionally substituted with a hydroxyl group. 9. Compound according to claim 8, in which: (i) R2 is H and R3 represents an ethyl, propyl, butyl or pentyl radical, optionally substituted with an OH and especially of structure —CH2-CH(OH)—R5 with R5 representing a linear C1-C4 alkyl radical; or a C2-C6 alkenyl radical, especially a radical —CH═CH—R4 with R4 representing a linear C1-C4 alkyl radical; or (ii) R2 represents CH3 and R3 represents (i) a C1-C10 alkyl radical, especially methyl, ethyl, propyl, butyl, pentyl or hexyl; (ii) a C2-C10 alkenyl radical, especially a radical —CH═CH—R4 with R4 representing a linear C1-C6 alkyl radical; or alternatively (iii) a hydroxyalkyl radical of structure —CH2-CH(OH)—R5 with R5 representing a linear C1-C10 and preferably C4-C10 alkyl radical. 10. Compound according to claim 8, of formula: 11. Cosmetic, dermatological or pharmaceutical composition comprising at least one compound of formula (I): in which: either R2 represents a hydrogen atom and R3 represents a linear C1-C6 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C6 alkenyl radical (C═C unsaturated), or alternatively a linear C2-C12 alkenyl radical optionally substituted with a hydroxyl group; or R2 represents a methyl or ethyl radical and R3 represents a linear C1-C12 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical (C═C unsaturated), optionally substituted with a hydroxyl group. 12. Nutraceutical or oral cosmetic composition comprising at least one compound of formula (I): in which: either R2 represents a hydrogen atom and R3 represents a linear C1-C6 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C6 alkenyl radical (C═C unsaturated), or alternatively a linear C2-C12 alkenyl radical optionally substituted with a hydroxyl group; or R2 represents a methyl or ethyl radical and R3 represents a linear C1-C12 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical (C═C unsaturated), optionally substituted with a hydroxyl group. 13. Composition according to claim 11, in which the compound of formula (I) is chosen from the compounds of formula (I′): in which: either R2 represents a hydrogen atom and R3 represents a linear C2-C6 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C6 alkenyl radical (C═C unsaturated), or alternatively a linear C2-C12 alkenyl radical optionally substituted with a hydroxyl group; or R2 represents a methyl or ethyl radical and R3 represents a linear C1-C12 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical (C═C unsaturated), optionally substituted with a hydroxyl group. 14. Composition according to claim 11, in which the compound of formula (I), alone or as a mixture, is present in a proportion of from 0.01% to 5% by weight and especially 0.1% to 2.5% by weight, relative to the weight of the composition. 15. Composition according to claim 11, comprising a physiologically acceptable medium which comprises at least one ingredient chosen from silicone fatty substances such as silicone oils, gums and waxes; non-silicone fatty substances such as oils, pastes and waxes of plant, mineral, animal and/or synthetic origin; fatty acids having from 8 to 32 carbon atoms; synthetic esters and ethers, in particular of formula R1COOR2 and R1OR2 in which R1 represents the residue of a fatty acid comprising from 8 to 29 carbon atoms, and R2 represents a branched or unbranched hydrocarbon-based chain containing from 3 to 30 carbon atoms; linear or branched hydrocarbons of mineral or synthetic origin; fatty alcohols having from 8 to 26 carbon atoms; water; C2-C6 alcohols; glycols such as propylene glycol, ketones; thickeners, emulsifiers, surfactants, gelling agents, active cosmetic agents, fragrances, fillers, dyestuffs, moisturizers, vitamins and polymers. 16. Composition according to claim 11, which is in the form of a product for making up the skin of the face, body or lips; an aftershave gel or lotion; a hair-removing cream; a body hygiene composition such as a shower gel or a shampoo; a pharmaceutical composition; a solid composition such as a soap or a cleansing bar; an aerosol composition also comprising a pressurized propellent; a hair-setting lotion, a hair-styling cream or gel, a dyeing composition, a hair-restructuring lotion, a permanent-wave composition, a lotion or a gel for combating hair loss; or a composition for oro-dental use. 17. Use according claim 2, in which the compound of formula (I) is chosen from the following compounds: 18. Use according to claim 2, in which the compound of formula (I), alone or as a mixture, is present in a proportion of from 0.01% to 5% by weight and especially 0.1% to 2.5% by weight, relative to the weight of the composition. 19. Use according to claim 3, in which the compound of formula (I), alone or as a mixture, is present in a proportion of from 0.01% to 5% by weight and especially 0.1% to 2.5% by weight, relative to the weight of the composition. 20. Use according to claim 2, in which the composition comprises a physiologically acceptable medium which comprises at least one ingredient chosen from silicone fatty substances such as silicone oils, gums and waxes; non-silicone fatty substances such as oils, pastes and waxes of plant, mineral, animal and/or synthetic origin; fatty acids having from 8 to 32 carbon atoms; synthetic esters and ethers, in particular of formula R1COOR2 and R1OR2 in which R1 represents the residue of a fatty acid comprising from 8 to 29 carbon atoms, and R2 represents a branched or unbranched hydrocarbon-based chain containing from 3 to 30 carbon atoms; linear or branched hydrocarbons of mineral or synthetic origin; fatty alcohols having from 8 to 26 carbon atoms; water; C2-C6 alcohols; glycols such as propylene glycol, ketones; thickeners, emulsifiers, surfactants, gelling agents, active cosmetic agents, fragrances, fillers, dyestuffs, moisturizers, vitamins and polymers.

The present invention relates to the use, in a cosmetic, dermatological or pharmaceutical composition, of at least one compound of formula (I): in which: R2 represents a hydrogen atom or a methyl or ethyl radical; R3 represents a linear C1-C12 alkyl radical, optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical, optionally substituted with a hydroxyl group; as a preserving agent. The invention also relates to certain novel compounds and to the cosmetic, dermatological or pharmaceutical compositions comprising them.1. Use as a preserving agent, in particular in a cosmetic, dermatological, pharmaceutical, nutraceutical or oral cosmetic composition, of at least one compound of formula (I): in which: either R2 represents a hydrogen atom and R3 represents a linear C1-C6 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C6 alkenyl radical (C═C unsaturated), or alternatively a linear C2-C12 alkenyl radical optionally substituted with a hydroxyl group; or R2 represents a methyl or ethyl radical and R3 represents a linear C1-C12 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical (C═C unsaturated), optionally substituted with a hydroxyl group. 2. Use according to claim 1, in which the compounds correspond to the formula (I), in which: (i) R2 is H and R3 represents a methyl, ethyl, propyl, butyl or pentyl radical, optionally substituted with an OH and especially of structure —CH2-CH(OH)—R5 with R5 representing a linear C1-C4 alkyl radical; or a C2-C6 alkenyl radical, especially a radical —CH═CH—R4 with R4 representing a linear C1-C4 alkyl radical; or (ii) R2 represents CH3 and R3 represents (i) a C1-C10 alkyl radical, especially methyl, ethyl, propyl, butyl, pentyl or hexyl; (ii) a C2-C10 alkenyl radical, especially a radical —CH═CH—R4 with R4 representing a linear C1-C6 alkyl radical; or alternatively (iii) a hydroxyalkyl radical of structure —CH2-CH(OH)—R5 with R5 representing a linear C1-C10 and preferably C4-C10 alkyl radical. 3. Use according to claim 1, in which the compound of formula (I) is chosen from the following compounds: 4. Use according to claim 1, in which the compound of formula (I), alone or as a mixture, is present in a proportion of from 0.01% to 5% by weight and especially 0.1% to 2.5% by weight, relative to the weight of the composition. 5. Use according to Claim 1, in which the composition comprises a physiologically acceptable medium which comprises at least one ingredient chosen from silicone fatty substances such as silicone oils, gums and waxes; non-silicone fatty substances such as oils, pastes and waxes of plant, mineral, animal and/or synthetic origin; fatty acids having from 8 to 32 carbon atoms; synthetic esters and ethers, in particular of formula R1COOR2 and R1OR2 in which R1 represents the residue of a fatty acid comprising from 8 to 29 carbon atoms, and R2 represents a branched or unbranched hydrocarbon-based chain containing from 3 to 30 carbon atoms; linear or branched hydrocarbons of mineral or synthetic origin; fatty alcohols having from 8 to 26 carbon atoms; water; C2-C6 alcohols; glycols such as propylene glycol, ketones; thickeners, emulsifiers, surfactants, gelling agents, active cosmetic agents, fragrances, fillers, dyestuffs, moisturizers, vitamins and polymers. 6. Use according to claim 1, in which the composition is in the form of a product for making up the skin of the face, body or lips; an aftershave gel or lotion; a hair-removing cream; a body hygiene composition such as a shower gel or a shampoo; a pharmaceutical composition; a solid composition such as a soap or a cleansing bar; an aerosol composition also comprising a pressurized propellent; a hair-setting lotion, a hair-styling cream or gel, a dyeing composition, a hair-restructuring lotion, a permanent-wave composition, a lotion or a gel for combating hair loss; or a composition for oro dental use. 7. Process for preserving a cosmetic, dermatological, pharmaceutical, nutraceutical or oral cosmetic composition, wherein it consists in incorporating into the said composition at least one compound of formula (I) as defined in claim 1. 8. Compound of formula (I′): in which: either R2 represents a hydrogen atom and R3 represents a linear C2-C6 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C6 alkenyl radical (C═C unsaturated), or alternatively a linear C2-C12 alkenyl radical optionally substituted with a hydroxyl group; or R2 represents a methyl or ethyl radical and R3 represents a linear C1-C12 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical (C═C unsaturated), optionally substituted with a hydroxyl group. 9. Compound according to claim 8, in which: (i) R2 is H and R3 represents an ethyl, propyl, butyl or pentyl radical, optionally substituted with an OH and especially of structure —CH2-CH(OH)—R5 with R5 representing a linear C1-C4 alkyl radical; or a C2-C6 alkenyl radical, especially a radical —CH═CH—R4 with R4 representing a linear C1-C4 alkyl radical; or (ii) R2 represents CH3 and R3 represents (i) a C1-C10 alkyl radical, especially methyl, ethyl, propyl, butyl, pentyl or hexyl; (ii) a C2-C10 alkenyl radical, especially a radical —CH═CH—R4 with R4 representing a linear C1-C6 alkyl radical; or alternatively (iii) a hydroxyalkyl radical of structure —CH2-CH(OH)—R5 with R5 representing a linear C1-C10 and preferably C4-C10 alkyl radical. 10. Compound according to claim 8, of formula: 11. Cosmetic, dermatological or pharmaceutical composition comprising at least one compound of formula (I): in which: either R2 represents a hydrogen atom and R3 represents a linear C1-C6 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C6 alkenyl radical (C═C unsaturated), or alternatively a linear C2-C12 alkenyl radical optionally substituted with a hydroxyl group; or R2 represents a methyl or ethyl radical and R3 represents a linear C1-C12 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical (C═C unsaturated), optionally substituted with a hydroxyl group. 12. Nutraceutical or oral cosmetic composition comprising at least one compound of formula (I): in which: either R2 represents a hydrogen atom and R3 represents a linear C1-C6 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C6 alkenyl radical (C═C unsaturated), or alternatively a linear C2-C12 alkenyl radical optionally substituted with a hydroxyl group; or R2 represents a methyl or ethyl radical and R3 represents a linear C1-C12 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical (C═C unsaturated), optionally substituted with a hydroxyl group. 13. Composition according to claim 11, in which the compound of formula (I) is chosen from the compounds of formula (I′): in which: either R2 represents a hydrogen atom and R3 represents a linear C2-C6 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C6 alkenyl radical (C═C unsaturated), or alternatively a linear C2-C12 alkenyl radical optionally substituted with a hydroxyl group; or R2 represents a methyl or ethyl radical and R3 represents a linear C1-C12 alkyl radical (saturated), optionally substituted with a hydroxyl group; or a linear C2-C12 alkenyl radical (C═C unsaturated), optionally substituted with a hydroxyl group. 14. Composition according to claim 11, in which the compound of formula (I), alone or as a mixture, is present in a proportion of from 0.01% to 5% by weight and especially 0.1% to 2.5% by weight, relative to the weight of the composition. 15. Composition according to claim 11, comprising a physiologically acceptable medium which comprises at least one ingredient chosen from silicone fatty substances such as silicone oils, gums and waxes; non-silicone fatty substances such as oils, pastes and waxes of plant, mineral, animal and/or synthetic origin; fatty acids having from 8 to 32 carbon atoms; synthetic esters and ethers, in particular of formula R1COOR2 and R1OR2 in which R1 represents the residue of a fatty acid comprising from 8 to 29 carbon atoms, and R2 represents a branched or unbranched hydrocarbon-based chain containing from 3 to 30 carbon atoms; linear or branched hydrocarbons of mineral or synthetic origin; fatty alcohols having from 8 to 26 carbon atoms; water; C2-C6 alcohols; glycols such as propylene glycol, ketones; thickeners, emulsifiers, surfactants, gelling agents, active cosmetic agents, fragrances, fillers, dyestuffs, moisturizers, vitamins and polymers. 16. Composition according to claim 11, which is in the form of a product for making up the skin of the face, body or lips; an aftershave gel or lotion; a hair-removing cream; a body hygiene composition such as a shower gel or a shampoo; a pharmaceutical composition; a solid composition such as a soap or a cleansing bar; an aerosol composition also comprising a pressurized propellent; a hair-setting lotion, a hair-styling cream or gel, a dyeing composition, a hair-restructuring lotion, a permanent-wave composition, a lotion or a gel for combating hair loss; or a composition for oro-dental use. 17. Use according claim 2, in which the compound of formula (I) is chosen from the following compounds: 18. Use according to claim 2, in which the compound of formula (I), alone or as a mixture, is present in a proportion of from 0.01% to 5% by weight and especially 0.1% to 2.5% by weight, relative to the weight of the composition. 19. Use according to claim 3, in which the compound of formula (I), alone or as a mixture, is present in a proportion of from 0.01% to 5% by weight and especially 0.1% to 2.5% by weight, relative to the weight of the composition. 20. Use according to claim 2, in which the composition comprises a physiologically acceptable medium which comprises at least one ingredient chosen from silicone fatty substances such as silicone oils, gums and waxes; non-silicone fatty substances such as oils, pastes and waxes of plant, mineral, animal and/or synthetic origin; fatty acids having from 8 to 32 carbon atoms; synthetic esters and ethers, in particular of formula R1COOR2 and R1OR2 in which R1 represents the residue of a fatty acid comprising from 8 to 29 carbon atoms, and R2 represents a branched or unbranched hydrocarbon-based chain containing from 3 to 30 carbon atoms; linear or branched hydrocarbons of mineral or synthetic origin; fatty alcohols having from 8 to 26 carbon atoms; water; C2-C6 alcohols; glycols such as propylene glycol, ketones; thickeners, emulsifiers, surfactants, gelling agents, active cosmetic agents, fragrances, fillers, dyestuffs, moisturizers, vitamins and polymers.

The invention is generally directed to reducing inflammation by means of cells that secrete factors that reduce leukocyte extravasation. Specifically, the invention is directed to methods using cells that secrete factors that downregulate the expression of cellular adhesion molecules in vascular endothelial cells. Downregulating expression of cellular adhesion molecules reduces leukocyte adhesion to the endothelial cells such that extravasation is reduced. The end result is a reduction of inflammation. The cells are non-embryonic non-germ cells that have pluripotent characteristics. These may include expression of pluripotential markers and broad differentiation potential.

1. (canceled) 2. A method to treat inflammation in a subject, the method comprising assessing cells in a preparation of cells for a desired potency for one or more of the following: (1) reduce leukocyte extravasation, (2) reduce leukocyte adhesion to vascular endothelium or to isolated endothelial cells, (3) reduce cytokine-mediated activation of endothelial cells, (4) reduce expression of one or more cell adhesion molecules on an endothelial cell, and, where the assessed cells are found to have the desired potency, administering cells from that preparation to the subject in a therapeutically effective amount and for a time sufficient to achieve a therapeutic result, the assessed and administered cells being non-embryonic, non-germ cells that express one or more of oct4, telomerase, rex-1, or rox-1 and/or can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers. 3. A method to construct a cell bank, the method comprising assessing cells in a preparation of cells for a desired potency for one or more of the following: (1) reduce leukocyte extravasation, (2) reduce leukocyte adhesion to vascular endothelium or to isolated endothelial cells, (3) reduce cytokine-mediated activation of endothelial cells, (4) reduce expression of one or more cell adhesion molecules on an endothelial cell, and, where the assessed cells are found to have the desired potency, expanding and storing cells from that preparation for future administration to a subject, the assessed, expanded, and stored cells being non-embryonic, non-germ cells that express one or more of oct4, telomerase, rex-1, or rox-1 and/or can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers. 4. A method for drug discovery, the method comprising assessing cells in a preparation of cells for a desired potency for one or more of the following: (1) reduce leukocyte extravasation, (2) reduce leukocyte adhesion to vascular endothelium or to isolated endothelial cells, (3) reduce cytokine-mediated activation of endothelial cells, (4) reduce expression of one or more cell adhesion molecules on an endothelial cell, and, where the assessed cells are found to have the desired potency, contacting cells from that preparation with an agent to assess the effect of the agent on the ability of the cells to effect any of (1)-(4), the assessed and contacted cells being non-embryonic, non-germ cells that express one or more of oct4, telomerase, rex-1, or rox-1 and/or can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers. 5. A composition comprising cells assessed and selected for the desired potency to achieve one or more of the following: 1) reduce leukocyte extravasation, (2) reduce leukocyte adhesion to vascular endothelium or to isolated endothelial cells, (3) reduce cytokine-mediated activation of endothelial cells, (4) reduce expression of one or more cell adhesion molecules on an endothelial cell; the cells being non-embryonic, non-germ cells that express one or more of oct4, telomerase, rex-1, or rox-1 and/or can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers. 6-8. (canceled) 9. A method to increase the potency of a cell to effect one of more of the following: 1) reduce leukocyte extravasation, (2) reduce leukocyte adhesion to vascular endothelium or to isolated endothelial cells, (3) reduce cytokine-mediated activation of endothelial cells, (4) reduce expression of one or more cell adhesion molecules on an endothelial cell; the method comprising contacting cells with IFN-γ, IL-1β, and TNF-α to produce cells that achieve one or more of effects (1)-(4); the cells being non-embryonic, non-germ cells that express one or more of oct4, telomerase, rex-1, or rox-1 and/or can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers. 10. The method of any of claims 2-4 and 9 wherein the cytokine is selected from the group consisting of TNF-α, and IL-1. 11. The method of any of claims 2-4 and 9 wherein cytokine-mediated activation produces an increase in expression of one or more of E-selectin, P-selectin, VCAM-1, ICAM, and VCAM-2 in the endothelial cell. 12. The methods of any of claims 2-4 and 9 wherein the cell adhesion molecule, the expression of which is reduced in the endothelial cell, is one or more of E-selectin, P-selectin, VCAM-1, ICAM, and VCAM-2.

The invention is generally directed to reducing inflammation by means of cells that secrete factors that reduce leukocyte extravasation. Specifically, the invention is directed to methods using cells that secrete factors that downregulate the expression of cellular adhesion molecules in vascular endothelial cells. Downregulating expression of cellular adhesion molecules reduces leukocyte adhesion to the endothelial cells such that extravasation is reduced. The end result is a reduction of inflammation. The cells are non-embryonic non-germ cells that have pluripotent characteristics. These may include expression of pluripotential markers and broad differentiation potential.1. (canceled) 2. A method to treat inflammation in a subject, the method comprising assessing cells in a preparation of cells for a desired potency for one or more of the following: (1) reduce leukocyte extravasation, (2) reduce leukocyte adhesion to vascular endothelium or to isolated endothelial cells, (3) reduce cytokine-mediated activation of endothelial cells, (4) reduce expression of one or more cell adhesion molecules on an endothelial cell, and, where the assessed cells are found to have the desired potency, administering cells from that preparation to the subject in a therapeutically effective amount and for a time sufficient to achieve a therapeutic result, the assessed and administered cells being non-embryonic, non-germ cells that express one or more of oct4, telomerase, rex-1, or rox-1 and/or can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers. 3. A method to construct a cell bank, the method comprising assessing cells in a preparation of cells for a desired potency for one or more of the following: (1) reduce leukocyte extravasation, (2) reduce leukocyte adhesion to vascular endothelium or to isolated endothelial cells, (3) reduce cytokine-mediated activation of endothelial cells, (4) reduce expression of one or more cell adhesion molecules on an endothelial cell, and, where the assessed cells are found to have the desired potency, expanding and storing cells from that preparation for future administration to a subject, the assessed, expanded, and stored cells being non-embryonic, non-germ cells that express one or more of oct4, telomerase, rex-1, or rox-1 and/or can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers. 4. A method for drug discovery, the method comprising assessing cells in a preparation of cells for a desired potency for one or more of the following: (1) reduce leukocyte extravasation, (2) reduce leukocyte adhesion to vascular endothelium or to isolated endothelial cells, (3) reduce cytokine-mediated activation of endothelial cells, (4) reduce expression of one or more cell adhesion molecules on an endothelial cell, and, where the assessed cells are found to have the desired potency, contacting cells from that preparation with an agent to assess the effect of the agent on the ability of the cells to effect any of (1)-(4), the assessed and contacted cells being non-embryonic, non-germ cells that express one or more of oct4, telomerase, rex-1, or rox-1 and/or can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers. 5. A composition comprising cells assessed and selected for the desired potency to achieve one or more of the following: 1) reduce leukocyte extravasation, (2) reduce leukocyte adhesion to vascular endothelium or to isolated endothelial cells, (3) reduce cytokine-mediated activation of endothelial cells, (4) reduce expression of one or more cell adhesion molecules on an endothelial cell; the cells being non-embryonic, non-germ cells that express one or more of oct4, telomerase, rex-1, or rox-1 and/or can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers. 6-8. (canceled) 9. A method to increase the potency of a cell to effect one of more of the following: 1) reduce leukocyte extravasation, (2) reduce leukocyte adhesion to vascular endothelium or to isolated endothelial cells, (3) reduce cytokine-mediated activation of endothelial cells, (4) reduce expression of one or more cell adhesion molecules on an endothelial cell; the method comprising contacting cells with IFN-γ, IL-1β, and TNF-α to produce cells that achieve one or more of effects (1)-(4); the cells being non-embryonic, non-germ cells that express one or more of oct4, telomerase, rex-1, or rox-1 and/or can differentiate into cell types of at least two of endodermal, ectodermal, and mesodermal germ layers. 10. The method of any of claims 2-4 and 9 wherein the cytokine is selected from the group consisting of TNF-α, and IL-1. 11. The method of any of claims 2-4 and 9 wherein cytokine-mediated activation produces an increase in expression of one or more of E-selectin, P-selectin, VCAM-1, ICAM, and VCAM-2 in the endothelial cell. 12. The methods of any of claims 2-4 and 9 wherein the cell adhesion molecule, the expression of which is reduced in the endothelial cell, is one or more of E-selectin, P-selectin, VCAM-1, ICAM, and VCAM-2.

Methods of treating gastrointestinal inflammatory disorders such as inflammatory bowel diseases including ulcerative colitis and Crohn's disease are provided. Also provided are methods of administering integrin beta7 antagonists, such as anti-beta7 antibodies. In addition, particular dosing regimens, including dosing regimens comprising subcutaneous administration and administration using self-inject devices are provided.

1-12. (canceled) 13. A method of treating a gastrointestinal inflammatory disorder in a patient, the method comprising administering to the patient a therapeutically effective amount of an integrin beta7 antagonist, wherein the integrin beta7 antagonist is administered subcutaneously at a flat dose. 14. The method of claim 13, wherein the integrin beta7 antagonist is a monoclonal anti-beta7 antibody. 15. The method of claim 14, wherein the anti-beta7 antibody is administered at a flat dose between 50 mg and 450 mg. 16. The method of claim 15, wherein the flat dose is 50 mg. 17. The method of claim 15, wherein the flat dose is 100 mg. 18. The method of claim 15, wherein the flat dose is 150 mg. 19. The method of claim 15, wherein the flat dose is 200 mg. 20. The method of claim 15, wherein the flat dose is 300 mg. 21. The method of claim 15, wherein the flat dose is 350 mg. 22. The method of claim 15, wherein the flat dose is 400 mg. 23. The method of claim 15, wherein the flat dose is 420 mg. 24. The method of claim 15, wherein the flat dose is 450 mg. 25. The method of claim 15, wherein the anti-beta7 antibody is administered once every week, or once or every two weeks, or once every four weeks, or once every six weeks, or once every eight weeks. 26. The method of claim 25, wherein the anti-beta7 antibody is administered for a period of two months, or three months, or six months, or 12 months, or 18 months, or 24 months, or for the lifetime of the patient. 27. The method of claim 13, wherein the patient is a human. 28. The method of claim 13, wherein the gastrointestinal inflammatory disorder is an inflammatory bowel disease. 29. The method of claim 28, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease. 30. The method of claim 14, wherein the anti-beta7 antibody is selected from a chimeric antibody, a human antibody, and a humanized antibody. 31. The method of claim 30, wherein the anti-beta7 antibody is an antibody fragment. 32. The method of claim 30, wherein the anti-beta7 antibody comprises six hypervariable regions (HVRs), wherein: (i) HVR-L1 comprises amino acid sequence A1-A11, wherein A1-A11 is RASESVDTYLH (SEQ ID NO:1); RASESVDSLLH (SEQ ID NO:7), RASESVDTLLH (SEQ ID NO:8), or RASESVDDLLH (SEQ ID NO:9) or a variant of SEQ ID NOs: 1, 7, 8 or 9 (SEQ ID NO:26) wherein amino acid A2 is selected from the group consisting of A, G, S, T, and V and/or amino acid A3 is selected from the group consisting of S, G, I, K, N, P, Q, R, and T, and/or A4 is selected from the group consisting of E, V, Q, A, D, G, H, I, K, L, N, and R, and/or amino acid A5 is selected from the group consisting of S, Y, A, D, G, H, I, K, N, P, R, T, and V, and/or amino acid A6 is selected from the group consisting of V, R, I, A, G, K, L, M, and Q, and/or amino acid A7 is selected from the group consisting of D, V, S, A, E, G, H, I, K, L, N, P, S, and T, and/or amino acid A8 is selected from the group consisting of D, G, N, E, T, P and S, and/or amino acid A9 is selected from the group consisting of L, Y, I and M, and/or amino acid A10 is selected from the group consisting of L, A, I, M, and V and/or amino acid A11 is selected from the group consisting of H, Y, F, and S; (ii) HVR-L2 comprises amino acid sequence B1-B8, wherein B1-B8 is KYASQSIS (SEQ ID NO:2), RYASQSIS (SEQ ID NO:20), or XaaYASQSIS (SEQ ID NO:21, where Xaa represents any amino acid) or a variant of SEQ ID NOs:2, 20 or 21 (SEQ ID NO:27) wherein amino acid B1 is selected from the group consisting of K, R, N, V, A, F, Q, H, P, I, L, Y and Xaa (where Xaa represents any amino acid), and/or amino acid B4 is selected from the group consisting of S and D, and/or amino acid B5 is selected from the group consisting of Q and S, and/or amino acid B6 is selected from the group consisting of S, D, L, and R, and/or amino acid B7 is selected from the group consisting of I, V, E, and K; (iii) HVR-L3 comprises amino acid sequence C1-C9, wherein C1-C9 is QQGNSLPNT (SEQ ID NO:3) or a variant of SEQ ID NO:3 (SEQ ID NO:28) wherein amino acid C8 is selected from the group consisting of N, V, W, Y, R, S, T, A, F, H, 1 L, and M; (iv) HVR-H1 comprises amino acid sequence D1-D10 wherein D1-D10 is GFFITNNYWG (SEQ ID NO:4); (v) HVR-H2 comprises amino acid sequence E1-E17 wherein E1-E17 is GYISYSGSTSYNPSLKS (SEQ ID NO:5), or a variant of SEQ ID NO:5 (SEQ ID NO:29) wherein amino acid E2 is selected from the group consisting of Y, F, V, and D, and/or amino acid E6 is selected from the group consisting of S and G, and/or amino acid E10 is selected from the group consisting of S and Y, and/or amino acid E12 is selected from the group consisting of N, T, A, and D, and/or amino acid 13 is selected from the group consisting of P, H, D, and A, and/or amino acid E15 is selected from the group consisting of L and V, and/or amino acid E11 is selected from the group consisting of S and G; and (vi) HVR-H3 comprises amino acid sequence F2-F 11 wherein F2-F 11 is MTGSSGYFDF (SEQ ID NO:6) or RTGSSGYFDF (SEQ ID NO:19); or comprises amino acid sequence F1-F11, wherein F1-F11 is AMTGSSGYFDF (SEQ ID NO:16), ARTGSSGYFDF (SEQ ID NO:17), or AQTGSSGYFDF (SEQ ID NO:18), or a variant of SEQ ID NOs:6, 16, 17, 18, or 19 (SEQ ID NO:30) wherein amino acid F2 is R, M, A, E, G, Q, S, and/or amino acid F11 is selected from the group consisting of F and Y. 33. The method of claim 32, wherein the anti-beta7 antibody comprises three heavy chain hypervariable region (HVR-H1-H3) sequences and three light chain hypervariable region (HVR-L1-L3) sequences, wherein: (i) HVR-L1 comprises SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; (ii) HVR-L2 comprises SEQ ID NO:2; (iii) HVR-L3 comprises SEQ ID NO:3; (iv) HVR-H1 comprises SEQ ID NO:4; (v) HVR-H2 comprises SEQ ID NO:5; and (vi) HVR-H3 comprises SEQ ID NO:6 or SEQ ID NO:16 or SEQ ID NO:17 or SEQ ID NO:19. 34. The method of claim 33, wherein the anti-beta7 antibody is administered at a flat dose of 100 mg every four weeks, wherein the patient suffers from ulcerative colitis, and wherein the patient experiences mucosal healing or a reduced rate of flare over time. 35. The method of claim 33, wherein the anti-beta7 antibody is administered at a flat dose of 300 mg every four weeks, wherein the patient suffers from ulcerative colitis, and wherein the patient experiences mucosal healing or a reduced rate of flare over time. 36-64. (canceled) 65. The method according to any one of claims 15, 25, or 32, wherein the integrin beta7 antagonist is administered with at least one additional compound selected from 5-aminosalicylic acid (5-ASA), azathioprine (AZA), 6-mercaptopurine (6-MP), and methotrexate. 66. The method according to any one of claims 15, 25, or 32, wherein the patient previously failed at least one biological agent. 67. The method of claim 66, wherein the biological agent is selected from adalimumab, etanercept, infliximab, golimumab, certolizumab pegol, natalizumab, and vedolizumab. 68. The method of any one of claim 15, 25, or 32, wherein the integrin beta7 antagonist is administered with a self-inject device. 69. The method of claim 68, wherein the self-inject device is selected from a prefilled syringe, microneedle device, autoinjector device and needle-free injection device. 70. An article of manufacture comprising a prefilled syringe comprising a liquid formulation of integrin beta7 antagonist, wherein the volume of the formulation is 2 ML and the amount of integrin beta7 antagonist is 150 mg. 71. The article of manufacture of claim 70, wherein the integrin beta7 antagonist is etrolizumab. 72. An article of manufacture comprising a prefilled syringe comprising a liquid formulation of integrin beta7 antagonist, wherein the volume of the formulation is 1 ML and the amount of integrin beta7 antagonist is 180 mg. 73. The article of manufacture of claim 72, wherein the integrin beta7 antagonist is etrolizumab. 74. A method of inducing remission in a patient suffering from ulcerative colitis comprising administering to the patient a therapeutically effective amount of etrolizumab, wherein etrolizumab is administered subcutaneously at a flat dose of either 100 mg or 300 mg every four weeks. 75. The method of claim 74, further comprising determining a Mayo Clinic Score and Mayo Clinic subscores, wherein the patient is determined to have an absolute Mayo Clinic Score ≦2 and no individual subscore >1. 76. The method of claim 75, wherein remission is induced at least by week 10 following the first administration of etrolizumab. 77. A method of inducing sustained remission in a patient suffering from ulcerative colitis, comprising administering to the patient a therapeutically effective amount of etrolizumab, wherein etrolizumab is administered subcutaneously at a flat dose of either 100 mg or 300 mg every four weeks, wherein remission is retained for a period of 8 weeks following induction of remission, or a period of 30 weeks following induction of remission, or a period of 50 weeks following induction of remission, or a period of 54 weeks or more following induction of remission. 78. The method of claim 77, wherein the remission is steroid-free remission. 79. The method of claim 78, wherein the steroid-free remission is for 20 weeks following induction of remission, or for 24 weeks or longer following induction of remission.

Methods of treating gastrointestinal inflammatory disorders such as inflammatory bowel diseases including ulcerative colitis and Crohn's disease are provided. Also provided are methods of administering integrin beta7 antagonists, such as anti-beta7 antibodies. In addition, particular dosing regimens, including dosing regimens comprising subcutaneous administration and administration using self-inject devices are provided.1-12. (canceled) 13. A method of treating a gastrointestinal inflammatory disorder in a patient, the method comprising administering to the patient a therapeutically effective amount of an integrin beta7 antagonist, wherein the integrin beta7 antagonist is administered subcutaneously at a flat dose. 14. The method of claim 13, wherein the integrin beta7 antagonist is a monoclonal anti-beta7 antibody. 15. The method of claim 14, wherein the anti-beta7 antibody is administered at a flat dose between 50 mg and 450 mg. 16. The method of claim 15, wherein the flat dose is 50 mg. 17. The method of claim 15, wherein the flat dose is 100 mg. 18. The method of claim 15, wherein the flat dose is 150 mg. 19. The method of claim 15, wherein the flat dose is 200 mg. 20. The method of claim 15, wherein the flat dose is 300 mg. 21. The method of claim 15, wherein the flat dose is 350 mg. 22. The method of claim 15, wherein the flat dose is 400 mg. 23. The method of claim 15, wherein the flat dose is 420 mg. 24. The method of claim 15, wherein the flat dose is 450 mg. 25. The method of claim 15, wherein the anti-beta7 antibody is administered once every week, or once or every two weeks, or once every four weeks, or once every six weeks, or once every eight weeks. 26. The method of claim 25, wherein the anti-beta7 antibody is administered for a period of two months, or three months, or six months, or 12 months, or 18 months, or 24 months, or for the lifetime of the patient. 27. The method of claim 13, wherein the patient is a human. 28. The method of claim 13, wherein the gastrointestinal inflammatory disorder is an inflammatory bowel disease. 29. The method of claim 28, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease. 30. The method of claim 14, wherein the anti-beta7 antibody is selected from a chimeric antibody, a human antibody, and a humanized antibody. 31. The method of claim 30, wherein the anti-beta7 antibody is an antibody fragment. 32. The method of claim 30, wherein the anti-beta7 antibody comprises six hypervariable regions (HVRs), wherein: (i) HVR-L1 comprises amino acid sequence A1-A11, wherein A1-A11 is RASESVDTYLH (SEQ ID NO:1); RASESVDSLLH (SEQ ID NO:7), RASESVDTLLH (SEQ ID NO:8), or RASESVDDLLH (SEQ ID NO:9) or a variant of SEQ ID NOs: 1, 7, 8 or 9 (SEQ ID NO:26) wherein amino acid A2 is selected from the group consisting of A, G, S, T, and V and/or amino acid A3 is selected from the group consisting of S, G, I, K, N, P, Q, R, and T, and/or A4 is selected from the group consisting of E, V, Q, A, D, G, H, I, K, L, N, and R, and/or amino acid A5 is selected from the group consisting of S, Y, A, D, G, H, I, K, N, P, R, T, and V, and/or amino acid A6 is selected from the group consisting of V, R, I, A, G, K, L, M, and Q, and/or amino acid A7 is selected from the group consisting of D, V, S, A, E, G, H, I, K, L, N, P, S, and T, and/or amino acid A8 is selected from the group consisting of D, G, N, E, T, P and S, and/or amino acid A9 is selected from the group consisting of L, Y, I and M, and/or amino acid A10 is selected from the group consisting of L, A, I, M, and V and/or amino acid A11 is selected from the group consisting of H, Y, F, and S; (ii) HVR-L2 comprises amino acid sequence B1-B8, wherein B1-B8 is KYASQSIS (SEQ ID NO:2), RYASQSIS (SEQ ID NO:20), or XaaYASQSIS (SEQ ID NO:21, where Xaa represents any amino acid) or a variant of SEQ ID NOs:2, 20 or 21 (SEQ ID NO:27) wherein amino acid B1 is selected from the group consisting of K, R, N, V, A, F, Q, H, P, I, L, Y and Xaa (where Xaa represents any amino acid), and/or amino acid B4 is selected from the group consisting of S and D, and/or amino acid B5 is selected from the group consisting of Q and S, and/or amino acid B6 is selected from the group consisting of S, D, L, and R, and/or amino acid B7 is selected from the group consisting of I, V, E, and K; (iii) HVR-L3 comprises amino acid sequence C1-C9, wherein C1-C9 is QQGNSLPNT (SEQ ID NO:3) or a variant of SEQ ID NO:3 (SEQ ID NO:28) wherein amino acid C8 is selected from the group consisting of N, V, W, Y, R, S, T, A, F, H, 1 L, and M; (iv) HVR-H1 comprises amino acid sequence D1-D10 wherein D1-D10 is GFFITNNYWG (SEQ ID NO:4); (v) HVR-H2 comprises amino acid sequence E1-E17 wherein E1-E17 is GYISYSGSTSYNPSLKS (SEQ ID NO:5), or a variant of SEQ ID NO:5 (SEQ ID NO:29) wherein amino acid E2 is selected from the group consisting of Y, F, V, and D, and/or amino acid E6 is selected from the group consisting of S and G, and/or amino acid E10 is selected from the group consisting of S and Y, and/or amino acid E12 is selected from the group consisting of N, T, A, and D, and/or amino acid 13 is selected from the group consisting of P, H, D, and A, and/or amino acid E15 is selected from the group consisting of L and V, and/or amino acid E11 is selected from the group consisting of S and G; and (vi) HVR-H3 comprises amino acid sequence F2-F 11 wherein F2-F 11 is MTGSSGYFDF (SEQ ID NO:6) or RTGSSGYFDF (SEQ ID NO:19); or comprises amino acid sequence F1-F11, wherein F1-F11 is AMTGSSGYFDF (SEQ ID NO:16), ARTGSSGYFDF (SEQ ID NO:17), or AQTGSSGYFDF (SEQ ID NO:18), or a variant of SEQ ID NOs:6, 16, 17, 18, or 19 (SEQ ID NO:30) wherein amino acid F2 is R, M, A, E, G, Q, S, and/or amino acid F11 is selected from the group consisting of F and Y. 33. The method of claim 32, wherein the anti-beta7 antibody comprises three heavy chain hypervariable region (HVR-H1-H3) sequences and three light chain hypervariable region (HVR-L1-L3) sequences, wherein: (i) HVR-L1 comprises SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; (ii) HVR-L2 comprises SEQ ID NO:2; (iii) HVR-L3 comprises SEQ ID NO:3; (iv) HVR-H1 comprises SEQ ID NO:4; (v) HVR-H2 comprises SEQ ID NO:5; and (vi) HVR-H3 comprises SEQ ID NO:6 or SEQ ID NO:16 or SEQ ID NO:17 or SEQ ID NO:19. 34. The method of claim 33, wherein the anti-beta7 antibody is administered at a flat dose of 100 mg every four weeks, wherein the patient suffers from ulcerative colitis, and wherein the patient experiences mucosal healing or a reduced rate of flare over time. 35. The method of claim 33, wherein the anti-beta7 antibody is administered at a flat dose of 300 mg every four weeks, wherein the patient suffers from ulcerative colitis, and wherein the patient experiences mucosal healing or a reduced rate of flare over time. 36-64. (canceled) 65. The method according to any one of claims 15, 25, or 32, wherein the integrin beta7 antagonist is administered with at least one additional compound selected from 5-aminosalicylic acid (5-ASA), azathioprine (AZA), 6-mercaptopurine (6-MP), and methotrexate. 66. The method according to any one of claims 15, 25, or 32, wherein the patient previously failed at least one biological agent. 67. The method of claim 66, wherein the biological agent is selected from adalimumab, etanercept, infliximab, golimumab, certolizumab pegol, natalizumab, and vedolizumab. 68. The method of any one of claim 15, 25, or 32, wherein the integrin beta7 antagonist is administered with a self-inject device. 69. The method of claim 68, wherein the self-inject device is selected from a prefilled syringe, microneedle device, autoinjector device and needle-free injection device. 70. An article of manufacture comprising a prefilled syringe comprising a liquid formulation of integrin beta7 antagonist, wherein the volume of the formulation is 2 ML and the amount of integrin beta7 antagonist is 150 mg. 71. The article of manufacture of claim 70, wherein the integrin beta7 antagonist is etrolizumab. 72. An article of manufacture comprising a prefilled syringe comprising a liquid formulation of integrin beta7 antagonist, wherein the volume of the formulation is 1 ML and the amount of integrin beta7 antagonist is 180 mg. 73. The article of manufacture of claim 72, wherein the integrin beta7 antagonist is etrolizumab. 74. A method of inducing remission in a patient suffering from ulcerative colitis comprising administering to the patient a therapeutically effective amount of etrolizumab, wherein etrolizumab is administered subcutaneously at a flat dose of either 100 mg or 300 mg every four weeks. 75. The method of claim 74, further comprising determining a Mayo Clinic Score and Mayo Clinic subscores, wherein the patient is determined to have an absolute Mayo Clinic Score ≦2 and no individual subscore >1. 76. The method of claim 75, wherein remission is induced at least by week 10 following the first administration of etrolizumab. 77. A method of inducing sustained remission in a patient suffering from ulcerative colitis, comprising administering to the patient a therapeutically effective amount of etrolizumab, wherein etrolizumab is administered subcutaneously at a flat dose of either 100 mg or 300 mg every four weeks, wherein remission is retained for a period of 8 weeks following induction of remission, or a period of 30 weeks following induction of remission, or a period of 50 weeks following induction of remission, or a period of 54 weeks or more following induction of remission. 78. The method of claim 77, wherein the remission is steroid-free remission. 79. The method of claim 78, wherein the steroid-free remission is for 20 weeks following induction of remission, or for 24 weeks or longer following induction of remission.

A method of identifying one or more biomarkers associated with one or more drugs effective to stop or repress proliferation of cancer cells, and a system for predicting effectiveness of the same. The method includes statistically analyzing (i) a first dataset of expression levels of proteins or glycoproteins in the cancer cells and (ii) a second dataset of responses of the cancer cells to drugs to identify at least one biomarker associated with effective repression of the cancer cells, and correlating or associating at least one protein or glycoprotein biomarker with a response of the cells to at least one of the drugs effective to stop or repress the proliferation of the cancer cells. The protein and/or glycoprotein expression level datasets may be generated experimentally or taken from published information. The method advantageously determines and/or predicts drug sensitivity of various cancer cells using protein and glycoprotein biomarkers.

1. A method of identifying one or more of a plurality of drugs effective to stop or repress proliferation of cancer cells, comprising: statistically analyzing (i) a first dataset of expression levels of a plurality of proteins or glycoproteins in said cancer cells and (ii) a second dataset of responses of said cancer cells to a plurality of drugs to identify one or more biomarkers associated with effective repression of said cancer cells; and correlating or associating at least one of said one or more biomarkers with a response of the cancer cells to at least one of said plurality of drugs effective to stop or repress the proliferation of the cancer cells. 2. A method according to claim 1, wherein said plurality of proteins or glycoproteins comprise glycoproteins. 3. A method according to claim 2, wherein said one or more biomarkers comprise one or more glycoprotein biomarkers. 4. A method according to claim 1, wherein said first and second datasets are statistically analyzed by lasso regression. 5. A method according to claim 1, wherein said cancer cells are selected from the group consisting of breast cancer cells, lung cancer cells, melanoma cells, prostate cancer cells, ovarian cancer cells, bladder cancer cells, endometrial cancer cells, kidney cancer cells, pancreatic cancer cells, colorectal cancer cells, lymphoma cells, CNS cancer cells, thyroid cancer cells, and leukemia cells. 6. A method according to claim 1, wherein said one or more biomarkers consist of one, two or three biomarkers. 7. A method according to claim 1, wherein said drugs effective to stop proliferation of cancer cells comprise (i) inhibitors of epidermal growth factor receptor and/or human epidermal growth factor receptor 2, (ii) agents that target microtubules, (iii) agents that target tubulin, (iv) agents that target nucleic acids, (v) mTOR inhibitors, (vi) PI3′ kinase inhibitors, and/or (vii) CDK inhibitors, and said biomarkers are selected from the group consisting of receptor tyrosine-protein kinase erbB-2 (PO4626), cathepsin B (P07858), cadherin-13 (P55290), bone marrow stromal antigen 2 (Q10589), neprilysin (P08473), large neutral amino acids transporter small subunit 1 (Q01650), integrin alpha-6 (P23229), dipeptidyl peptidase 1 (P53634), collagen alpha-1 (VI) chain (P12109), neutral amino acid transporter B (Q15758), transcobalamin-1 (P20061), sushi domain-containing protein 2 (Q9UGT4), podocalyxin (000592), laminin subunit beta-1 (P07942), dipeptidyl peptidase 1 (P53634), gamma-interferon-inducible lysosomal thiol reductase (P13284), neuroplalstin (Q9Y639), CD44 antigen (P16070), ubiquitin carboxyl-terminal hydrolase 5 (P45974), solute carrier family 2, facilitated glucose transporter membrane 1 (P11166), and alpha-aminoadipic semialdehyde dehydrogenase (P49419), CD276 antigen (Q5ZPR3), cathepsin Z (Q9UBR2), and serpin H1 (P50454); lysosome membrane protein 2 (Q14108), alpha-aminoadipic semialdehyde dehydrogenase (P49419), isochorismatase domain-containing protein 1 (Q96CN7), beta-mannosidase (000462), glucose-6-phosphate 1-dehydrogenase (P11413), ribonuclease UK114 (P52758), tropomyosin alpha-4 chain (P67936), ganglioside GM2 activator (P17900), granulins (P28799), steryl-sulfatase (P08842), insulin-like growth factor-binding protein 7 (Q16270), lysosomal pro-x carboxypeptidase (P42785), receptor tyrosine-protein kinase erbB-2 (PO4626), transmembrane emp24 domain-containing protein 7 (Q9Y3B3), arylsulfatase A (P15289), mucin-1 (P15941), G2/mitotic-specific cyclin-B1 (P14635), G1/S-specific cyclin-E1 (P24864), thioredoxin-dependent peroxide reductase, mitochondrial (P30048), acylaminoacyl-peptidase, putative (ApeH-1; Q97YB2), and importin subunit alpha-1 (P52292). 8. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells comprises an inhibitor of EGFR and/or HER2 selected from the group consisting of (i) afatinib, and said one or more glycoprotein biomarkers includes one or more of receptor tyrosine-protein kinase erbB-2 (PO4626), cathepsin B (P07858), cadherin-13 (P55290), bone marrow stromal antigen 2 (Q10589), and sushi domain-containing protein 2 (Q9UGT4), (ii) erlotinib, and said one or more glycoprotein biomarkers includes one or more of sushi domain-containing protein 2 (Q9UGT4), neprilysin (P08473), large neutral amino acids transporter small subunit 1 (Q01650), integrin alpha-6 (P23229), dipeptidyl peptidase 1 (P53634), collagen alpha-1 (VI) chain (P12109), and neutral amino acid transporter B (Q15758), (iii) gefitinib, and said one or more glycoprotein biomarkers includes one or more of transcobalamin-1 (P20061), sushi domain-containing protein 2 (Q9UGT4), podocalyxin (000592), large neutral amino acids transporter small subunit 1 (Q01650), laminin subunit beta-1 (P07942), and dipeptidyl peptidase 1 (P53634), and (iv) lapatinib, and said one or more glycoprotein biomarkers includes one or more of receptor tyrosine-protein kinase erbB-2 (PO4626), gamma-interferon-inducible lysosomal thiol reductase (P13284), neuroplalstin (Q9Y639), cathepsin B (P07858), CD44 antigen (P16070), and bone marrow stromal antigen 2 (Q10589). 9. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells targets tubulin or microtubules and is selected from the group consisting of (i) paclitaxel, and said one or more protein biomarkers includes one or more of ubiquitin carboxyl-terminal hydrolase 5 (P45974), solute carrier family 2, facilitated glucose transporter membrane 1 (P11166), and alpha-aminoadipic semialdehyde dehydrogenase (P49419), and said one or more glycoprotein biomarkers includes one or more of CD276 antigen (Q5ZPR3), cathepsin Z (Q9UBR2), and serpin H1 (P50454), (ii) docetaxel, and said one or more protein biomarkers includes one or more of lysosome membrane protein 2 (Q14108), alpha-aminoadipic semialdehyde dehydrogenase (P49419), and isochorismatase domain-containing protein 1 (Q96CN7), and said one or more glycoprotein biomarker includes one or more of beta-mannosidase (000462), cathepsin Z (Q9UBR2), and serpin H1 (P50454), and (iii) vinorelbine, and said one or more protein biomarkers includes one or more of glucose-6-phosphate 1-dehydrogenase (P11413), ribonuclease UK114 (P52758), and tropomyosin alpha-4 chain (P67936). 10. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells targets nucleic acids and is selected from the group consisting of gemcitabine, and said one or more glycoprotein biomarkers includes one or more of ganglioside GM2 activator (P17900), granulins (P28799), and steryl-sulfatase (P08842). 11. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells comprises an inhibitor of mTOR selected from the group consisting of (i) everolimus, and said one or more glycoprotein biomarkers includes one or more of insulin-like growth factor-binding protein 7 (Q16270), lysosomal pro-x carboxypeptidase (P42785), and receptor tyrosine-protein kinase erbB-2 (PO4626), and (ii) temsirolimus, and said one or more glycoprotein biomarkers includes one or more of transmembrane emp24 domain-containing protein 7 (Q9Y3B3), arylsulfatase A (P15289), and receptor tyrosine-protein kinase erbB-2 (PO4626). 12. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells comprises an inhibitor of PI3′ kinase, and said one or more glycoprotein biomarkers includes one or more of collagen alpha-1 (VI) chain (P12109), large neutral amino acids transporter small subunit 1 (Q01650), mucin-1 (P15941), and receptor tyrosine-protein kinase erbB-2 (PO4626). 13. A method according to claim 12, wherein said inhibitor of PI3′ kinase is BEZ235. 14. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells comprises an inhibitor of CDK, and said one or more protein biomarkers includes one or more of G2/mitotic-specific cyclin-B1 (P14635), G1/S-specific cyclin-E1 (P24864), thioredoxin-dependent peroxide reductase, mitochondrial (P30048), acylaminoacyl-peptidase, putative (ApeH-1; Q97YB2), and importin subunit alpha-1 (P52292). 15. A method of treating cancer, comprising: identifying at least one protein or glycoprotein biomarker in cancer cells from a patient; identifying one or more of a plurality of drugs that effectively stop or repress proliferation of said cancer cells from a correlation or association of said at least one protein or glycoprotein biomarker with effectiveness of said one or more drugs to stop or repress said proliferation of cancer cell lines expressing said at least one protein or glycoprotein biomarker; and administering said one or more of said plurality of drugs in a pharmaceutically acceptable carrier or excipient to said patient having said cancer cells in an amount effective to stop or repress said proliferation of said cancer cells. 16. A method according to claim 15, wherein said at least one biomarker comprises a glycoprotein biomarker. 17. A method according to claim 15, wherein said one or more of said plurality of drugs is administered orally, intravenously, or by chemotherapy infusion. 18. A system configured to predict effectiveness of one or more of a plurality of drugs to stop or repress proliferation of cancer cells, comprising: a memory storing (i) a first dataset including expression levels of a plurality of proteins or glycoproteins in said plurality of said cancer cell lines, and (ii) a second dataset including an effectiveness of each of said plurality of drugs to stop or repress proliferation of said cancer cell lines; and a computer configured to statistically analyze said first and second datasets to (i) identify and/or select at least one biomarker for each of said cancer cell lines and (ii) correlate or associate at least one of said plurality of drugs that effectively stops or represses proliferation of said cancer cells in each of said cancer cell lines with said at least one biomarker for each of said cancer cell lines. 19. The system of claim 18, wherein said computer is configured to statistically analyze said first and second datasets using lasso regression. 20. The system according to claim 18, wherein said first dataset includes expression levels of a plurality of glycoproteins, and said at least one biomarker comprises at least one glycoprotein biomarker.

A method of identifying one or more biomarkers associated with one or more drugs effective to stop or repress proliferation of cancer cells, and a system for predicting effectiveness of the same. The method includes statistically analyzing (i) a first dataset of expression levels of proteins or glycoproteins in the cancer cells and (ii) a second dataset of responses of the cancer cells to drugs to identify at least one biomarker associated with effective repression of the cancer cells, and correlating or associating at least one protein or glycoprotein biomarker with a response of the cells to at least one of the drugs effective to stop or repress the proliferation of the cancer cells. The protein and/or glycoprotein expression level datasets may be generated experimentally or taken from published information. The method advantageously determines and/or predicts drug sensitivity of various cancer cells using protein and glycoprotein biomarkers.1. A method of identifying one or more of a plurality of drugs effective to stop or repress proliferation of cancer cells, comprising: statistically analyzing (i) a first dataset of expression levels of a plurality of proteins or glycoproteins in said cancer cells and (ii) a second dataset of responses of said cancer cells to a plurality of drugs to identify one or more biomarkers associated with effective repression of said cancer cells; and correlating or associating at least one of said one or more biomarkers with a response of the cancer cells to at least one of said plurality of drugs effective to stop or repress the proliferation of the cancer cells. 2. A method according to claim 1, wherein said plurality of proteins or glycoproteins comprise glycoproteins. 3. A method according to claim 2, wherein said one or more biomarkers comprise one or more glycoprotein biomarkers. 4. A method according to claim 1, wherein said first and second datasets are statistically analyzed by lasso regression. 5. A method according to claim 1, wherein said cancer cells are selected from the group consisting of breast cancer cells, lung cancer cells, melanoma cells, prostate cancer cells, ovarian cancer cells, bladder cancer cells, endometrial cancer cells, kidney cancer cells, pancreatic cancer cells, colorectal cancer cells, lymphoma cells, CNS cancer cells, thyroid cancer cells, and leukemia cells. 6. A method according to claim 1, wherein said one or more biomarkers consist of one, two or three biomarkers. 7. A method according to claim 1, wherein said drugs effective to stop proliferation of cancer cells comprise (i) inhibitors of epidermal growth factor receptor and/or human epidermal growth factor receptor 2, (ii) agents that target microtubules, (iii) agents that target tubulin, (iv) agents that target nucleic acids, (v) mTOR inhibitors, (vi) PI3′ kinase inhibitors, and/or (vii) CDK inhibitors, and said biomarkers are selected from the group consisting of receptor tyrosine-protein kinase erbB-2 (PO4626), cathepsin B (P07858), cadherin-13 (P55290), bone marrow stromal antigen 2 (Q10589), neprilysin (P08473), large neutral amino acids transporter small subunit 1 (Q01650), integrin alpha-6 (P23229), dipeptidyl peptidase 1 (P53634), collagen alpha-1 (VI) chain (P12109), neutral amino acid transporter B (Q15758), transcobalamin-1 (P20061), sushi domain-containing protein 2 (Q9UGT4), podocalyxin (000592), laminin subunit beta-1 (P07942), dipeptidyl peptidase 1 (P53634), gamma-interferon-inducible lysosomal thiol reductase (P13284), neuroplalstin (Q9Y639), CD44 antigen (P16070), ubiquitin carboxyl-terminal hydrolase 5 (P45974), solute carrier family 2, facilitated glucose transporter membrane 1 (P11166), and alpha-aminoadipic semialdehyde dehydrogenase (P49419), CD276 antigen (Q5ZPR3), cathepsin Z (Q9UBR2), and serpin H1 (P50454); lysosome membrane protein 2 (Q14108), alpha-aminoadipic semialdehyde dehydrogenase (P49419), isochorismatase domain-containing protein 1 (Q96CN7), beta-mannosidase (000462), glucose-6-phosphate 1-dehydrogenase (P11413), ribonuclease UK114 (P52758), tropomyosin alpha-4 chain (P67936), ganglioside GM2 activator (P17900), granulins (P28799), steryl-sulfatase (P08842), insulin-like growth factor-binding protein 7 (Q16270), lysosomal pro-x carboxypeptidase (P42785), receptor tyrosine-protein kinase erbB-2 (PO4626), transmembrane emp24 domain-containing protein 7 (Q9Y3B3), arylsulfatase A (P15289), mucin-1 (P15941), G2/mitotic-specific cyclin-B1 (P14635), G1/S-specific cyclin-E1 (P24864), thioredoxin-dependent peroxide reductase, mitochondrial (P30048), acylaminoacyl-peptidase, putative (ApeH-1; Q97YB2), and importin subunit alpha-1 (P52292). 8. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells comprises an inhibitor of EGFR and/or HER2 selected from the group consisting of (i) afatinib, and said one or more glycoprotein biomarkers includes one or more of receptor tyrosine-protein kinase erbB-2 (PO4626), cathepsin B (P07858), cadherin-13 (P55290), bone marrow stromal antigen 2 (Q10589), and sushi domain-containing protein 2 (Q9UGT4), (ii) erlotinib, and said one or more glycoprotein biomarkers includes one or more of sushi domain-containing protein 2 (Q9UGT4), neprilysin (P08473), large neutral amino acids transporter small subunit 1 (Q01650), integrin alpha-6 (P23229), dipeptidyl peptidase 1 (P53634), collagen alpha-1 (VI) chain (P12109), and neutral amino acid transporter B (Q15758), (iii) gefitinib, and said one or more glycoprotein biomarkers includes one or more of transcobalamin-1 (P20061), sushi domain-containing protein 2 (Q9UGT4), podocalyxin (000592), large neutral amino acids transporter small subunit 1 (Q01650), laminin subunit beta-1 (P07942), and dipeptidyl peptidase 1 (P53634), and (iv) lapatinib, and said one or more glycoprotein biomarkers includes one or more of receptor tyrosine-protein kinase erbB-2 (PO4626), gamma-interferon-inducible lysosomal thiol reductase (P13284), neuroplalstin (Q9Y639), cathepsin B (P07858), CD44 antigen (P16070), and bone marrow stromal antigen 2 (Q10589). 9. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells targets tubulin or microtubules and is selected from the group consisting of (i) paclitaxel, and said one or more protein biomarkers includes one or more of ubiquitin carboxyl-terminal hydrolase 5 (P45974), solute carrier family 2, facilitated glucose transporter membrane 1 (P11166), and alpha-aminoadipic semialdehyde dehydrogenase (P49419), and said one or more glycoprotein biomarkers includes one or more of CD276 antigen (Q5ZPR3), cathepsin Z (Q9UBR2), and serpin H1 (P50454), (ii) docetaxel, and said one or more protein biomarkers includes one or more of lysosome membrane protein 2 (Q14108), alpha-aminoadipic semialdehyde dehydrogenase (P49419), and isochorismatase domain-containing protein 1 (Q96CN7), and said one or more glycoprotein biomarker includes one or more of beta-mannosidase (000462), cathepsin Z (Q9UBR2), and serpin H1 (P50454), and (iii) vinorelbine, and said one or more protein biomarkers includes one or more of glucose-6-phosphate 1-dehydrogenase (P11413), ribonuclease UK114 (P52758), and tropomyosin alpha-4 chain (P67936). 10. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells targets nucleic acids and is selected from the group consisting of gemcitabine, and said one or more glycoprotein biomarkers includes one or more of ganglioside GM2 activator (P17900), granulins (P28799), and steryl-sulfatase (P08842). 11. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells comprises an inhibitor of mTOR selected from the group consisting of (i) everolimus, and said one or more glycoprotein biomarkers includes one or more of insulin-like growth factor-binding protein 7 (Q16270), lysosomal pro-x carboxypeptidase (P42785), and receptor tyrosine-protein kinase erbB-2 (PO4626), and (ii) temsirolimus, and said one or more glycoprotein biomarkers includes one or more of transmembrane emp24 domain-containing protein 7 (Q9Y3B3), arylsulfatase A (P15289), and receptor tyrosine-protein kinase erbB-2 (PO4626). 12. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells comprises an inhibitor of PI3′ kinase, and said one or more glycoprotein biomarkers includes one or more of collagen alpha-1 (VI) chain (P12109), large neutral amino acids transporter small subunit 1 (Q01650), mucin-1 (P15941), and receptor tyrosine-protein kinase erbB-2 (PO4626). 13. A method according to claim 12, wherein said inhibitor of PI3′ kinase is BEZ235. 14. A method according to claim 7, wherein said drug effective to stop proliferation of cancer cells comprises an inhibitor of CDK, and said one or more protein biomarkers includes one or more of G2/mitotic-specific cyclin-B1 (P14635), G1/S-specific cyclin-E1 (P24864), thioredoxin-dependent peroxide reductase, mitochondrial (P30048), acylaminoacyl-peptidase, putative (ApeH-1; Q97YB2), and importin subunit alpha-1 (P52292). 15. A method of treating cancer, comprising: identifying at least one protein or glycoprotein biomarker in cancer cells from a patient; identifying one or more of a plurality of drugs that effectively stop or repress proliferation of said cancer cells from a correlation or association of said at least one protein or glycoprotein biomarker with effectiveness of said one or more drugs to stop or repress said proliferation of cancer cell lines expressing said at least one protein or glycoprotein biomarker; and administering said one or more of said plurality of drugs in a pharmaceutically acceptable carrier or excipient to said patient having said cancer cells in an amount effective to stop or repress said proliferation of said cancer cells. 16. A method according to claim 15, wherein said at least one biomarker comprises a glycoprotein biomarker. 17. A method according to claim 15, wherein said one or more of said plurality of drugs is administered orally, intravenously, or by chemotherapy infusion. 18. A system configured to predict effectiveness of one or more of a plurality of drugs to stop or repress proliferation of cancer cells, comprising: a memory storing (i) a first dataset including expression levels of a plurality of proteins or glycoproteins in said plurality of said cancer cell lines, and (ii) a second dataset including an effectiveness of each of said plurality of drugs to stop or repress proliferation of said cancer cell lines; and a computer configured to statistically analyze said first and second datasets to (i) identify and/or select at least one biomarker for each of said cancer cell lines and (ii) correlate or associate at least one of said plurality of drugs that effectively stops or represses proliferation of said cancer cells in each of said cancer cell lines with said at least one biomarker for each of said cancer cell lines. 19. The system of claim 18, wherein said computer is configured to statistically analyze said first and second datasets using lasso regression. 20. The system according to claim 18, wherein said first dataset includes expression levels of a plurality of glycoproteins, and said at least one biomarker comprises at least one glycoprotein biomarker.

A method of determining the susceptibility of a cancer in a subject to treatment with an antimetabolite includes obtaining a sample of cancer cells from the subject, measuring the level of UDG expression in the cancer cells, and comparing the measured levels of UDG expression in the cancer cells to a control level.

1. A method of determining the susceptibility of a cancer in a subject to treatment with an antimetabolite that induces or promotes incorporation of a UDG substrate into DNA of cancer cells, comprising: obtaining a sample of cancer cells from the subject; measuring the level of UDG in the cancer cells; and comparing the measured levels of UDG in the cancer cells to a control level; wherein an increase in the measured levels of UDG in the cancer cells compared to a control level indicates that the cancer is less susceptible to treatment with the antimetabolite agent. 2. The method of claim 1, wherein the cancer is human lung cancer, breast cancer, colorectal cancer, cervical cancer, leukemia, or non-Hodgkin's lymphoma. 3. The method of claim 1, wherein the cancer is human non-small cell lung cancer. 4. The method of claim 1, further comprising treating the cancer in the subject with an antimetabolite agent and an AP endonuclease inhibitor if the measured level of UDG activity is increased relative to the control level. 5. The method of claim 4, wherein the antimetabolite agent promotes introduction of uracil or a UDG substrate into the cancer cell DNA. 6. The method of claim 5, wherein the antimetabolite agent comprises at least one of a thymidylate synthase inhibitor, antifolate agent, or a pyrmidine analogue. 7. The method of claim 6, wherein the antimetabolite is selected from the group consisting of pemetrexed, fludarabine, 5-fluorouracil, raltitrexed, nolatrexed, and floxuridine. 8. A method of treating cancer in a subject comprising: obtaining a sample of cancer cells from the subject; measuring the level of UDG expression in the cancer cells; comparing the measured levels of UDG expression in the cancer cells to a control level; and administering an antimetabolite agent that induces or promotes incorporation of a UDG substrate into DNA of cancer cells to the subject if the measured level of UDG expression is decreased compared to a control level or administering the antimetabolite agent in combination with an AP endonuclease inhibitor if the measured level of UDG expression is increased compared to a control level. 9. The method of claim 8, wherein the cancer is carcinomas, melanomas, sarcomas, lymphomas, leukemias, astrocytomas, gliomas, malignant melanomas, chronic lymphocytic leukemia, lung cancers, colorectal cancers, ovarian cancers, pancreatic cancers, renal cancers, endometrial cancers, gastric cancers, liver cancers, head and neck cancers, and breast cancers. 10. The method of claim 8, wherein the cancer is human non-small cell lung cancer. 11. The method of claim 8, wherein the antimetabolite agent comprises at least one of a thymidylate synthase inhibitor, antifolate agent, or a pyrmidine analogue. 12. The method of claim 11, wherein the antimetabolite is selected from the group consisting of pemetrexed, fludarabine, 5-fluorouracil, raltitrexed, nolatrexed, and floxuridine. 13. The method of claim 12, wherein the AP endonuclease inhibitor is administered at an amount effective to potentiate the cytotoxicity of the antimetabolite agent administered to the cancer cells. 14. The method of claim 8, wherein the AP endonuclease inhibitor is selected from group consisting of methoxyamine, O-benzylohydroxylamine; ethyl aminooxyacetate; aminooxyacetic acid; ethyl aminooxyacetate; H2NOCHMeCO2H; carboxymethoxyamine; aminooxyacetic acid; HN═C(NH2)SCH2CH2ONH2; H2NO(CH2)3SC(NH2)═NH; MeOC(O)CH(NH2)CH2ONH2; H2NOCH2CH(NH2)CO2H; canaline; H2NO(CH2)4ONH2; O-(p-nitrobenzyl)hydroxylamine; 2-amino-4-(aminooxymethyl)thiazole; 4-(aminooxymethyl)thiazole; O,O′-(o-phenylenedimethylene)dihydroxylamine; 2,4-dinitrophenoxyamine; O,O′-(m-phenylenedimethylene)dihydroxylamine; O,O′-(p-phenylenedimethylene)dihydroxylamine; H2C═CHCH2ONH2; H2NO(CH2)4ONH2; H3C—(CH2)15—O—NH2, 2,2′-(1,2-ethanediyl)bis(3-aminooxy)butenedioic acid dimethyl diethyl ester; a compound having a structure of Formula I: wherein X is O or NH, Y is O, S, or NH, Z is absent or represents O, S, or NH, and R represents a hydrogen or a hydrocarbon moiety, and pharmaceutically acceptable salts thereof. 15. The method of claim 8, the AP endonuclease inhibitor comprising methoxyamine. 16. The method of claim 8, wherein the amount of methoxyamine is an amount sufficient to sensitize the cancer cells to the antimetabolite without causing undue sensitization of normal cells. 17. A method of treating human lung cancer in a subject with an antimetabolite agent that induces or promotes incorporation of a UDG substrate into DNA of cancer cells comprising: obtaining a sample of lung cancer cells from the subject; measuring the level of UDG expression in the lung cancer cells; comparing the measured levels of UDG expression in the lung cancer cells to a control level; and administering the antimetabolite agent to the subject if the measured level of UDG expression is decreased compared to a control level or administering an antimetabolite agent in combination with an AP endonuclease inhibitor if the measured level of UDG expression is increased compared to a control level. 18. The method of claim 17, wherein the lung cancer is human non-small cell lung cancer. 19. The method of claim 17, wherein the antimetabolite agent comprises at least one of a thymidylate synthase inhibitor, antifolate agent, or a pyrmidine analogue. 20. The method of claim 19, wherein the antimetabolite is selected from the group consisting of pemetrexed, fludarabine, 5-fluorouracil, raltitrexed, nolatrexed, and floxuridine. 21. The method of claim 17, wherein the AP endonuclease inhibitor is selected from group consisting of methoxyamine, O-benzylohydroxylamine; ethyl aminooxyacetate; aminooxyacetic acid; ethyl aminooxyacetate; H2NOCHMeCO2H; carboxymethoxyamine; aminooxyacetic acid; HN═C(NH2)SCH2CH2ONH2; H2NO(CH2)3SC(NH2)═NH; MeOC(O)CH(NH2)CH2ONH2; H2NOCH2CH(NH2)CO2H; canaline; H2NO(CH2)4ONH2; O-(p-nitrobenzyl)hydroxylamine;2-amino-4-(aminooxymethyl)thiazole; 4-(aminooxymethyl)thiazole; O,O′-(o-phenylenedimethylene)dihydroxylamine; 2,4-dinitrophenoxyamine; O,O′-(m-phenylenedimethylene)dihydroxylamine; O,O′-(p-phenylenedimethylene)dihydroxylamine; H2C═CHCH2ONH2; H2NO(CH2)4ONH2; H3C—(CH2)15—O—NH2, 2,2′-(1,2-ethanediyl)bis(3-aminooxy)butenedioic acid dimethyl diethyl ester; a compound having a structure of Formula I: wherein X is O or NH, Y is O, S, or NH, Z is absent or represents O, S, or NH, and R represents a hydrogen or a hydrocarbon moiety, and pharmaceutically acceptable salts thereof. 22. The method of claim 17, the AP endonuclease inhibitor is methoxyamine.

A method of determining the susceptibility of a cancer in a subject to treatment with an antimetabolite includes obtaining a sample of cancer cells from the subject, measuring the level of UDG expression in the cancer cells, and comparing the measured levels of UDG expression in the cancer cells to a control level.1. A method of determining the susceptibility of a cancer in a subject to treatment with an antimetabolite that induces or promotes incorporation of a UDG substrate into DNA of cancer cells, comprising: obtaining a sample of cancer cells from the subject; measuring the level of UDG in the cancer cells; and comparing the measured levels of UDG in the cancer cells to a control level; wherein an increase in the measured levels of UDG in the cancer cells compared to a control level indicates that the cancer is less susceptible to treatment with the antimetabolite agent. 2. The method of claim 1, wherein the cancer is human lung cancer, breast cancer, colorectal cancer, cervical cancer, leukemia, or non-Hodgkin's lymphoma. 3. The method of claim 1, wherein the cancer is human non-small cell lung cancer. 4. The method of claim 1, further comprising treating the cancer in the subject with an antimetabolite agent and an AP endonuclease inhibitor if the measured level of UDG activity is increased relative to the control level. 5. The method of claim 4, wherein the antimetabolite agent promotes introduction of uracil or a UDG substrate into the cancer cell DNA. 6. The method of claim 5, wherein the antimetabolite agent comprises at least one of a thymidylate synthase inhibitor, antifolate agent, or a pyrmidine analogue. 7. The method of claim 6, wherein the antimetabolite is selected from the group consisting of pemetrexed, fludarabine, 5-fluorouracil, raltitrexed, nolatrexed, and floxuridine. 8. A method of treating cancer in a subject comprising: obtaining a sample of cancer cells from the subject; measuring the level of UDG expression in the cancer cells; comparing the measured levels of UDG expression in the cancer cells to a control level; and administering an antimetabolite agent that induces or promotes incorporation of a UDG substrate into DNA of cancer cells to the subject if the measured level of UDG expression is decreased compared to a control level or administering the antimetabolite agent in combination with an AP endonuclease inhibitor if the measured level of UDG expression is increased compared to a control level. 9. The method of claim 8, wherein the cancer is carcinomas, melanomas, sarcomas, lymphomas, leukemias, astrocytomas, gliomas, malignant melanomas, chronic lymphocytic leukemia, lung cancers, colorectal cancers, ovarian cancers, pancreatic cancers, renal cancers, endometrial cancers, gastric cancers, liver cancers, head and neck cancers, and breast cancers. 10. The method of claim 8, wherein the cancer is human non-small cell lung cancer. 11. The method of claim 8, wherein the antimetabolite agent comprises at least one of a thymidylate synthase inhibitor, antifolate agent, or a pyrmidine analogue. 12. The method of claim 11, wherein the antimetabolite is selected from the group consisting of pemetrexed, fludarabine, 5-fluorouracil, raltitrexed, nolatrexed, and floxuridine. 13. The method of claim 12, wherein the AP endonuclease inhibitor is administered at an amount effective to potentiate the cytotoxicity of the antimetabolite agent administered to the cancer cells. 14. The method of claim 8, wherein the AP endonuclease inhibitor is selected from group consisting of methoxyamine, O-benzylohydroxylamine; ethyl aminooxyacetate; aminooxyacetic acid; ethyl aminooxyacetate; H2NOCHMeCO2H; carboxymethoxyamine; aminooxyacetic acid; HN═C(NH2)SCH2CH2ONH2; H2NO(CH2)3SC(NH2)═NH; MeOC(O)CH(NH2)CH2ONH2; H2NOCH2CH(NH2)CO2H; canaline; H2NO(CH2)4ONH2; O-(p-nitrobenzyl)hydroxylamine; 2-amino-4-(aminooxymethyl)thiazole; 4-(aminooxymethyl)thiazole; O,O′-(o-phenylenedimethylene)dihydroxylamine; 2,4-dinitrophenoxyamine; O,O′-(m-phenylenedimethylene)dihydroxylamine; O,O′-(p-phenylenedimethylene)dihydroxylamine; H2C═CHCH2ONH2; H2NO(CH2)4ONH2; H3C—(CH2)15—O—NH2, 2,2′-(1,2-ethanediyl)bis(3-aminooxy)butenedioic acid dimethyl diethyl ester; a compound having a structure of Formula I: wherein X is O or NH, Y is O, S, or NH, Z is absent or represents O, S, or NH, and R represents a hydrogen or a hydrocarbon moiety, and pharmaceutically acceptable salts thereof. 15. The method of claim 8, the AP endonuclease inhibitor comprising methoxyamine. 16. The method of claim 8, wherein the amount of methoxyamine is an amount sufficient to sensitize the cancer cells to the antimetabolite without causing undue sensitization of normal cells. 17. A method of treating human lung cancer in a subject with an antimetabolite agent that induces or promotes incorporation of a UDG substrate into DNA of cancer cells comprising: obtaining a sample of lung cancer cells from the subject; measuring the level of UDG expression in the lung cancer cells; comparing the measured levels of UDG expression in the lung cancer cells to a control level; and administering the antimetabolite agent to the subject if the measured level of UDG expression is decreased compared to a control level or administering an antimetabolite agent in combination with an AP endonuclease inhibitor if the measured level of UDG expression is increased compared to a control level. 18. The method of claim 17, wherein the lung cancer is human non-small cell lung cancer. 19. The method of claim 17, wherein the antimetabolite agent comprises at least one of a thymidylate synthase inhibitor, antifolate agent, or a pyrmidine analogue. 20. The method of claim 19, wherein the antimetabolite is selected from the group consisting of pemetrexed, fludarabine, 5-fluorouracil, raltitrexed, nolatrexed, and floxuridine. 21. The method of claim 17, wherein the AP endonuclease inhibitor is selected from group consisting of methoxyamine, O-benzylohydroxylamine; ethyl aminooxyacetate; aminooxyacetic acid; ethyl aminooxyacetate; H2NOCHMeCO2H; carboxymethoxyamine; aminooxyacetic acid; HN═C(NH2)SCH2CH2ONH2; H2NO(CH2)3SC(NH2)═NH; MeOC(O)CH(NH2)CH2ONH2; H2NOCH2CH(NH2)CO2H; canaline; H2NO(CH2)4ONH2; O-(p-nitrobenzyl)hydroxylamine;2-amino-4-(aminooxymethyl)thiazole; 4-(aminooxymethyl)thiazole; O,O′-(o-phenylenedimethylene)dihydroxylamine; 2,4-dinitrophenoxyamine; O,O′-(m-phenylenedimethylene)dihydroxylamine; O,O′-(p-phenylenedimethylene)dihydroxylamine; H2C═CHCH2ONH2; H2NO(CH2)4ONH2; H3C—(CH2)15—O—NH2, 2,2′-(1,2-ethanediyl)bis(3-aminooxy)butenedioic acid dimethyl diethyl ester; a compound having a structure of Formula I: wherein X is O or NH, Y is O, S, or NH, Z is absent or represents O, S, or NH, and R represents a hydrogen or a hydrocarbon moiety, and pharmaceutically acceptable salts thereof. 22. The method of claim 17, the AP endonuclease inhibitor is methoxyamine.

The present disclosure provides methods and compositions for reducing the risk of pathological effects of traumatic brain injury.

1. A method for reducing the risk of pathological effects of traumatic brain injury, comprising: (a) administering to a subject who is at risk of traumatic brain injury a composition comprising at least about 35 wt % docosahexaenoate (DHA), wherein the composition is administered in a prophylactically effective amount for a sufficient time period prior to engagement in an activity associated with a risk of traumatic brain injury to reduce the risk of pathological effects of traumatic brain injury, and wherein the composition has an eicosapentaenoate (EPA) content of less than about 2 wt % of the total fatty acid content. 2. (canceled) 3. The method of claim 1 in which the DHA is in the form of a triglyceride. 4. The method of claim 3 in which the DHA is in the form of an alkylester. 5. The method of claim 4 in which the DHA alkylester is a ethyl ester. 6. The method of claim 5 in which the DHA alkylester is at least about 85 wt % of the total fatty acid content of the composition. 7. The method of claim 6 in which the DHA alkylester is about 85 to about 96 wt % of the total fatty acid content of the composition. 8. The method of claim 1 in which the DHA is at least about 40 wt % of the total fatty acid content of the composition. 9. The method of claim 8 in which the DHA is about 40 to about 50 wt % of the total fatty acid content of the composition. 10. The method of claim 1 in which the DHA is at least about 55 wt % of the total fatty acid content of the composition. 11. The method of claim 10 in which the DHA is about 55 to 65 wt % of the total fatty acid content of the composition. 12. The method of claim 1 in which the DHA to EPA ratio is at least 10:1. 13. The method of claim 1 in which the DHA to EPA ratio is at least 100:1. 14-17. (canceled) 18. The method of claim 1 in which the traumatic brain injury is from a closed head injury. 19-21. (canceled) 22. The method of claim 1 in which the composition is administered for at least 28 days prior to engaging in the activity associated with a risk of traumatic brain injury. 23. The method of claim 1 in which the composition is administered for at least 6 weeks prior to engaging in the activity associated with a risk of for traumatic brain injury. 24. The method of claim 1 in which the effective amount is a dose of about 10 mg/kg body weight/day to about 40 mg/kg body weight/day of DHA. 25. The method of claim 1 in which the composition is an oral dosage form. 26. The method of claim 25 in which the oral dosage form is a gelatin capsule. 27. The method of claim 26 in which the gelatin capsule comprises from about 200 mg to about 1 g of DHA, and a pharmaceutically acceptable excipient. 28. (canceled) 29. A method of protecting the brain of a human subject, the method comprising: administering to the subject, before an activity associated with a potential traumatic brain injuring event, an oral dosage form comprising at least 900 mg of DHA, wherein the dosage form comprises at least about 35 wt % docosahexaenoate (DHA) of the total fatty acid content, wherein the dosage form has an eicosapentaenoate (EPA) content of less than about 2 wt % of the total fatty acid content. 30. The method of claim 29, wherein the activity associated with a potential traumatic brain injuring event is selected from the group consisting of boxing, football, soccer, hockey, aimed conflict, or brain surgery. 31. The method of claim 29, wherein protecting the brain of a subject is the prevention of the pathological effects of a concussion, or the reduction of the pathological effects associated with a concussion 32. (canceled)

The present disclosure provides methods and compositions for reducing the risk of pathological effects of traumatic brain injury.1. A method for reducing the risk of pathological effects of traumatic brain injury, comprising: (a) administering to a subject who is at risk of traumatic brain injury a composition comprising at least about 35 wt % docosahexaenoate (DHA), wherein the composition is administered in a prophylactically effective amount for a sufficient time period prior to engagement in an activity associated with a risk of traumatic brain injury to reduce the risk of pathological effects of traumatic brain injury, and wherein the composition has an eicosapentaenoate (EPA) content of less than about 2 wt % of the total fatty acid content. 2. (canceled) 3. The method of claim 1 in which the DHA is in the form of a triglyceride. 4. The method of claim 3 in which the DHA is in the form of an alkylester. 5. The method of claim 4 in which the DHA alkylester is a ethyl ester. 6. The method of claim 5 in which the DHA alkylester is at least about 85 wt % of the total fatty acid content of the composition. 7. The method of claim 6 in which the DHA alkylester is about 85 to about 96 wt % of the total fatty acid content of the composition. 8. The method of claim 1 in which the DHA is at least about 40 wt % of the total fatty acid content of the composition. 9. The method of claim 8 in which the DHA is about 40 to about 50 wt % of the total fatty acid content of the composition. 10. The method of claim 1 in which the DHA is at least about 55 wt % of the total fatty acid content of the composition. 11. The method of claim 10 in which the DHA is about 55 to 65 wt % of the total fatty acid content of the composition. 12. The method of claim 1 in which the DHA to EPA ratio is at least 10:1. 13. The method of claim 1 in which the DHA to EPA ratio is at least 100:1. 14-17. (canceled) 18. The method of claim 1 in which the traumatic brain injury is from a closed head injury. 19-21. (canceled) 22. The method of claim 1 in which the composition is administered for at least 28 days prior to engaging in the activity associated with a risk of traumatic brain injury. 23. The method of claim 1 in which the composition is administered for at least 6 weeks prior to engaging in the activity associated with a risk of for traumatic brain injury. 24. The method of claim 1 in which the effective amount is a dose of about 10 mg/kg body weight/day to about 40 mg/kg body weight/day of DHA. 25. The method of claim 1 in which the composition is an oral dosage form. 26. The method of claim 25 in which the oral dosage form is a gelatin capsule. 27. The method of claim 26 in which the gelatin capsule comprises from about 200 mg to about 1 g of DHA, and a pharmaceutically acceptable excipient. 28. (canceled) 29. A method of protecting the brain of a human subject, the method comprising: administering to the subject, before an activity associated with a potential traumatic brain injuring event, an oral dosage form comprising at least 900 mg of DHA, wherein the dosage form comprises at least about 35 wt % docosahexaenoate (DHA) of the total fatty acid content, wherein the dosage form has an eicosapentaenoate (EPA) content of less than about 2 wt % of the total fatty acid content. 30. The method of claim 29, wherein the activity associated with a potential traumatic brain injuring event is selected from the group consisting of boxing, football, soccer, hockey, aimed conflict, or brain surgery. 31. The method of claim 29, wherein protecting the brain of a subject is the prevention of the pathological effects of a concussion, or the reduction of the pathological effects associated with a concussion 32. (canceled)

The present invention relates to a composition for ameliorating viral infections in nursery pigs. The composition contains the polyether ionophore narasin, and is supplied to the nursery pigs in an orally-acceptable form. The composition is effective in reducing viral shedding and the severity of diarrhea after challenge of nursery pigs with Porcine Epidemic Diarrhea Virus (PEDV).

1. A method of treating porcine epidemic diarrhea virus (PEDV) infection, comprising administering to a nursery pig narasin with an orally-acceptable carrier. 2. The method of claim 1, wherein the orally-acceptable carrier is selected from the group comprising an animal feed, a liquid composition other than an animal feed, and a solid composition other than an animal feed. 3. The method of claim 1, wherein the concentration of narasin is selected from the group of about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, and about 60 mg/kg of the composition. 4. A method of treating porcine epidemic diarrhea virus (PEDV) infection, comprising administering to a nursery pig narasin with an animal feed, wherein narasin is present at a concentration of about 60 mg/kg of the animal feed. 5. (canceled) 6. (canceled) 7. (canceled) 8. A composition comprising a concentration of narasin and an orally-acceptable carrier; wherein the composition provides an antiviral effect to a nursery pie. 9. The composition of claim 8, wherein the orally-acceptable carrier is selected from the group comprising an animal feed, a liquid composition other than an animal feed, and a solid composition other than an animal feed. 10. The composition of claim 8, wherein the concentration of narasin is selected from the group of about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, and about 60 mg/kg of the composition. 11. The method of claim 2, wherein the concentration of narasin is selected from the group of about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, and about 60 mg/kg of the composition.

The present invention relates to a composition for ameliorating viral infections in nursery pigs. The composition contains the polyether ionophore narasin, and is supplied to the nursery pigs in an orally-acceptable form. The composition is effective in reducing viral shedding and the severity of diarrhea after challenge of nursery pigs with Porcine Epidemic Diarrhea Virus (PEDV).1. A method of treating porcine epidemic diarrhea virus (PEDV) infection, comprising administering to a nursery pig narasin with an orally-acceptable carrier. 2. The method of claim 1, wherein the orally-acceptable carrier is selected from the group comprising an animal feed, a liquid composition other than an animal feed, and a solid composition other than an animal feed. 3. The method of claim 1, wherein the concentration of narasin is selected from the group of about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, and about 60 mg/kg of the composition. 4. A method of treating porcine epidemic diarrhea virus (PEDV) infection, comprising administering to a nursery pig narasin with an animal feed, wherein narasin is present at a concentration of about 60 mg/kg of the animal feed. 5. (canceled) 6. (canceled) 7. (canceled) 8. A composition comprising a concentration of narasin and an orally-acceptable carrier; wherein the composition provides an antiviral effect to a nursery pie. 9. The composition of claim 8, wherein the orally-acceptable carrier is selected from the group comprising an animal feed, a liquid composition other than an animal feed, and a solid composition other than an animal feed. 10. The composition of claim 8, wherein the concentration of narasin is selected from the group of about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, and about 60 mg/kg of the composition. 11. The method of claim 2, wherein the concentration of narasin is selected from the group of about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, and about 60 mg/kg of the composition.

The invention provides methods and compositions for regulating weight and size in animals.

1. A method for reducing white adipose tissue in an animal comprising administering a high glycine diet comprising about 10% to about 30% glycine to the animal. 2. The method of claim 1, wherein the high glycine diet comprises glycine analogs or a combination of glycine and glycine analogs. 3. A method for inducing apoptosis in white adipose tissue of an animal comprising administering a high glycine diet comprising about 10% to about 30% glycine to the animal. 4. The method of claim 3, wherein the high glycine diet comprises glycine analogs or a combination of glycine and glycine analogs. 5. A method for reducing phosphorylation of BAD at amino acid position 136 in white adipose tissue of an animal comprising administering a high glycine diet comprising about 10 to about 30% glycine to the animal. 6. The method of claim 5, wherein the high glycine diet comprises glycine analogs or a combination of glycine and glycine analogs. 7. A method of claim 1, wherein the method of reducing white adipose tissue comprises reducing white adipose cell size. 8. A method for producing weight loss in an animal comprising administering a high glycine diet comprising about 10% to about 30% glycine to the animal. 9. The method of claim 8, wherein the method for producing weight loss comprises reducing abdominal fat content in the animal. 10. The method of claim 8, wherein the high glycine diet comprises glycine analogs or a combination of glycine and glycine analogs. 11. The method of claim 8, wherein the method further comprises an adjunctive weight loss therapy such as an exercise regimen, a low-fat diet, a low-calorie diet, a low-carbohydrate diet, surgical intervention, behavioral therapy, pharmacotherapy, or a combination thereof. 12. A method for producing a reduced-sized or reduced-weight animal and then returning it to a normal size or normal weight comprising administering a high glycine diet to an immature animal to produce a reduced-sized or reduced-weight animal and then administering a normal diet to the animal to produce a normal-sized or normal-weight animal. 13. A diet composition for weight reduction in an animal comprising about 10% to about 30% glycine, glycine analogs or combinations thereof and about 70% to about 90% of a low-calorie diet, a low-fat diet, a low-carbohydrate diet, or a combination thereof. 14. The diet composition of claim 13, wherein the low-fat diet is a low-saturated fat diet. 15. A method for producing a reduced-size or reduced-weight or both reduced-sized and reduced-weight animal comprising administering a high glycine diet comprising about 10 to about 30% glycine to an immature animal. 16. The method of claim 15, wherein the high glycine diet comprises glycine analogs or a combination of glycine and glycine analogs. 17. A method of inducing apoptosis in a white adipocyte comprising administering one or more glycine analogs or a combination of glycine and glycine analogs to the adipocyte. 18. A method of reducing phosphorylation of BAD at amino acid position 136 in a white adipocyte comprising administering a one or more glycine analogs or a combination of glycine and glycine analogs to the adipocyte.

The invention provides methods and compositions for regulating weight and size in animals.1. A method for reducing white adipose tissue in an animal comprising administering a high glycine diet comprising about 10% to about 30% glycine to the animal. 2. The method of claim 1, wherein the high glycine diet comprises glycine analogs or a combination of glycine and glycine analogs. 3. A method for inducing apoptosis in white adipose tissue of an animal comprising administering a high glycine diet comprising about 10% to about 30% glycine to the animal. 4. The method of claim 3, wherein the high glycine diet comprises glycine analogs or a combination of glycine and glycine analogs. 5. A method for reducing phosphorylation of BAD at amino acid position 136 in white adipose tissue of an animal comprising administering a high glycine diet comprising about 10 to about 30% glycine to the animal. 6. The method of claim 5, wherein the high glycine diet comprises glycine analogs or a combination of glycine and glycine analogs. 7. A method of claim 1, wherein the method of reducing white adipose tissue comprises reducing white adipose cell size. 8. A method for producing weight loss in an animal comprising administering a high glycine diet comprising about 10% to about 30% glycine to the animal. 9. The method of claim 8, wherein the method for producing weight loss comprises reducing abdominal fat content in the animal. 10. The method of claim 8, wherein the high glycine diet comprises glycine analogs or a combination of glycine and glycine analogs. 11. The method of claim 8, wherein the method further comprises an adjunctive weight loss therapy such as an exercise regimen, a low-fat diet, a low-calorie diet, a low-carbohydrate diet, surgical intervention, behavioral therapy, pharmacotherapy, or a combination thereof. 12. A method for producing a reduced-sized or reduced-weight animal and then returning it to a normal size or normal weight comprising administering a high glycine diet to an immature animal to produce a reduced-sized or reduced-weight animal and then administering a normal diet to the animal to produce a normal-sized or normal-weight animal. 13. A diet composition for weight reduction in an animal comprising about 10% to about 30% glycine, glycine analogs or combinations thereof and about 70% to about 90% of a low-calorie diet, a low-fat diet, a low-carbohydrate diet, or a combination thereof. 14. The diet composition of claim 13, wherein the low-fat diet is a low-saturated fat diet. 15. A method for producing a reduced-size or reduced-weight or both reduced-sized and reduced-weight animal comprising administering a high glycine diet comprising about 10 to about 30% glycine to an immature animal. 16. The method of claim 15, wherein the high glycine diet comprises glycine analogs or a combination of glycine and glycine analogs. 17. A method of inducing apoptosis in a white adipocyte comprising administering one or more glycine analogs or a combination of glycine and glycine analogs to the adipocyte. 18. A method of reducing phosphorylation of BAD at amino acid position 136 in a white adipocyte comprising administering a one or more glycine analogs or a combination of glycine and glycine analogs to the adipocyte.

In one aspect, the present disclosure provides microparticles that are configured to release a first drug over a first time period and to release a second drug over a second time period, wherein a lag period of substantially no drug release occurs between the first and second time periods. In other aspects, the present disclosure pertains to the use of such microparticles in delivery systems and methods of treatment. In another aspect, the present disclosure pertains to drug delivery systems that comprising a folded inflatable drug delivery balloon that comprises folds and microparticles positioned within the folds.

1. A method of treatment comprising delivering therapeutic-agent releasing microparticles into a feeder artery of a tumor, wherein the microparticles release a first drug over a first time period, wherein the microparticles release a second drug over a second time period, wherein a lag period of substantially no drug release occurs between the first and second time periods, and wherein the first drug and the second drug may be the same or different. 2. The method of claim 1, wherein the first and second drugs are anti-cancer drugs. 3. The method of claim 1, wherein the first and second drugs are the same. 4. The method of claim 1, wherein the lag period ranges from 2 to 6 weeks. 5. The method of claim 4, wherein the first time period is a period of five days or less and wherein the second time is a period of five days or less. 6. The method of claim 1, wherein 30 to 70% of the total cumulative drug release from the microparticles occurs over the first time period, wherein 30 to 70% of the total cumulative drug release from the microparticles occurs over the second time period, and wherein less than 5% of the total cumulative drug release occurs over the lag period. 7. The method of claim 1, wherein the microparticles comprise a bioerodible core that comprises the second drug, a first bioerodible layer surrounding the bioerodible core that comprises substantially no drug, and a second bioerodible layer surrounding the first bioerodible layer that comprises the first drug, or wherein the microparticles comprise first microparticles that comprise the first drug but not the second drug and release the first drug over the first time period and second microparticles that comprise the second drug but not the first drug and release the second drug over the second time period. 8. The method of claim 1, wherein the microparticles are released into the artery from a balloon. 9. The method of claim 1, wherein a folded balloon that comprises folds and microparticles positioned within the folds is delivered to the artery, after which the balloon is inflated thereby releasing the microparticles from the folds into the artery. 10. A delivery system comprising: a delivery device configured to release microparticles into a feeder artery within a body of a subject and microparticles that are configured to release a first drug over a first time period and to release a second drug over a second time period, wherein a lag period of substantially no drug release occurs between the first and second time periods, and wherein the first drug and the second drug may be the same or different. 11. The delivery system of claim 10, wherein the first and second drugs are anti-cancer drugs. 12. The delivery system of claim 10, wherein the first and second drugs are the same. 13. The delivery system of claim 10, wherein the lag period ranges from 2 to 6 weeks. 14. The delivery system of claim 13, wherein the first time period is a period of five days or less and wherein the second time is a period of five days or less. 15. The delivery system of claim 10, wherein the microparticles comprise a bioerodible core that comprises the second drug, a first bioerodible layer surrounding the bioerodible core that comprises substantially no drug, and a second bioerodible layer surrounding the first bioerodible layer that comprises the first drug, or wherein the microparticles comprise first microparticles that comprise the first drug but not the second drug and release the first drug over the first time period and second microparticles that comprise the second drug but not the first drug and release the second drug over the second time period. 16. The delivery system of claim 10, wherein the delivery device comprises a balloon. 17. The delivery system of claim 16, wherein the microparticles are provided on a surface of the balloon. 18. The delivery system of claim 16, wherein the balloon comprises folds and the microparticles are positioned within the folds. 19. A drug delivery system comprising a folded inflatable drug delivery balloon that comprises folds and microparticles positioned within the folds, wherein the microparticles are printed on a surface of the balloon before forming the folds or wherein the microparticles are injected into the folds after forming the folds. 20. The drug delivery system of claim 19, wherein the microparticles are printed on a surface of the balloon by a process selected from an ink jet printing process and a three-dimensional printing process.

In one aspect, the present disclosure provides microparticles that are configured to release a first drug over a first time period and to release a second drug over a second time period, wherein a lag period of substantially no drug release occurs between the first and second time periods. In other aspects, the present disclosure pertains to the use of such microparticles in delivery systems and methods of treatment. In another aspect, the present disclosure pertains to drug delivery systems that comprising a folded inflatable drug delivery balloon that comprises folds and microparticles positioned within the folds.1. A method of treatment comprising delivering therapeutic-agent releasing microparticles into a feeder artery of a tumor, wherein the microparticles release a first drug over a first time period, wherein the microparticles release a second drug over a second time period, wherein a lag period of substantially no drug release occurs between the first and second time periods, and wherein the first drug and the second drug may be the same or different. 2. The method of claim 1, wherein the first and second drugs are anti-cancer drugs. 3. The method of claim 1, wherein the first and second drugs are the same. 4. The method of claim 1, wherein the lag period ranges from 2 to 6 weeks. 5. The method of claim 4, wherein the first time period is a period of five days or less and wherein the second time is a period of five days or less. 6. The method of claim 1, wherein 30 to 70% of the total cumulative drug release from the microparticles occurs over the first time period, wherein 30 to 70% of the total cumulative drug release from the microparticles occurs over the second time period, and wherein less than 5% of the total cumulative drug release occurs over the lag period. 7. The method of claim 1, wherein the microparticles comprise a bioerodible core that comprises the second drug, a first bioerodible layer surrounding the bioerodible core that comprises substantially no drug, and a second bioerodible layer surrounding the first bioerodible layer that comprises the first drug, or wherein the microparticles comprise first microparticles that comprise the first drug but not the second drug and release the first drug over the first time period and second microparticles that comprise the second drug but not the first drug and release the second drug over the second time period. 8. The method of claim 1, wherein the microparticles are released into the artery from a balloon. 9. The method of claim 1, wherein a folded balloon that comprises folds and microparticles positioned within the folds is delivered to the artery, after which the balloon is inflated thereby releasing the microparticles from the folds into the artery. 10. A delivery system comprising: a delivery device configured to release microparticles into a feeder artery within a body of a subject and microparticles that are configured to release a first drug over a first time period and to release a second drug over a second time period, wherein a lag period of substantially no drug release occurs between the first and second time periods, and wherein the first drug and the second drug may be the same or different. 11. The delivery system of claim 10, wherein the first and second drugs are anti-cancer drugs. 12. The delivery system of claim 10, wherein the first and second drugs are the same. 13. The delivery system of claim 10, wherein the lag period ranges from 2 to 6 weeks. 14. The delivery system of claim 13, wherein the first time period is a period of five days or less and wherein the second time is a period of five days or less. 15. The delivery system of claim 10, wherein the microparticles comprise a bioerodible core that comprises the second drug, a first bioerodible layer surrounding the bioerodible core that comprises substantially no drug, and a second bioerodible layer surrounding the first bioerodible layer that comprises the first drug, or wherein the microparticles comprise first microparticles that comprise the first drug but not the second drug and release the first drug over the first time period and second microparticles that comprise the second drug but not the first drug and release the second drug over the second time period. 16. The delivery system of claim 10, wherein the delivery device comprises a balloon. 17. The delivery system of claim 16, wherein the microparticles are provided on a surface of the balloon. 18. The delivery system of claim 16, wherein the balloon comprises folds and the microparticles are positioned within the folds. 19. A drug delivery system comprising a folded inflatable drug delivery balloon that comprises folds and microparticles positioned within the folds, wherein the microparticles are printed on a surface of the balloon before forming the folds or wherein the microparticles are injected into the folds after forming the folds. 20. The drug delivery system of claim 19, wherein the microparticles are printed on a surface of the balloon by a process selected from an ink jet printing process and a three-dimensional printing process.

The present disclosure relates to recombinantly engineered cells that contain a chlorite dismutase polypeptide and methods for culturing such cells in a culture medium containing chlorite in an amount sufficient to reduce the growth rate or kill contaminating microorganisms without killing the recombinantly engineered cells. Also provided are methods for the production of a fermentation product using the recombinantly engineered cells.

1-49. (canceled) 50. A method of culturing a cell, comprising: a) culturing a cell recombinantly engineered to express a chlorite dismutase polypeptide in a culture medium under conditions whereby the chlorite dismutase polypeptide is expressed in said cell, wherein said culture medium comprises one or more contaminating microorganisms; and b) treating said culture medium with chlorite in an amount sufficient to reduce the growth rate or kill said one or more contaminating microorganisms without killing said cell. 51. The method of claim 50, wherein the chlorite dismutase polypeptide is expressed in the cell from a chlorite dismutase gene (cld) heterologous to the cell. 52. The method of claim 50, wherein the chlorite dismutase polypeptide is expressed in the cell from a chlorite dismutase gene (cld) heterologous to the cell, wherein the gene is of a perchlorate reducing bacterium. 53. The method of claim 50, wherein the cell does not naturally express a chlorite dismutase. 54. The method of claim 50, wherein the cell is a bacterial call selected from Acidobacteria, Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia. 55. The method of claim 51, wherein the cell is a bacterial call selected from Acidobacteria, Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia. 56. The method of claim 52, wherein the cell is a bacterial call selected from Acidobacteria, Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia. 57. The method of claim 50, wherein the cell is a fungal cell selected from Ascomycota and Basidiomycota. 58. The method of claim 51, wherein the cell is a fungal cell selected from Ascomycota and Basidiomycota. 59. The method of claim 52, wherein the cell is a fungal cell selected from Ascomycota and Basidiomycota. 60. The method of claim 50, wherein the cell is selected from Saccharomyces sp., Trichoderma reesei, Neurospora sp., Kluyveromyces sp., Pichia sp., Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia sp., and Bacillus sp. 61. The method of claim 51, wherein the cell is selected from Saccharomyces sp., Trichoderma reesei, Neurospora sp., Kluyveromyces sp., Pichia sp., Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia sp., and Bacillus sp. 62. The method of claim 52, wherein the cell is selected from Saccharomyces sp., Trichoderma reesei, Neurospora sp., Kluyveromyces sp., Pichia sp., Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia sp., and Bacillus sp. 63. The method of claim 50, wherein the cell is selected from Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Trichoderma reesei, Neurospora crassa, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Pichia pastoris, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium saccharoperbutylacetonicum, Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Clostridium botulinum, Clostridium butyricum, Clostridium diolis, Clostridium ljungdahlii, Clostridium aerotolerans, Clostridium cellulolyticum, Clostridium tyrobutyricum, Clostridium pasteurianum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia lipolytica, and Bacillus subtilis. 64. The method of claim 51, wherein the cell is selected from Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Trichoderma reesei, Neurospora crassa, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Pichia pastoris, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium saccharoperbutylacetonicum, Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Clostridium botulinum, Clostridium butyricum, Clostridium diolis, Clostridium ljungdahlii, Clostridium aerotolerans, Clostridium cellulolyticum, Clostridium tyrobutyricum, Clostridium pasteurianum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia lipolytica, and Bacillus subtilis. 65. The method of claim 52, wherein the cell is selected from Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Trichoderma reesei, Neurospora crassa, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Pichia pastoris, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium saccharoperbutylacetonicum, Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Clostridium botulinum, Clostridium butyricum, Clostridium diolis, Clostridium ljungdahlii, Clostridium aerotolerans, Clostridium cellulolyticum, Clostridium tyrobutyricum, Clostridium pasteurianum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia lipolytica, and Bacillus subtilis. 66. The method of claim 50 wherein the cell is further recombinantly engineered to express one or more proteins necessary for heme biosynthesis selected from the group consisting of uroporphyrinogen III decarboxylase, coproporphyrinogen III oxidase, protoporphyrinogen IX oxidase, and ferrochelatase. 67. The method of claim 50, wherein the treating step comprises producing the chlorite in the culture medium by electrochemical generation. 68. The method of claim 50, wherein the treating step is performed intermittently. 69. The method of claim 50 further comprising culturing the cell under conditions sufficient for the cell to produce a fermentation product.

The present disclosure relates to recombinantly engineered cells that contain a chlorite dismutase polypeptide and methods for culturing such cells in a culture medium containing chlorite in an amount sufficient to reduce the growth rate or kill contaminating microorganisms without killing the recombinantly engineered cells. Also provided are methods for the production of a fermentation product using the recombinantly engineered cells.1-49. (canceled) 50. A method of culturing a cell, comprising: a) culturing a cell recombinantly engineered to express a chlorite dismutase polypeptide in a culture medium under conditions whereby the chlorite dismutase polypeptide is expressed in said cell, wherein said culture medium comprises one or more contaminating microorganisms; and b) treating said culture medium with chlorite in an amount sufficient to reduce the growth rate or kill said one or more contaminating microorganisms without killing said cell. 51. The method of claim 50, wherein the chlorite dismutase polypeptide is expressed in the cell from a chlorite dismutase gene (cld) heterologous to the cell. 52. The method of claim 50, wherein the chlorite dismutase polypeptide is expressed in the cell from a chlorite dismutase gene (cld) heterologous to the cell, wherein the gene is of a perchlorate reducing bacterium. 53. The method of claim 50, wherein the cell does not naturally express a chlorite dismutase. 54. The method of claim 50, wherein the cell is a bacterial call selected from Acidobacteria, Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia. 55. The method of claim 51, wherein the cell is a bacterial call selected from Acidobacteria, Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia. 56. The method of claim 52, wherein the cell is a bacterial call selected from Acidobacteria, Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia. 57. The method of claim 50, wherein the cell is a fungal cell selected from Ascomycota and Basidiomycota. 58. The method of claim 51, wherein the cell is a fungal cell selected from Ascomycota and Basidiomycota. 59. The method of claim 52, wherein the cell is a fungal cell selected from Ascomycota and Basidiomycota. 60. The method of claim 50, wherein the cell is selected from Saccharomyces sp., Trichoderma reesei, Neurospora sp., Kluyveromyces sp., Pichia sp., Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia sp., and Bacillus sp. 61. The method of claim 51, wherein the cell is selected from Saccharomyces sp., Trichoderma reesei, Neurospora sp., Kluyveromyces sp., Pichia sp., Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia sp., and Bacillus sp. 62. The method of claim 52, wherein the cell is selected from Saccharomyces sp., Trichoderma reesei, Neurospora sp., Kluyveromyces sp., Pichia sp., Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia sp., and Bacillus sp. 63. The method of claim 50, wherein the cell is selected from Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Trichoderma reesei, Neurospora crassa, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Pichia pastoris, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium saccharoperbutylacetonicum, Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Clostridium botulinum, Clostridium butyricum, Clostridium diolis, Clostridium ljungdahlii, Clostridium aerotolerans, Clostridium cellulolyticum, Clostridium tyrobutyricum, Clostridium pasteurianum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia lipolytica, and Bacillus subtilis. 64. The method of claim 51, wherein the cell is selected from Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Trichoderma reesei, Neurospora crassa, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Pichia pastoris, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium saccharoperbutylacetonicum, Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Clostridium botulinum, Clostridium butyricum, Clostridium diolis, Clostridium ljungdahlii, Clostridium aerotolerans, Clostridium cellulolyticum, Clostridium tyrobutyricum, Clostridium pasteurianum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia lipolytica, and Bacillus subtilis. 65. The method of claim 52, wherein the cell is selected from Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Trichoderma reesei, Neurospora crassa, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Pichia pastoris, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium saccharoperbutylacetonicum, Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Clostridium botulinum, Clostridium butyricum, Clostridium diolis, Clostridium ljungdahlii, Clostridium aerotolerans, Clostridium cellulolyticum, Clostridium tyrobutyricum, Clostridium pasteurianum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia lipolytica, and Bacillus subtilis. 66. The method of claim 50 wherein the cell is further recombinantly engineered to express one or more proteins necessary for heme biosynthesis selected from the group consisting of uroporphyrinogen III decarboxylase, coproporphyrinogen III oxidase, protoporphyrinogen IX oxidase, and ferrochelatase. 67. The method of claim 50, wherein the treating step comprises producing the chlorite in the culture medium by electrochemical generation. 68. The method of claim 50, wherein the treating step is performed intermittently. 69. The method of claim 50 further comprising culturing the cell under conditions sufficient for the cell to produce a fermentation product.

The present invention relates to novel granular silicas for use as support material, especially as support for catalysts for fixed bed reactors, and to the production and use thereof.

1. Granular silica having an Hg pore volume (<4 μm) of more than 0.90 ml/g, a dQ3=10% of more than 400 μm with, at the same time, a dQ3=90% of less than 3000 μm, and a ratio of the d50 without ultrasound exposure to d50 after 3 min of ultrasound exposure of <4.00, the measurement being effected on a fraction of particles from 400 to 500 μm. 2. Granular silica according to claim 1, wherein it has a pH in the range from 5 to 8.5. 3. Granular silica according to claim 1, wherein it has a ratio of the d50 without ultrasound exposure to d50 after 3 min of ultrasound exposure of 1.00 to 3.00, with the measurement is effected on a fraction of particles from 400 to 500 μm. 4. Granular silica according to claim 1, wherein it has an Hg pore volume (<4 μm) of more than 35 ml/g. 5. Granular silica according to claim 1, wherein it has an Hg pore volume (<4 μm) of 0.9 to 1.34 ml/g. 6. Process for producing granular silicas, comprising the steps of a) providing a precipitated or fumed silica, in dried and/or ground form, having a mean particle size d50 without ultrasound treatment of 0.1 to 350 μm, a BET surface area of 30 to 800 m2/g, and a DBP number of 140 to 400 g/100 g; b) moistening the silica from step a) according to the shaping process employed to a drying loss of 30-80% by weight; c) shaping the silica from step b) by extrusion, granulation, compaction, or other customary shaping processes; d) drying the shaped silica bodies in drying units suitable therefor; and e) screen granulation or screening of the granules at a screen size of 3000 μm and screening off the fines with a screen mesh size of 400 μm. 7. Process according to claim 6, wherein a water-containing filtercake with a drying loss of 30-80% by weight is used as the starting material for step c). 8. Process according to claim 6, wherein the silica from step a) is, in step c), compacted and granulated in high-speed intensive mixers. 9. Process for producing granular silicas, comprising the steps of i) providing a precipitated or fumed silica, in dried and/or ground form, having a drying loss of <30% by weight, and having a mean particle size d50 without ultrasound treatment of 0.1 to 350 μm, a BET surface area of 30 to 800 m2/g, and a DBP number of 140 to 400 g/100 g; ii) shaping the silica from step i) by dry compaction, preferably between two rotating rollers, at a specific contact pressure of 0.5 kN/cm of roller width to 12 kN/cm of roller width to give slugs, and iii) screen granulation or screening of the granules at a screen size of 3000 μm and screening off the fines with a screen mesh size of 400 μm. 10. Process according to claim 6, wherein all screen fractions smaller than 400 μm are removed. 11. Process according to claim 6, wherein shaping steps c) and ii) are performed without the addition of binders. 12. A method for producing absorbates comprising combining the granular silicas according to claim 1 with an additional absorbate material. 13. Absorbates comprising at least one of the granular silicas according to claim 1. 14. Absorbates according to claim 13, wherein they comprise at least one catalytically active substance. 15. Absorbate according to claim 13, wherein the absorbed substances are applied in proportions of 1 to 70% by weight. 16. Absorbate according to claim 14, wherein the active substance or active substance mixture is applied in proportions of 1 to 20% by weight. 17. Process for producing absorbates according to claim 13, wherein a granular silica according to claim 1 is contacted with at least one liquid selected from the group consisting of hardening agents or initiators, crosslinking agents, catalysts, active pharmaceutical ingredients and excipients, active cosmetic ingredients and excipients, cleansing and/or care compositions, flavourings, aromas and fragrances, animal feeds or animal feed additives, foods or food additives, dyes and/or pigments, amino acids, oxidizing or bleaching agents, additives with microbicidal, especially fungicidal or bactericidal, action, chemicals for agriculture and forestry, and/or a concrete admixture. 18. A catalytic process comprising utilizing the absorbate according to claim 13 in the catalytic process. 19. Process according to claim 18, wherein the process is performed in a fixed bed reactor, a fluidized bed reactor, or by suspending the absorbates in a reaction mixture. 20. Process according to claim 18, wherein the absorbate comprises an enzyme as a catalyst on a granular silica according to claim 1. 21. Process according to claim 9, wherein all screen fractions smaller than 400 μm are removed. 22. Process according to claim 9, wherein shaping steps c) and ii) are performed without the addition of binders.

The present invention relates to novel granular silicas for use as support material, especially as support for catalysts for fixed bed reactors, and to the production and use thereof.1. Granular silica having an Hg pore volume (<4 μm) of more than 0.90 ml/g, a dQ3=10% of more than 400 μm with, at the same time, a dQ3=90% of less than 3000 μm, and a ratio of the d50 without ultrasound exposure to d50 after 3 min of ultrasound exposure of <4.00, the measurement being effected on a fraction of particles from 400 to 500 μm. 2. Granular silica according to claim 1, wherein it has a pH in the range from 5 to 8.5. 3. Granular silica according to claim 1, wherein it has a ratio of the d50 without ultrasound exposure to d50 after 3 min of ultrasound exposure of 1.00 to 3.00, with the measurement is effected on a fraction of particles from 400 to 500 μm. 4. Granular silica according to claim 1, wherein it has an Hg pore volume (<4 μm) of more than 35 ml/g. 5. Granular silica according to claim 1, wherein it has an Hg pore volume (<4 μm) of 0.9 to 1.34 ml/g. 6. Process for producing granular silicas, comprising the steps of a) providing a precipitated or fumed silica, in dried and/or ground form, having a mean particle size d50 without ultrasound treatment of 0.1 to 350 μm, a BET surface area of 30 to 800 m2/g, and a DBP number of 140 to 400 g/100 g; b) moistening the silica from step a) according to the shaping process employed to a drying loss of 30-80% by weight; c) shaping the silica from step b) by extrusion, granulation, compaction, or other customary shaping processes; d) drying the shaped silica bodies in drying units suitable therefor; and e) screen granulation or screening of the granules at a screen size of 3000 μm and screening off the fines with a screen mesh size of 400 μm. 7. Process according to claim 6, wherein a water-containing filtercake with a drying loss of 30-80% by weight is used as the starting material for step c). 8. Process according to claim 6, wherein the silica from step a) is, in step c), compacted and granulated in high-speed intensive mixers. 9. Process for producing granular silicas, comprising the steps of i) providing a precipitated or fumed silica, in dried and/or ground form, having a drying loss of <30% by weight, and having a mean particle size d50 without ultrasound treatment of 0.1 to 350 μm, a BET surface area of 30 to 800 m2/g, and a DBP number of 140 to 400 g/100 g; ii) shaping the silica from step i) by dry compaction, preferably between two rotating rollers, at a specific contact pressure of 0.5 kN/cm of roller width to 12 kN/cm of roller width to give slugs, and iii) screen granulation or screening of the granules at a screen size of 3000 μm and screening off the fines with a screen mesh size of 400 μm. 10. Process according to claim 6, wherein all screen fractions smaller than 400 μm are removed. 11. Process according to claim 6, wherein shaping steps c) and ii) are performed without the addition of binders. 12. A method for producing absorbates comprising combining the granular silicas according to claim 1 with an additional absorbate material. 13. Absorbates comprising at least one of the granular silicas according to claim 1. 14. Absorbates according to claim 13, wherein they comprise at least one catalytically active substance. 15. Absorbate according to claim 13, wherein the absorbed substances are applied in proportions of 1 to 70% by weight. 16. Absorbate according to claim 14, wherein the active substance or active substance mixture is applied in proportions of 1 to 20% by weight. 17. Process for producing absorbates according to claim 13, wherein a granular silica according to claim 1 is contacted with at least one liquid selected from the group consisting of hardening agents or initiators, crosslinking agents, catalysts, active pharmaceutical ingredients and excipients, active cosmetic ingredients and excipients, cleansing and/or care compositions, flavourings, aromas and fragrances, animal feeds or animal feed additives, foods or food additives, dyes and/or pigments, amino acids, oxidizing or bleaching agents, additives with microbicidal, especially fungicidal or bactericidal, action, chemicals for agriculture and forestry, and/or a concrete admixture. 18. A catalytic process comprising utilizing the absorbate according to claim 13 in the catalytic process. 19. Process according to claim 18, wherein the process is performed in a fixed bed reactor, a fluidized bed reactor, or by suspending the absorbates in a reaction mixture. 20. Process according to claim 18, wherein the absorbate comprises an enzyme as a catalyst on a granular silica according to claim 1. 21. Process according to claim 9, wherein all screen fractions smaller than 400 μm are removed. 22. Process according to claim 9, wherein shaping steps c) and ii) are performed without the addition of binders.

Described herein are toothpastes comprising a first dentifrice comprising a calcium carbonate abrasive and a second dentifrice comprising a zinc ion source in a gel base, wherein the second dentifrice is entrained as a stripe in the first dentifrice, as well as products comprising the toothpaste and methods of making and using the same.

1. A toothpaste product comprising a first dentifrice comprising a calcium carbonate abrasive, a second dentifrice comprising a zinc ion source in a gel base, and a container which holds the first dentifrice physically separate from the second dentifrice until the dentifrices are dispensed. 2. The product of claim 1, wherein the product is capable of dispensing the second dentifrice as a stripe entrained in the first dentifrice. 3. The toothpaste product of claim 1, wherein the zinc ion source is selected from zinc oxide, zinc citrate, and mixtures thereof. 4. The toothpaste product of claim 1, wherein the amount of first dentifrice relative to the second dentifrice is from 3:1 to 5:1. 5. The toothpaste product of claim 1 wherein the second dentifrice contains zinc in an amount of 0.1 to 10% by weight of the second dentifrice. 6. The toothpaste product of claim 1 wherein the second dentifrice contains a zinc ion source selected from (i) zinc oxide in an amount of 0.1 to 6%; (ii) zinc citrate in an amount of 0.1 to 10%; and (iii) zinc oxide in an amount of 0.1 to 6% and zinc citrate in an amount of 0.1 to 10%. 7. The toothpaste product of claim 1 wherein the ratio of the first dentifrice to the second dentifrice is about 4:1 and the second dentifrice contains about 5% zinc oxide. 8. The toothpaste product of claim 1 wherein the second dentifrice comprises a viscosity-modifying amount of one or more thickening agents selected from carboxymethyl cellulose, xanthan, thickening silica, and mixtures thereof. 9. The toothpaste product of claim 1 wherein the calcium carbonate abrasive in the first dentifrice is a mixture of precipitated calcium carbonate and natural calcium carbonate in an amount of 35-45% by weight of the first dentifrice. 10. The toothpaste product of claim 1 wherein the second dentifrice comprises silica abrasive in an amount of 10-40% by weight of the second dentifrice. 11. The toothpaste product of claim 1 wherein the first dentifrice is an opaque white paste and the second abrasive is a colored translucent gel. 12. A striped toothpaste comprising a first dentifrice comprising a calcium carbonate abrasive, second dentifrice comprising a zinc ion source and a silica gel portion, wherein the second dentifrice is entrained as a stripe in the first dentifrice, and wherein the zinc ion source is concentrated in the silica gel portion. 13. A method of making a dentifrice comprising a first dentifrice comprising a calcium carbonate abrasive and a second dentifrice comprising a zinc ion source and a silica gel portion, comprising entraining the second dentifrice as a stripe in the first dentifrice, and wherein the zinc ion source is concentrated in the silica gel portion. 14. A method of treating or preventing a condition of the oral cavity, comprising administering a composition according to claim 1, to the oral cavity of a subject in need thereof. 15. (canceled) 16. (canceled)

Described herein are toothpastes comprising a first dentifrice comprising a calcium carbonate abrasive and a second dentifrice comprising a zinc ion source in a gel base, wherein the second dentifrice is entrained as a stripe in the first dentifrice, as well as products comprising the toothpaste and methods of making and using the same.1. A toothpaste product comprising a first dentifrice comprising a calcium carbonate abrasive, a second dentifrice comprising a zinc ion source in a gel base, and a container which holds the first dentifrice physically separate from the second dentifrice until the dentifrices are dispensed. 2. The product of claim 1, wherein the product is capable of dispensing the second dentifrice as a stripe entrained in the first dentifrice. 3. The toothpaste product of claim 1, wherein the zinc ion source is selected from zinc oxide, zinc citrate, and mixtures thereof. 4. The toothpaste product of claim 1, wherein the amount of first dentifrice relative to the second dentifrice is from 3:1 to 5:1. 5. The toothpaste product of claim 1 wherein the second dentifrice contains zinc in an amount of 0.1 to 10% by weight of the second dentifrice. 6. The toothpaste product of claim 1 wherein the second dentifrice contains a zinc ion source selected from (i) zinc oxide in an amount of 0.1 to 6%; (ii) zinc citrate in an amount of 0.1 to 10%; and (iii) zinc oxide in an amount of 0.1 to 6% and zinc citrate in an amount of 0.1 to 10%. 7. The toothpaste product of claim 1 wherein the ratio of the first dentifrice to the second dentifrice is about 4:1 and the second dentifrice contains about 5% zinc oxide. 8. The toothpaste product of claim 1 wherein the second dentifrice comprises a viscosity-modifying amount of one or more thickening agents selected from carboxymethyl cellulose, xanthan, thickening silica, and mixtures thereof. 9. The toothpaste product of claim 1 wherein the calcium carbonate abrasive in the first dentifrice is a mixture of precipitated calcium carbonate and natural calcium carbonate in an amount of 35-45% by weight of the first dentifrice. 10. The toothpaste product of claim 1 wherein the second dentifrice comprises silica abrasive in an amount of 10-40% by weight of the second dentifrice. 11. The toothpaste product of claim 1 wherein the first dentifrice is an opaque white paste and the second abrasive is a colored translucent gel. 12. A striped toothpaste comprising a first dentifrice comprising a calcium carbonate abrasive, second dentifrice comprising a zinc ion source and a silica gel portion, wherein the second dentifrice is entrained as a stripe in the first dentifrice, and wherein the zinc ion source is concentrated in the silica gel portion. 13. A method of making a dentifrice comprising a first dentifrice comprising a calcium carbonate abrasive and a second dentifrice comprising a zinc ion source and a silica gel portion, comprising entraining the second dentifrice as a stripe in the first dentifrice, and wherein the zinc ion source is concentrated in the silica gel portion. 14. A method of treating or preventing a condition of the oral cavity, comprising administering a composition according to claim 1, to the oral cavity of a subject in need thereof. 15. (canceled) 16. (canceled)

Compounds and methods in the fields of chemistry and medicine are disclosed. Some of the disclosed embodiments include compounds, compositions and methods of using imidazole-fused heterocycle amines. Some of the disclosed embodiments include imizazo-fused heterocycle compounds useful to treat leukemia and other hematopoietic disorders.

1. A compound of Formula I: or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof, wherein X is selected from N or CR4a; X′ is selected from N or CR4b; Y is selected from N or CR5a; Y′ is selected from N or CR5b; provided at least one of X, X′, Y, and Y′ is N; R1 is selected from the group consisting of hydrogen, halogen, —OR6, —CN, —NR7R8, —CH2OR6, —CH2NR7R8, an optionally substituted C1-6 alkyl, an optionally substituted C1-6 haloalkyl, an optionally substituted C2-6 alkenyl, an optionally substituted C2-6 alkynyl, an optionally substituted (5 to 7 membered heterocyclyl)alkyl, an optionally substituted 5 to 7 membered heterocyclyl, an optionally substituted aralkyl; an optionally substituted (5 or 6 membered heteroaryl)alkyl, an optionally substituted C1-6 heteroalkyl, —C(═O)R6, —C(═O)OR6, —C(═O)NR7R8, —NHC(═O)R6, —SO2R6, and —SO2NR7R8; each of R2, R3, R4a and R4b is independently selected from the group consisting of hydrogen, halogen, C1-6 alkyl, OH, and C1-6 alkoxy; each of R5a and R5b is independently selected from the group consisting of hydrogen, halogen, —OR6, —CN, —NR7R8, —CH2OR6, an optionally substituted aryl, an optionally substituted 5 to 10 membered heteroaryl, an optionally substituted 5-10 membered heterocyclyl, an optionally substituted C3-7 carbocyclyl, an optionally substituted C1-6 alkyl, an optionally substituted C1-6 haloalkyl, an optionally substituted C1-6 heteroalkyl, —C(═O)R6, —C(═O)OR6, —C(═O)NR7R8, —NHC(═O)R6, —SO2R6, and —SO2NR7R8; each R6 is independently selected from hydrogen, an optionally substituted C1-10 alkyl, an optionally substituted C1-10 haloalkyl, or an optionally substituted C1-6 heteroalkyl; and each R7 and R8 is independently selected from hydrogen; an optionally substituted C1-10 alkyl; an optionally substituted C1-10 haloalkyl; or an optionally substituted C1-6 heteroalkyl; or R7 and R8 are joined together with the nitrogen atom to which they are attached to form an optionally substituted C3-7 cycloalkyl or 3 to 7 membered heterocyclyl ring. 2. The compound of claim 1, wherein the compound of Formula (I) is also represented by Formula (II): 3. The compound of claim 2, wherein each R4a and R4b is hydrogen. 4. The compound of claim 1, wherein the compound of Formula (I) is also represented by Formula (III): 5. The compound of claim 4, wherein each R4a and R5a is hydrogen. 6. The compound of claim 1, wherein R1 is an optionally substituted (5 to 7 membered heterocyclyl)alkyl. 7. The compound of claim 6, wherein R1 is an optionally substituted (5 membered heterocyclyl)alkyl. 8. The compound of claim 7, wherein R1 is pyrrolidyl-CH2—. 9. The compound of claim 6, wherein R1 is an optionally substituted (6 membered heterocyclyl)alkyl. 10. The compound of claim 9, wherein R1 is selected from piperidinyl-CH2— or morpholine-CH2—. 11. The compound of claim 1, wherein R1 is an optionally substituted 5 to 7 membered heterocyclyl. 12. The compound of claim 11, wherein R1 is an optionally substituted 6 membered heterocyclyl. 13. The compound of claim 12, wherein R1 is selected from optionally substituted morpholinyl, optionally substituted piperazinyl, or optionally substituted piperidinyl. 14. The compound of claim 1, wherein R2 is hydrogen. 15. The compound of claim 1, wherein R3 is hydrogen. 16. The compound of claim 1, wherein R5b is an optionally substituted 5 to 10 membered heteroaryl. 17. The compound of claim 16, wherein R5b is an optionally substituted 6 membered heteroaryl. 18. The compound of claim 17, wherein R5b is selected from pyridyl, pyrazinyl, pyridazinyl, or pyrimidyl. 19. The compound of claim 16, wherein R5b is an optionally substituted 5 membered heteroaryl. 20. The compound of claim 19, wherein R5b is pyrazolyl. 21. The compound claim 1, selected from or pharmaceutically acceptable salts, esters, amides, or prodrugs thereof. 22. A pharmaceutical composition comprising an effective amount of a compound of claim 1, or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof, and a pharmaceutically acceptable carrier, diluent, excipient or combinations thereof. 23. A method of treating a hematopoietic disorder, comprising administering a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof, to a subject in need thereof. 24. The method of claim 23, wherein the hematopoietic disorder is leukemia. 25. The method of claim 24, wherein said leukemia is acute myelogenous leukemia.

Compounds and methods in the fields of chemistry and medicine are disclosed. Some of the disclosed embodiments include compounds, compositions and methods of using imidazole-fused heterocycle amines. Some of the disclosed embodiments include imizazo-fused heterocycle compounds useful to treat leukemia and other hematopoietic disorders.1. A compound of Formula I: or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof, wherein X is selected from N or CR4a; X′ is selected from N or CR4b; Y is selected from N or CR5a; Y′ is selected from N or CR5b; provided at least one of X, X′, Y, and Y′ is N; R1 is selected from the group consisting of hydrogen, halogen, —OR6, —CN, —NR7R8, —CH2OR6, —CH2NR7R8, an optionally substituted C1-6 alkyl, an optionally substituted C1-6 haloalkyl, an optionally substituted C2-6 alkenyl, an optionally substituted C2-6 alkynyl, an optionally substituted (5 to 7 membered heterocyclyl)alkyl, an optionally substituted 5 to 7 membered heterocyclyl, an optionally substituted aralkyl; an optionally substituted (5 or 6 membered heteroaryl)alkyl, an optionally substituted C1-6 heteroalkyl, —C(═O)R6, —C(═O)OR6, —C(═O)NR7R8, —NHC(═O)R6, —SO2R6, and —SO2NR7R8; each of R2, R3, R4a and R4b is independently selected from the group consisting of hydrogen, halogen, C1-6 alkyl, OH, and C1-6 alkoxy; each of R5a and R5b is independently selected from the group consisting of hydrogen, halogen, —OR6, —CN, —NR7R8, —CH2OR6, an optionally substituted aryl, an optionally substituted 5 to 10 membered heteroaryl, an optionally substituted 5-10 membered heterocyclyl, an optionally substituted C3-7 carbocyclyl, an optionally substituted C1-6 alkyl, an optionally substituted C1-6 haloalkyl, an optionally substituted C1-6 heteroalkyl, —C(═O)R6, —C(═O)OR6, —C(═O)NR7R8, —NHC(═O)R6, —SO2R6, and —SO2NR7R8; each R6 is independently selected from hydrogen, an optionally substituted C1-10 alkyl, an optionally substituted C1-10 haloalkyl, or an optionally substituted C1-6 heteroalkyl; and each R7 and R8 is independently selected from hydrogen; an optionally substituted C1-10 alkyl; an optionally substituted C1-10 haloalkyl; or an optionally substituted C1-6 heteroalkyl; or R7 and R8 are joined together with the nitrogen atom to which they are attached to form an optionally substituted C3-7 cycloalkyl or 3 to 7 membered heterocyclyl ring. 2. The compound of claim 1, wherein the compound of Formula (I) is also represented by Formula (II): 3. The compound of claim 2, wherein each R4a and R4b is hydrogen. 4. The compound of claim 1, wherein the compound of Formula (I) is also represented by Formula (III): 5. The compound of claim 4, wherein each R4a and R5a is hydrogen. 6. The compound of claim 1, wherein R1 is an optionally substituted (5 to 7 membered heterocyclyl)alkyl. 7. The compound of claim 6, wherein R1 is an optionally substituted (5 membered heterocyclyl)alkyl. 8. The compound of claim 7, wherein R1 is pyrrolidyl-CH2—. 9. The compound of claim 6, wherein R1 is an optionally substituted (6 membered heterocyclyl)alkyl. 10. The compound of claim 9, wherein R1 is selected from piperidinyl-CH2— or morpholine-CH2—. 11. The compound of claim 1, wherein R1 is an optionally substituted 5 to 7 membered heterocyclyl. 12. The compound of claim 11, wherein R1 is an optionally substituted 6 membered heterocyclyl. 13. The compound of claim 12, wherein R1 is selected from optionally substituted morpholinyl, optionally substituted piperazinyl, or optionally substituted piperidinyl. 14. The compound of claim 1, wherein R2 is hydrogen. 15. The compound of claim 1, wherein R3 is hydrogen. 16. The compound of claim 1, wherein R5b is an optionally substituted 5 to 10 membered heteroaryl. 17. The compound of claim 16, wherein R5b is an optionally substituted 6 membered heteroaryl. 18. The compound of claim 17, wherein R5b is selected from pyridyl, pyrazinyl, pyridazinyl, or pyrimidyl. 19. The compound of claim 16, wherein R5b is an optionally substituted 5 membered heteroaryl. 20. The compound of claim 19, wherein R5b is pyrazolyl. 21. The compound claim 1, selected from or pharmaceutically acceptable salts, esters, amides, or prodrugs thereof. 22. A pharmaceutical composition comprising an effective amount of a compound of claim 1, or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof, and a pharmaceutically acceptable carrier, diluent, excipient or combinations thereof. 23. A method of treating a hematopoietic disorder, comprising administering a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof, to a subject in need thereof. 24. The method of claim 23, wherein the hematopoietic disorder is leukemia. 25. The method of claim 24, wherein said leukemia is acute myelogenous leukemia.

Described herein are enhanced compositions and methods for storing biomaterials. In certain aspects, these biomaterials include natural and engineered eukaryotic tissues. The methods described herein include storing these biomaterials in such a manner that reduces or prevents the loss of biomaterial properties (e.g., extracellular matrix integrity, cell viability, or a combination thereof) occurring either during storage or after removal of the biomaterial from storage. In certain aspects, these biomaterials will be stored in animal product-free solutions containing an agent that prevents or reduces the loss of extracellular matrix integrity.

1. A composition comprising a biomaterial placed in a solution that includes at least one agent that reduces or prevents a loss of biomaterial properties, wherein the solution is an animal product-free solution, the biomaterial comprises chondrocytes in an extracellular matrix or cartilage, and the at least one agent comprises an enzyme inhibitor of a matrix metalloproteinase having a concentration ranging from 1.0 nM to 1 mM. 2. The composition of claim 1, wherein the biomaterial properties comprise extracellular matrix integrity, cell viability, or a combination thereof. 3. The composition of claim 2, wherein extracellular matrix integrity comprises extracellular matrix permeability, extracellular matrix water content, extracellular matrix glycosaminoglycan content, or any combination thereof. 4. The composition of claim 1, wherein the biomaterial comprises cartilage. 5. The composition of claim 1, wherein the biomaterial comprises chondrocytes in an extracellular matrix. 6. The composition of claim 1, wherein the solution does not include fetal bovine serum. 7. The composition of claim 1, wherein the animal product-free solution is an extracellular-type solution that is isotonic. 8. The composition of claim 1, wherein the animal product-free solution is an intracellular-type solution that is isotonic. 9. The composition of claim 1, wherein the enzyme inhibitor minimizes an enzymatic activity to reduce or prevent the loss of biomaterial properties, wherein the biomaterial properties include extracellular matrix integrity. 10. The composition of claim 1, wherein the matrix metalloproteinase comprises one or more members selected from the group consisting of MMP 1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP 11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP 19, MMP 20, MMP 21, MMP 23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. 11. The composition of claim 1, wherein the enzyme inhibitor is selected from the group consisting of doxycycline, TIMPs, a compound that up-regulates endogenous TIMPs, PCK3145, BB-2516, and BB-94. 12. A method for storing a biomaterial comprising preparing the composition of claim 1, wherein preparing the composition of claim 1 includes placing the biomaterial in a solution that includes at least one agent that reduces or prevents a loss of biomaterial properties. 13. The method of claim 12, further comprising storing the biomaterial placed in the solution at a temperature ranging from −25° C. to +35° C. 14. A composition comprising an animal product-free solution, wherein the solution includes at least one matrix metalloproteinase inhibitor at concentrations ranging from 1.0 nM to 1000 μM, the animal product-free solution is an intracellular-type solution that does not include a cell culture media, and the matrix metalloproteinase inhibitor reduces or inhibits enzymatic activity of at least one of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP 14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28 or any combination thereof. 15. The composition of claim 14, wherein the at least one matrix metalloproteinase inhibitor reduces or inhibits enzymatic activity of at least two of MMP1, MMP8, MMP9, and MMP13. 16. The composition of claim 14, wherein the at least one matrix metalloproteinase inhibitor reduces or inhibits enzymatic activity of at least three of MMP1, MMP8, MMP9, and MMP13. 17. The composition of claim 14, wherein the at least one matrix metalloproteinase inhibitor is selected from the group consisting of doxycycline, TIMPs, a compound that up-regulates endogenous TIMPs, PCK3145, BB-2516, and BB-94. 18. The composition of claim 14, wherein doxycycline is the at least one matrix metalloproteinase inhibitor and is present at a concentration of from 1.0 nM to 1000 μM. 19. The composition of claim 18, wherein the intracellular-type solution further comprises a nutrient cocktail that includes at least one of the following components: D-glucose, glycine, L-arginine hydrochloride, L-cystine hydrochloride, L-glutamine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, choline, D-calcium pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, inositol, any salt thereof, or any combination thereof. 20. A method comprising storing a biomaterial at hypothermic temperatures in the composition of claim 14, wherein the composition includes at least one additive that promotes retention of extracellular matrix integrity and cell viability, and the at least one additive comprises an enzyme inhibitor, an amino acid, a plurality of amino acids, a sugar, a plurality of sugars, a lipid, a plurality of lipids, a vitamin, a plurality of vitamins, or any combination thereof.

Described herein are enhanced compositions and methods for storing biomaterials. In certain aspects, these biomaterials include natural and engineered eukaryotic tissues. The methods described herein include storing these biomaterials in such a manner that reduces or prevents the loss of biomaterial properties (e.g., extracellular matrix integrity, cell viability, or a combination thereof) occurring either during storage or after removal of the biomaterial from storage. In certain aspects, these biomaterials will be stored in animal product-free solutions containing an agent that prevents or reduces the loss of extracellular matrix integrity.1. A composition comprising a biomaterial placed in a solution that includes at least one agent that reduces or prevents a loss of biomaterial properties, wherein the solution is an animal product-free solution, the biomaterial comprises chondrocytes in an extracellular matrix or cartilage, and the at least one agent comprises an enzyme inhibitor of a matrix metalloproteinase having a concentration ranging from 1.0 nM to 1 mM. 2. The composition of claim 1, wherein the biomaterial properties comprise extracellular matrix integrity, cell viability, or a combination thereof. 3. The composition of claim 2, wherein extracellular matrix integrity comprises extracellular matrix permeability, extracellular matrix water content, extracellular matrix glycosaminoglycan content, or any combination thereof. 4. The composition of claim 1, wherein the biomaterial comprises cartilage. 5. The composition of claim 1, wherein the biomaterial comprises chondrocytes in an extracellular matrix. 6. The composition of claim 1, wherein the solution does not include fetal bovine serum. 7. The composition of claim 1, wherein the animal product-free solution is an extracellular-type solution that is isotonic. 8. The composition of claim 1, wherein the animal product-free solution is an intracellular-type solution that is isotonic. 9. The composition of claim 1, wherein the enzyme inhibitor minimizes an enzymatic activity to reduce or prevent the loss of biomaterial properties, wherein the biomaterial properties include extracellular matrix integrity. 10. The composition of claim 1, wherein the matrix metalloproteinase comprises one or more members selected from the group consisting of MMP 1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP 11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP 19, MMP 20, MMP 21, MMP 23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. 11. The composition of claim 1, wherein the enzyme inhibitor is selected from the group consisting of doxycycline, TIMPs, a compound that up-regulates endogenous TIMPs, PCK3145, BB-2516, and BB-94. 12. A method for storing a biomaterial comprising preparing the composition of claim 1, wherein preparing the composition of claim 1 includes placing the biomaterial in a solution that includes at least one agent that reduces or prevents a loss of biomaterial properties. 13. The method of claim 12, further comprising storing the biomaterial placed in the solution at a temperature ranging from −25° C. to +35° C. 14. A composition comprising an animal product-free solution, wherein the solution includes at least one matrix metalloproteinase inhibitor at concentrations ranging from 1.0 nM to 1000 μM, the animal product-free solution is an intracellular-type solution that does not include a cell culture media, and the matrix metalloproteinase inhibitor reduces or inhibits enzymatic activity of at least one of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP 14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28 or any combination thereof. 15. The composition of claim 14, wherein the at least one matrix metalloproteinase inhibitor reduces or inhibits enzymatic activity of at least two of MMP1, MMP8, MMP9, and MMP13. 16. The composition of claim 14, wherein the at least one matrix metalloproteinase inhibitor reduces or inhibits enzymatic activity of at least three of MMP1, MMP8, MMP9, and MMP13. 17. The composition of claim 14, wherein the at least one matrix metalloproteinase inhibitor is selected from the group consisting of doxycycline, TIMPs, a compound that up-regulates endogenous TIMPs, PCK3145, BB-2516, and BB-94. 18. The composition of claim 14, wherein doxycycline is the at least one matrix metalloproteinase inhibitor and is present at a concentration of from 1.0 nM to 1000 μM. 19. The composition of claim 18, wherein the intracellular-type solution further comprises a nutrient cocktail that includes at least one of the following components: D-glucose, glycine, L-arginine hydrochloride, L-cystine hydrochloride, L-glutamine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, choline, D-calcium pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, inositol, any salt thereof, or any combination thereof. 20. A method comprising storing a biomaterial at hypothermic temperatures in the composition of claim 14, wherein the composition includes at least one additive that promotes retention of extracellular matrix integrity and cell viability, and the at least one additive comprises an enzyme inhibitor, an amino acid, a plurality of amino acids, a sugar, a plurality of sugars, a lipid, a plurality of lipids, a vitamin, a plurality of vitamins, or any combination thereof.

A topical composition includes a nanoemulsion of a plurality of hydrophobic particles having a hydrophilic coating therein. The hydrophobic particles are derived from the same or different hydrophobic material and each hydrophobic particle has a melting point below the melting point of the respective hydrophobic material. The hydrophobic particles comprise a mean particle size of less than about 10 nm, and the nanoemulsion further includes one or more pharmaceutically active agents.

1. A topical composition comprising a nanoemulsion of a plurality of hydrophobic particles having a hydrophilic coating therein, wherein the hydrophobic particles are derived from the same or different hydrophobic material and each hydrophobic particle has a melting point less than the melting point of the respective hydrophobic material, the hydrophobic particles comprise a mean particle size of less than about 20 nm with a polydispersity of less than about 10%, and the nanoemulsion further comprises at least one pharmaceutically active agent. 2. The topical composition of claim 1, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm with a polydispersity of less than about 10%. 3. The topical composition of claim 1, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm with a polydispersity of less than about 5%. 4. The topical composition of claim 1, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm with a polydispersity of less than about 3%. 5. The topical composition of claim 1, wherein the hydrophobic particles have a melting point of from about 21° C. to about 33° C. 6. The topical composition of claim 5, wherein the hydrophobic material is selected from the group consisting of a wax, butter and combinations thereof. 7. The topical composition of claim 5, wherein the hydrophobic material is selected from the group consisting paraffin wax, beeswax white, candellila wax, mango butter, aloe butter, olive butter, coffea butter, macadamia nut butter, avocado butter, cocoa butter, seed hemp seed butter, illipe seed butter, kokum seed butter, pistachio nut butter, shea butter, sweet almond butter, grape seed butter, mowrah butter, apricot butter, sal butter, soy butter, wheat germ butter, palm kernel flakes and mixtures thereof. 8. The topical composition of claim 1, wherein the hydrophilic coating comprises a surfactant. 9. The topical composition of claim 8, wherein the surfactant is a non-ionic surfactant selected from the group consisting of a fatty alcohol, a polyoxyalkylene alkyl ether, a polyoxyalkylene alkenyl ether, a polyoxyethylene glycol octylphenol ether, a polyethylene glycol alkyl ether, a polyglyceryl fatty acid ester, a fatty acid mono- or diethanolamide, a polyoxyethylene hydrogenated castor oil, a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene fatty acid ester, a sorbitan ester, a sucrose ester, an alkyl-saccharide-based surfactant, an alkylamine oxide, an alkylamidoamine oxide and mixtures thereof. 10. A topical composition comprising a nanoemulsion of a plurality of hydrophobic particles having a hydrophilic coating therein, wherein the hydrophobic particles are derived from the same or different hydrophobic material and each hydrophobic particle has a melting point less than the melting point of the respective hydrophobic material, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm, and wherein the nanoemulsion further comprises at least one pharmaceutically active agent relating to hirsutism or alopecia. 11. The topical composition of claim 10, wherein the hydrophobic particles comprise a mean particle size of less than about 8 nm. 12. The topical composition of claim 10, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm with a polydispersity of less than about 10%. 13. The topical composition of claim 10, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm with a polydispersity of less than about 5%. 14. The topical composition of claim 10, wherein the at least one pharmaceutically active agent relating to hirsutism or alopecia comprises cyclosporine, FK506, minoxidil, or mixtures thereof. 15. The topical composition of claim 10, wherein the hydrophobic particles have a melting point of from about 21° C. to about 33° C. 16. The topical composition of claim 15, wherein the hydrophobic material is selected from the group consisting of a wax, butter and combinations thereof. 17. The topical composition of claim 15, wherein the hydrophobic material is selected from the group consisting paraffin wax, beeswax white, candellila wax, mango butter, aloe butter, olive butter, coffea butter, macadamia nut butter, avocado butter, cocoa butter, seed hemp seed butter, illipe seed butter, kokum seed butter, pistachio nut butter, shea butter, sweet almond butter, grape seed butter, mowrah butter, apricot butter, sal butter, soy butter, wheat germ butter, palm kernel flakes and mixtures thereof. 18. The topical composition of claim 10, wherein the hydrophilic coating comprises a surfactant. 19. A method for treating one of hirsutism and alopecia, the method comprising contacting an area of a host with a therapeutically effective amount of a topical composition comprising a nanoemulsion of a plurality of hydrophobic particles having a hydrophilic coating therein, wherein the hydrophobic particles are derived from the same or different hydrophobic material and each hydrophobic particle has a melting point less than the melting point of the respective hydrophobic material, the hydrophobic particles comprise a mean particle size of less than about 10 nm, and the nanoemulsion further comprises at least one pharmaceutically active agent relating to hirsutism or alopecia. 20. The method of claim 19, wherein the at least one pharmaceutically active agent relating to hirsutism or alopecia comprises cyclosporine, FK506, minoxidil, or mixtures thereof.

A topical composition includes a nanoemulsion of a plurality of hydrophobic particles having a hydrophilic coating therein. The hydrophobic particles are derived from the same or different hydrophobic material and each hydrophobic particle has a melting point below the melting point of the respective hydrophobic material. The hydrophobic particles comprise a mean particle size of less than about 10 nm, and the nanoemulsion further includes one or more pharmaceutically active agents.1. A topical composition comprising a nanoemulsion of a plurality of hydrophobic particles having a hydrophilic coating therein, wherein the hydrophobic particles are derived from the same or different hydrophobic material and each hydrophobic particle has a melting point less than the melting point of the respective hydrophobic material, the hydrophobic particles comprise a mean particle size of less than about 20 nm with a polydispersity of less than about 10%, and the nanoemulsion further comprises at least one pharmaceutically active agent. 2. The topical composition of claim 1, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm with a polydispersity of less than about 10%. 3. The topical composition of claim 1, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm with a polydispersity of less than about 5%. 4. The topical composition of claim 1, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm with a polydispersity of less than about 3%. 5. The topical composition of claim 1, wherein the hydrophobic particles have a melting point of from about 21° C. to about 33° C. 6. The topical composition of claim 5, wherein the hydrophobic material is selected from the group consisting of a wax, butter and combinations thereof. 7. The topical composition of claim 5, wherein the hydrophobic material is selected from the group consisting paraffin wax, beeswax white, candellila wax, mango butter, aloe butter, olive butter, coffea butter, macadamia nut butter, avocado butter, cocoa butter, seed hemp seed butter, illipe seed butter, kokum seed butter, pistachio nut butter, shea butter, sweet almond butter, grape seed butter, mowrah butter, apricot butter, sal butter, soy butter, wheat germ butter, palm kernel flakes and mixtures thereof. 8. The topical composition of claim 1, wherein the hydrophilic coating comprises a surfactant. 9. The topical composition of claim 8, wherein the surfactant is a non-ionic surfactant selected from the group consisting of a fatty alcohol, a polyoxyalkylene alkyl ether, a polyoxyalkylene alkenyl ether, a polyoxyethylene glycol octylphenol ether, a polyethylene glycol alkyl ether, a polyglyceryl fatty acid ester, a fatty acid mono- or diethanolamide, a polyoxyethylene hydrogenated castor oil, a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene fatty acid ester, a sorbitan ester, a sucrose ester, an alkyl-saccharide-based surfactant, an alkylamine oxide, an alkylamidoamine oxide and mixtures thereof. 10. A topical composition comprising a nanoemulsion of a plurality of hydrophobic particles having a hydrophilic coating therein, wherein the hydrophobic particles are derived from the same or different hydrophobic material and each hydrophobic particle has a melting point less than the melting point of the respective hydrophobic material, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm, and wherein the nanoemulsion further comprises at least one pharmaceutically active agent relating to hirsutism or alopecia. 11. The topical composition of claim 10, wherein the hydrophobic particles comprise a mean particle size of less than about 8 nm. 12. The topical composition of claim 10, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm with a polydispersity of less than about 10%. 13. The topical composition of claim 10, wherein the hydrophobic particles comprise a mean particle size of less than about 10 nm with a polydispersity of less than about 5%. 14. The topical composition of claim 10, wherein the at least one pharmaceutically active agent relating to hirsutism or alopecia comprises cyclosporine, FK506, minoxidil, or mixtures thereof. 15. The topical composition of claim 10, wherein the hydrophobic particles have a melting point of from about 21° C. to about 33° C. 16. The topical composition of claim 15, wherein the hydrophobic material is selected from the group consisting of a wax, butter and combinations thereof. 17. The topical composition of claim 15, wherein the hydrophobic material is selected from the group consisting paraffin wax, beeswax white, candellila wax, mango butter, aloe butter, olive butter, coffea butter, macadamia nut butter, avocado butter, cocoa butter, seed hemp seed butter, illipe seed butter, kokum seed butter, pistachio nut butter, shea butter, sweet almond butter, grape seed butter, mowrah butter, apricot butter, sal butter, soy butter, wheat germ butter, palm kernel flakes and mixtures thereof. 18. The topical composition of claim 10, wherein the hydrophilic coating comprises a surfactant. 19. A method for treating one of hirsutism and alopecia, the method comprising contacting an area of a host with a therapeutically effective amount of a topical composition comprising a nanoemulsion of a plurality of hydrophobic particles having a hydrophilic coating therein, wherein the hydrophobic particles are derived from the same or different hydrophobic material and each hydrophobic particle has a melting point less than the melting point of the respective hydrophobic material, the hydrophobic particles comprise a mean particle size of less than about 10 nm, and the nanoemulsion further comprises at least one pharmaceutically active agent relating to hirsutism or alopecia. 20. The method of claim 19, wherein the at least one pharmaceutically active agent relating to hirsutism or alopecia comprises cyclosporine, FK506, minoxidil, or mixtures thereof.

Synthetic composite materials for use, for example, as orthopedic implants are described herein. In one example, a composite material for use as a scaffold includes a thermoplastic polymer forming a porous matrix that has continuous porosity and a plurality of pores. The porosity and the size of the pores are selectively formed during synthesis of the composite material. The example composite material also includes a plurality of a anisometric calcium phosphate particles integrally formed, embedded in, or exposed on a surface of the porous matrix. The calcium phosphate particles provide one or more of reinforcement, bioactivity, or bioresorption.

1. A porous reinforced composite scaffold material, comprising: a thermoplastic polymer material having essentially uniform porosity and comprising a plurality of anisometric calcium phosphate particles distributed essentially uniformly throughout, wherein the composite comprises a plurality pores that are defined by voids interconnected by struts, the size of the pores being either homogenous or heterogeneous and within the range from about 10 to 500 μm, and wherein the calcium phosphate particles comprise single crystals, dense polycrystals or combinations thereof, and wherein the calcium phosphate particles are embedded within the thermoplastic polymer material and exposed on the struts within the pore voids. 2. A porous reinforced composite scaffold material as described in claim 1, wherein the porosity ranges between 1 to 95 percent by volume. 3. A porous reinforced composite scaffold material as described in claim 1, wherein the anisometric calcium phosphate comprises one or more of hydroxyapatite, calcium-deficient hydroxyapatite, carbonated calcium hydroxyapatite, beta-tricalcium phosphate (beta-TCP), alpha-tricalcium phosphate (alpha-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), tetracalcium phosphate, biphasic calcium phosphate (BCP), anhydrous dicalcium phosphate (DCPA), dicalcium phosphate dihydrate (DCPD), anhydrous monocalcium phosphate (MCPA), monocalcium phosphate monohydrate (MCPM), and combinations thereof, and, wherein the polymer comprises one or more of polyaryletherketone, polyetheretherketone (PEEK), polyetherketonekteone (PEKK), polyetherketone (PEK), polyethylene, high density polyethylene (HDPE), ultra high molecular weight polyethylene (UHMWPE), low density polyethylene (LDPE), polyethylene oxide (PEO), polyurethane, polypropylene, polypropylene oxide (PPO), polysulfone, polymethylmethacrylate (PMMA), and other polyacrylics from monomers such as bisphenol a hydroxypropylmethacrylate (bis-GMA) or tri(ethylene glycol) dimethacrylate (TEG-DMA), polypropylene, poly(e-caprolactone) (PCL), poly(dioxanone) (PDO), poly(glyconate), poly(hydroxybutyrate) (PHB), poly(hydroxyvalerate (PHV), poly(orthoesters), poly(carboxylates), poly(propylene fumarate), poly(phosphates), poly(carbonates), poly(anhydrides), poly(iminocarbonates), poly(phosphazenes), poly(DL-lactide) (PDLA), poly(L-lactide) (PLLA), poly(glycolide) (PGA), copolymers thereof, and blends thereof, and wherein the porogen is selected from NaCl particles, wax, polysaccharides, and cellulose. 4. A porous reinforced composite scaffold material as described in claim 3, wherein the anisometric calcium phosphate particles comprise hydroxyapatite whiskers, and wherein the scaffold comprises between 1 to 50 percent by volume of hydroxyapatite whiskers. 5. A porous reinforced composite scaffold material as described in claim 1, wherein the reinforcement particles have a mean aspect ratio (length along c-axis/length along a-axis) of greater than 1 and less than 100. 6. A porous reinforced composite scaffold material as described in claim 1, wherein the size of the reinforcement particles ranges between 20 nm and 2 mm. 7. A porous reinforced composite scaffold material as described in claim 1, comprising one or more additives selected from growth factors, transcription factors, matrix metalloproteinases, peptides, proteins, bone cells, progenitor cells, blood plasma, bone marrow aspirate, and combinations thereof, and wherein the one or more additives coat the scaffold material, or are contained within some or all of the pore void space, or combinations of these. 8. A porous reinforced composite scaffold material as described in claim 7, comprising a Bone Morphogenetic Protein (BMP). 9. A porous reinforced composite scaffold material as described in claim 1, wherein the scaffold material is part of a synthetic bone graft, a bone ingrowth surface applied to an existing implant, or tissue scaffold. 10. A porous reinforced composite scaffold material as described in claim 1, wherein the porosity is either variable or uniform from a center of the material to an exterior surface. 11. A porous reinforced composite scaffold material as described in claim 10, wherein the porosity is either functionally graded radially from a highly porous center to a relatively dense exterior surface or functionally graded radially from a relatively dense center to a highly porous exterior surface. 12. A porous reinforced composite scaffold material as described in claim 1, further comprising one or a plurality of any one or combination of features selected from holes, notches, pins, radiographic markers, gripping features for positioning of the implantable device by surgical tools. 13. A porous reinforced composite scaffold material as described in claim 1, wherein the material is formed according to the method set forth in claim 14. 14. A method of forming a reinforced composite scaffold material, the method comprising the steps of: providing a thermoplastic polymer and anisometric calcium phosphate particles; uniformly mixing the polymer and the calcium phosphate particles; providing a selection of porogen materials having homogenous or heterogenous particle size within the range from about 10 to 500 μm selecting a porogen material to achieve desired scaffold porosity, and uniformly mixing the selected porogen with the polymer and calcium phosphate mixture; processing the mixture to form a solid composite material; leaching the porogen material from the solid composite form; whereby, a composite material of essentially uniform continuous porosity is formed, the composite material comprising pores sized from about 10 to 500 μm. 15. A method as described in claim 14, wherein the anisometric calcium phosphate comprises one or more of hydroxyapatite, calcium-deficient hydroxyapatite, carbonated calcium hydroxyapatite, beta-tricalcium phosphate (beta-TCP), alpha-tricalcium phosphate (alpha-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), tetracalcium phosphate, biphasic calcium phosphate (BCP), anhydrous dicalcium phosphate (DCPA), dicalcium phosphate dihydrate (DCPD), anhydrous monocalcium phosphate (MCPA), monocalcium phosphate monohydrate (MCPM), and combinations thereof, and, wherein the polymer comprises one or more of polyaryletherketone, polyetheretherketone (PEEK), polyetherketonekteone (PEKK), polyetherketone (PEK), polyethylene, high density polyethylene (HDPE), ultra high molecular weight polyethylene (UHMWPE), low density polyethylene (LDPE), polyethylene oxide (PEO), polyurethane, polypropylene, polypropylene oxide (PPO), polysulfone, polymethylmethacrylate (PMMA), and other polyacrylics from monomers such as bisphenol a hydroxypropylmethacrylate (bis-GMA) or tri(ethylene glycol) dimethacrylate (TEG-DMA), polypropylene, poly(e-caprolactone) (PCL), poly(dioxanone) (PDO), poly(glyconate), poly(hydroxybutyrate) (PHB), poly(hydroxyvalerate (PHV), poly(orthoesters), poly(carboxylates), polypropylene fumarate), poly(phosphates), poly(carbonates), poly(anhydrides), poly(iminocarbonates), poly(phosphazenes), poly(DL-lactide) (PDLA), poly(L-lactide) (PLLA), poly(glycolide) (PGA), copolymers thereof, and blends thereof, and wherein the porogen is selected from NaCl particles, wax, polysaccharides, and cellulose. 16. A method as described in claim 14, wherein processing the mixture comprises one or more steps of vacuum filtering, drying and densifying under uniaxial compression. 17. A method as described in claim 14, wherein processing includes one or a combination of injection molding, reaction injection molding, compression molding, transfer molding, extrusion, blow molding, pultrusion, casting/potting, solvent casting, sintering, and rapid prototyping processes 18. A method as described in claim 14, wherein mixing the polymer and the calcium phosphate particles comprises using ultrasonication under constant stirring, and the method further comprising the step of sintering the composite material at elevated temperatures while the composite material is under uniaxial compression, wherein sintering is at a temperature range between approximately 25° C. to approximately 1000° C. 19. A method as described in claim 14, wherein leaching of the porogen is achieved by a method selected from dissolution in a solvent and heating. 20. An implantable orthopedic device, comprising: an interbody spinal fusion cage, bone ingrowth scaffolding for implant fixation, fracture fixation, synthetic bone graft substitute, or a tissue engineering scaffold, that is completely or at least partially formed with a porous reinforced composite scaffold that comprises a thermoplastic polymer material having essentially uniform porosity and comprising a plurality of reinforcing anisometric calcium phosphate particles distributed essentially uniformly throughout, wherein the composite comprises a plurality pores that are defined by voids interconnected by struts, the size of the pores being either homogenous or heterogeneous and within the range from about 10 to 500 μm, and wherein the calcium phosphate particles comprise single crystals, dense polycrystals or combinations thereof, and wherein the calcium phosphate particles are embedded within the thermoplastic polymer material and exposed on the struts within the pore voids.

Synthetic composite materials for use, for example, as orthopedic implants are described herein. In one example, a composite material for use as a scaffold includes a thermoplastic polymer forming a porous matrix that has continuous porosity and a plurality of pores. The porosity and the size of the pores are selectively formed during synthesis of the composite material. The example composite material also includes a plurality of a anisometric calcium phosphate particles integrally formed, embedded in, or exposed on a surface of the porous matrix. The calcium phosphate particles provide one or more of reinforcement, bioactivity, or bioresorption.1. A porous reinforced composite scaffold material, comprising: a thermoplastic polymer material having essentially uniform porosity and comprising a plurality of anisometric calcium phosphate particles distributed essentially uniformly throughout, wherein the composite comprises a plurality pores that are defined by voids interconnected by struts, the size of the pores being either homogenous or heterogeneous and within the range from about 10 to 500 μm, and wherein the calcium phosphate particles comprise single crystals, dense polycrystals or combinations thereof, and wherein the calcium phosphate particles are embedded within the thermoplastic polymer material and exposed on the struts within the pore voids. 2. A porous reinforced composite scaffold material as described in claim 1, wherein the porosity ranges between 1 to 95 percent by volume. 3. A porous reinforced composite scaffold material as described in claim 1, wherein the anisometric calcium phosphate comprises one or more of hydroxyapatite, calcium-deficient hydroxyapatite, carbonated calcium hydroxyapatite, beta-tricalcium phosphate (beta-TCP), alpha-tricalcium phosphate (alpha-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), tetracalcium phosphate, biphasic calcium phosphate (BCP), anhydrous dicalcium phosphate (DCPA), dicalcium phosphate dihydrate (DCPD), anhydrous monocalcium phosphate (MCPA), monocalcium phosphate monohydrate (MCPM), and combinations thereof, and, wherein the polymer comprises one or more of polyaryletherketone, polyetheretherketone (PEEK), polyetherketonekteone (PEKK), polyetherketone (PEK), polyethylene, high density polyethylene (HDPE), ultra high molecular weight polyethylene (UHMWPE), low density polyethylene (LDPE), polyethylene oxide (PEO), polyurethane, polypropylene, polypropylene oxide (PPO), polysulfone, polymethylmethacrylate (PMMA), and other polyacrylics from monomers such as bisphenol a hydroxypropylmethacrylate (bis-GMA) or tri(ethylene glycol) dimethacrylate (TEG-DMA), polypropylene, poly(e-caprolactone) (PCL), poly(dioxanone) (PDO), poly(glyconate), poly(hydroxybutyrate) (PHB), poly(hydroxyvalerate (PHV), poly(orthoesters), poly(carboxylates), poly(propylene fumarate), poly(phosphates), poly(carbonates), poly(anhydrides), poly(iminocarbonates), poly(phosphazenes), poly(DL-lactide) (PDLA), poly(L-lactide) (PLLA), poly(glycolide) (PGA), copolymers thereof, and blends thereof, and wherein the porogen is selected from NaCl particles, wax, polysaccharides, and cellulose. 4. A porous reinforced composite scaffold material as described in claim 3, wherein the anisometric calcium phosphate particles comprise hydroxyapatite whiskers, and wherein the scaffold comprises between 1 to 50 percent by volume of hydroxyapatite whiskers. 5. A porous reinforced composite scaffold material as described in claim 1, wherein the reinforcement particles have a mean aspect ratio (length along c-axis/length along a-axis) of greater than 1 and less than 100. 6. A porous reinforced composite scaffold material as described in claim 1, wherein the size of the reinforcement particles ranges between 20 nm and 2 mm. 7. A porous reinforced composite scaffold material as described in claim 1, comprising one or more additives selected from growth factors, transcription factors, matrix metalloproteinases, peptides, proteins, bone cells, progenitor cells, blood plasma, bone marrow aspirate, and combinations thereof, and wherein the one or more additives coat the scaffold material, or are contained within some or all of the pore void space, or combinations of these. 8. A porous reinforced composite scaffold material as described in claim 7, comprising a Bone Morphogenetic Protein (BMP). 9. A porous reinforced composite scaffold material as described in claim 1, wherein the scaffold material is part of a synthetic bone graft, a bone ingrowth surface applied to an existing implant, or tissue scaffold. 10. A porous reinforced composite scaffold material as described in claim 1, wherein the porosity is either variable or uniform from a center of the material to an exterior surface. 11. A porous reinforced composite scaffold material as described in claim 10, wherein the porosity is either functionally graded radially from a highly porous center to a relatively dense exterior surface or functionally graded radially from a relatively dense center to a highly porous exterior surface. 12. A porous reinforced composite scaffold material as described in claim 1, further comprising one or a plurality of any one or combination of features selected from holes, notches, pins, radiographic markers, gripping features for positioning of the implantable device by surgical tools. 13. A porous reinforced composite scaffold material as described in claim 1, wherein the material is formed according to the method set forth in claim 14. 14. A method of forming a reinforced composite scaffold material, the method comprising the steps of: providing a thermoplastic polymer and anisometric calcium phosphate particles; uniformly mixing the polymer and the calcium phosphate particles; providing a selection of porogen materials having homogenous or heterogenous particle size within the range from about 10 to 500 μm selecting a porogen material to achieve desired scaffold porosity, and uniformly mixing the selected porogen with the polymer and calcium phosphate mixture; processing the mixture to form a solid composite material; leaching the porogen material from the solid composite form; whereby, a composite material of essentially uniform continuous porosity is formed, the composite material comprising pores sized from about 10 to 500 μm. 15. A method as described in claim 14, wherein the anisometric calcium phosphate comprises one or more of hydroxyapatite, calcium-deficient hydroxyapatite, carbonated calcium hydroxyapatite, beta-tricalcium phosphate (beta-TCP), alpha-tricalcium phosphate (alpha-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), tetracalcium phosphate, biphasic calcium phosphate (BCP), anhydrous dicalcium phosphate (DCPA), dicalcium phosphate dihydrate (DCPD), anhydrous monocalcium phosphate (MCPA), monocalcium phosphate monohydrate (MCPM), and combinations thereof, and, wherein the polymer comprises one or more of polyaryletherketone, polyetheretherketone (PEEK), polyetherketonekteone (PEKK), polyetherketone (PEK), polyethylene, high density polyethylene (HDPE), ultra high molecular weight polyethylene (UHMWPE), low density polyethylene (LDPE), polyethylene oxide (PEO), polyurethane, polypropylene, polypropylene oxide (PPO), polysulfone, polymethylmethacrylate (PMMA), and other polyacrylics from monomers such as bisphenol a hydroxypropylmethacrylate (bis-GMA) or tri(ethylene glycol) dimethacrylate (TEG-DMA), polypropylene, poly(e-caprolactone) (PCL), poly(dioxanone) (PDO), poly(glyconate), poly(hydroxybutyrate) (PHB), poly(hydroxyvalerate (PHV), poly(orthoesters), poly(carboxylates), polypropylene fumarate), poly(phosphates), poly(carbonates), poly(anhydrides), poly(iminocarbonates), poly(phosphazenes), poly(DL-lactide) (PDLA), poly(L-lactide) (PLLA), poly(glycolide) (PGA), copolymers thereof, and blends thereof, and wherein the porogen is selected from NaCl particles, wax, polysaccharides, and cellulose. 16. A method as described in claim 14, wherein processing the mixture comprises one or more steps of vacuum filtering, drying and densifying under uniaxial compression. 17. A method as described in claim 14, wherein processing includes one or a combination of injection molding, reaction injection molding, compression molding, transfer molding, extrusion, blow molding, pultrusion, casting/potting, solvent casting, sintering, and rapid prototyping processes 18. A method as described in claim 14, wherein mixing the polymer and the calcium phosphate particles comprises using ultrasonication under constant stirring, and the method further comprising the step of sintering the composite material at elevated temperatures while the composite material is under uniaxial compression, wherein sintering is at a temperature range between approximately 25° C. to approximately 1000° C. 19. A method as described in claim 14, wherein leaching of the porogen is achieved by a method selected from dissolution in a solvent and heating. 20. An implantable orthopedic device, comprising: an interbody spinal fusion cage, bone ingrowth scaffolding for implant fixation, fracture fixation, synthetic bone graft substitute, or a tissue engineering scaffold, that is completely or at least partially formed with a porous reinforced composite scaffold that comprises a thermoplastic polymer material having essentially uniform porosity and comprising a plurality of reinforcing anisometric calcium phosphate particles distributed essentially uniformly throughout, wherein the composite comprises a plurality pores that are defined by voids interconnected by struts, the size of the pores being either homogenous or heterogeneous and within the range from about 10 to 500 μm, and wherein the calcium phosphate particles comprise single crystals, dense polycrystals or combinations thereof, and wherein the calcium phosphate particles are embedded within the thermoplastic polymer material and exposed on the struts within the pore voids.

The present invention relates to the use of a known compound as an agent for promoting and/or accelerating fibroblast proliferation and/or differentiation and, consequently, cicatrization. This compound is a copolymer of a 2-methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulphonic acid salt and of propenoic acid 2-hydroxyethyl ester. It may be used alone or in combination with other active substances for inducing or accelerating cicatrization. This compound, alone or in combination with another active substance, may be administered directly on the wound and the surrounding area or the mucous membranes, by topical application. It may also be used ex vivo, in particular for generating cells for skin grafts.

1-12. (canceled) 13. A method for promoting and/or accelerating fibroblast proliferation in vivo or ex vivo, comprising: providing a composition comprising an effective amount of a copolymer of a salt of 2-methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid and of 2-hydroxyethylpropenoate ester. 14. The method as claimed in claim 13, wherein said composition is administered to promote healing. 15. The method as claimed in claim 13, wherein said copolymer is combined with one or more active substances. 16. The method as claimed in claim 13, wherein said copolymer is combined with a sulfated saccharide. 17. The method as claimed in claim 13, wherein said salt of 2-methyl-2-[(1-oxo-2-propenyl)amino]-1-propane-sulfonic acid is a sodium salt. 18. The method as claimed in claim 15, wherein said copolymer is combined with a sulfated saccharide. 19. The method as claimed in claim 13, wherein said copolymer is combined with potassium sucrose octasulfate.

The present invention relates to the use of a known compound as an agent for promoting and/or accelerating fibroblast proliferation and/or differentiation and, consequently, cicatrization. This compound is a copolymer of a 2-methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulphonic acid salt and of propenoic acid 2-hydroxyethyl ester. It may be used alone or in combination with other active substances for inducing or accelerating cicatrization. This compound, alone or in combination with another active substance, may be administered directly on the wound and the surrounding area or the mucous membranes, by topical application. It may also be used ex vivo, in particular for generating cells for skin grafts.1-12. (canceled) 13. A method for promoting and/or accelerating fibroblast proliferation in vivo or ex vivo, comprising: providing a composition comprising an effective amount of a copolymer of a salt of 2-methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid and of 2-hydroxyethylpropenoate ester. 14. The method as claimed in claim 13, wherein said composition is administered to promote healing. 15. The method as claimed in claim 13, wherein said copolymer is combined with one or more active substances. 16. The method as claimed in claim 13, wherein said copolymer is combined with a sulfated saccharide. 17. The method as claimed in claim 13, wherein said salt of 2-methyl-2-[(1-oxo-2-propenyl)amino]-1-propane-sulfonic acid is a sodium salt. 18. The method as claimed in claim 15, wherein said copolymer is combined with a sulfated saccharide. 19. The method as claimed in claim 13, wherein said copolymer is combined with potassium sucrose octasulfate.

This disclosure provides a retroviral replicating vector for gene delivery comprising a therapeutic cassette containing at least one mini-promoter linked to a gene to be expressed.

1. A recombinant replication competent gammaretrovirus comprising: a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′ end of the retroviral polynucleotide sequence, a promoter sequence at the 5′ end of the retroviral polynucleotide, said promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain; a therapeutic cassette comprising at least one mini-promoter cassette having a mini-promoter operably linked to a heterologous polynucleotide, wherein the therapeutic cassette is positioned 5′ to the 3′ LTR and 3′ to the env nucleic acid domain encoding the retroviral envelope, and wherein when only one mini-promoter cassette is present the heterologous polynucleotide is 1.2 kb to 2.0 kb in length; and cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell. 2. The recombinant replicating retroviral vector of claim 1, wherein the mini-promoter comprises an RSV promoter. 3. The retrovirus of claim 1, wherein the retroviral polynucleotide sequence is derived from a virus selected from the group consisting of murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), Feline leukemia virus (FeLV), Baboon endogenous retrovirus (BEV), porcine endogenous virus (PERV), the cat derived retrovirus RD114, squirrel monkey retrovirus, avian reticuloendotheliosis virus(REV), or Gibbon ape leukemia virus (GALV). 4. The retrovirus of claim 1, wherein the retroviral envelope is an amphotropic MLV envelope. 5. The retrovirus of claim 1, wherein the target cell is a cancer cell. 6. The retrovirus of claim 1, wherein the target cell is a neoplastic cell. 7. The retrovirus of claim 5, wherein the cancer cell is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer, lymphoma, leukemia, and rheumatoid arthritis or other autoimmune disease. 8. The retrovirus of claim 1, wherein the promoter sequence is associated with a growth regulatory gene. 9. The retrovirus of claim 1, wherein the promoter sequence comprises a tissue-specific promoter sequence. 10-11. (canceled) 12. The retrovirus of claim 1, wherein the promoter comprises a CMV-R-U5 domain polynucleotide. 13-15. (canceled) 16. The retrovirus of claim 1, wherein the mini-promoter is a core promoter. 17. The retrovirus of claim 1, wherein the mini-promoter is an optimized core promoter. 18. The retrovirus of claim 1, wherein the therapeutic cassette comprises (a) at least two mini-promoter cassettes, (b) at least one minipromoter cassette and a polIII promoter cassette or (c) at least one mini-promoter cassette and an IRES cassette. 19. The retrovirus of claim 1, wherein the mini-promoter is from about 70-500 bp in length. 20. The retrovirus of claim 1, wherein the mini promoter comprises a core promoter and further comprises an enhancer element. 21. The retrovirus of claim 1, wherein the mini-promoter comprises a TATA box, and initiator site, a Motif Ten Element (MTE), a Downstream promoter element (DPE) and at least one additional element selected from the group consisting of: (a) TFIIB recognition element, upstream (BREu); (b) TFIIB recognition element downstream (BREd); (c) HBV X core promoter element 1 (XCPE1); (d) HBV X core promoter element 2 (XCPE2); (d) downstream core element site I (CDE SI); (e) downstream core element site II (CDE SII); and (f) downstream core element site III (CDE SIII). 22. The retrovirus of claim 21, wherein the mini-promoter further comprises an enhance element. 23. The retrovirus of claim 1, wherein the heterologous nucleic acid comprises a polynucleotide having a sequence as set forth in SEQ ID NO:3, 5, 11, 13, 15 or 17. 24. (canceled) 25. The retrovirus of claim 1, wherein the heterologous nucleic acid is human codon optimized and encodes a polypeptide as set forth in SEQ ID NO:4. 26. The retrovirus of claim 1, wherein the 3′ LTR is derived from a gammaretrovirus. 27. The retrovirus of claim 26, wherein the 3′ LTR comprises a U3-R-U5 domain. 28. The retrovirus of claim 1, wherein the heterologous nucleic acid sequence encodes a biological response modifier or an immunopotentiating cytokine. 29. The retrovirus according to claim 28, wherein the immunopotentiating cytokine is selected from the group consisting of interleukins 1 through 15, interferon, tumor necrosis factor (TNF), and granulocyte-macrophage-colony stimulating factor (GM-CSF). 30. The retrovirus according to claim 28, wherein the immunopotentiating cytokine is interferon gamma. 31. The retrovirus according to claim 1, wherein the heterologous nucleic acid encodes a polypeptide that converts a nontoxic prodrug in to a toxic drug. 32. The retrovirus according to claim 31, wherein the polypeptide that converts a nontoxic prodrug in to a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP), or cytosine deaminase. 33. (canceled) 34. The retrovirus according to claim 1, wherein the heterologous nucleic acid sequence comprises an inhibitory polynucleotide. 35. The retrovirus according to claim 34, wherein the inhibitory polynucleotide comprises an miRNA, siRNA sequence or other RNAi construct. 36. The retrovirus of claim 35, wherein the therapeutic cassette comprises a mini-promoter operably linked to heterologous nucleic acid and a polIII promoter operably linked to the miRNA, miRNA, siRNA sequence or other RNAi coding domain. 37. A recombinant retroviral polynucleotide genome for producing a retrovirus of claim 1. 38. A method of delivering a therapeutic molecule to a subject comprising contact the subject with a retrovirus of claim 1. 39. A method of treating a cell proliferative disorder comprising contacting the subject with a retrovirus of claim 23 under conditions such that the polynucleotide is expressed and contacting the subject with 5-fluorocytosine. 40. The method of claim 39, wherein the cell proliferative disorder is glioblastoma multiforme. 41. The method of claim 39, wherein the cell proliferative disorder is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer. 42. A method of treating a cell proliferative disorder in a subject comprising contacting the subject with a retrovirus of claim 1, wherein the heterologous nucleic acid sequence encodes a therapeutic protein that inhibits proliferation of a neoplastic cell. 43. The method of claim 42, wherein the therapeutic protein comprises a polypeptide that converts a non-cytotoxic drug to a cytotoxic drug. 44. The method of claim 43, wherein the polypeptide has cytosine deaminase activity. 45. The method of claim 44, wherein the polypeptide comprises a sequence as set forth in SEQ ID NO:4, 12, 14, 16, or 18. 46. The method of claim 43, wherein the non-cytotoxic drug is 5-fluorocytosine. 47. A method of treating a cell proliferative disorder comprising administering a retrovirus of claim 1 to a subject having a cell proliferative disorder under conditions such that the retrovirus infects cells with the disorder and contacting the subject with an anti-cancer agent or chemotherapeutic agent. 48. The method of claim 47, wherein the anti-cancer agent is selected from the group consisting of bevacizumab, pegaptanib, ranibizumab, sorafenib, sunitinib, AE-941, VEGF Trap, pazopanib, vandetanib, vatalanib, cediranib, fenretinide, squalamine, INGN-241, oral tetrathiomolybdate, tetrathiomolybdate, Panzem NCD, 2-methoxyestradiol, AEE-788, AG-013958, bevasiranib sodium, AMG-706, axitinib, BIBF-1120, CDP-791, CP-547632, PI-88, SU-14813, SU-6668, XL-647, XL-999, IMC-1121B, ABT-869, BAY-57-9352, BAY-73-4506, BMS-582664, CEP-7055, CHIR-265, CT-322, CX-3542, E-7080, ENMD-1198, OSI-930, PTC-299, Sirna-027, TKI-258, Veglin, XL-184, and ZK-304709. 49. The method of claim 47, wherein the retrovirus is administered from about 103 to 107 TU/g brain weight. 50. The method of claim 49, wherein the retrovirus is administered from about 104 to 106 TU/g brain weight. 51. A recombinant retroviral replicating vector (RRV) comprising: a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′ end of the retroviral polynucleotide sequence, a promoter sequence at the 5′ end of the retroviral polynucleotide, said promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain; a therapeutic cassette comprising a mini-promoter cassette operably linked to a heterologous polynucleotide and a miRNA cassette comprising a polIII promoter linked to a primary precursor miRNA (pri-miRNA) for an miRNA or siRNA sequence; and cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell. 52. The RRV of claim 51, wherein the miRNA is selected from the group consisting of miR-142-3p, miR-181, miR-223, miR 128-1 and miR 128-2.

This disclosure provides a retroviral replicating vector for gene delivery comprising a therapeutic cassette containing at least one mini-promoter linked to a gene to be expressed.1. A recombinant replication competent gammaretrovirus comprising: a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′ end of the retroviral polynucleotide sequence, a promoter sequence at the 5′ end of the retroviral polynucleotide, said promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain; a therapeutic cassette comprising at least one mini-promoter cassette having a mini-promoter operably linked to a heterologous polynucleotide, wherein the therapeutic cassette is positioned 5′ to the 3′ LTR and 3′ to the env nucleic acid domain encoding the retroviral envelope, and wherein when only one mini-promoter cassette is present the heterologous polynucleotide is 1.2 kb to 2.0 kb in length; and cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell. 2. The recombinant replicating retroviral vector of claim 1, wherein the mini-promoter comprises an RSV promoter. 3. The retrovirus of claim 1, wherein the retroviral polynucleotide sequence is derived from a virus selected from the group consisting of murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), Feline leukemia virus (FeLV), Baboon endogenous retrovirus (BEV), porcine endogenous virus (PERV), the cat derived retrovirus RD114, squirrel monkey retrovirus, avian reticuloendotheliosis virus(REV), or Gibbon ape leukemia virus (GALV). 4. The retrovirus of claim 1, wherein the retroviral envelope is an amphotropic MLV envelope. 5. The retrovirus of claim 1, wherein the target cell is a cancer cell. 6. The retrovirus of claim 1, wherein the target cell is a neoplastic cell. 7. The retrovirus of claim 5, wherein the cancer cell is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer, lymphoma, leukemia, and rheumatoid arthritis or other autoimmune disease. 8. The retrovirus of claim 1, wherein the promoter sequence is associated with a growth regulatory gene. 9. The retrovirus of claim 1, wherein the promoter sequence comprises a tissue-specific promoter sequence. 10-11. (canceled) 12. The retrovirus of claim 1, wherein the promoter comprises a CMV-R-U5 domain polynucleotide. 13-15. (canceled) 16. The retrovirus of claim 1, wherein the mini-promoter is a core promoter. 17. The retrovirus of claim 1, wherein the mini-promoter is an optimized core promoter. 18. The retrovirus of claim 1, wherein the therapeutic cassette comprises (a) at least two mini-promoter cassettes, (b) at least one minipromoter cassette and a polIII promoter cassette or (c) at least one mini-promoter cassette and an IRES cassette. 19. The retrovirus of claim 1, wherein the mini-promoter is from about 70-500 bp in length. 20. The retrovirus of claim 1, wherein the mini promoter comprises a core promoter and further comprises an enhancer element. 21. The retrovirus of claim 1, wherein the mini-promoter comprises a TATA box, and initiator site, a Motif Ten Element (MTE), a Downstream promoter element (DPE) and at least one additional element selected from the group consisting of: (a) TFIIB recognition element, upstream (BREu); (b) TFIIB recognition element downstream (BREd); (c) HBV X core promoter element 1 (XCPE1); (d) HBV X core promoter element 2 (XCPE2); (d) downstream core element site I (CDE SI); (e) downstream core element site II (CDE SII); and (f) downstream core element site III (CDE SIII). 22. The retrovirus of claim 21, wherein the mini-promoter further comprises an enhance element. 23. The retrovirus of claim 1, wherein the heterologous nucleic acid comprises a polynucleotide having a sequence as set forth in SEQ ID NO:3, 5, 11, 13, 15 or 17. 24. (canceled) 25. The retrovirus of claim 1, wherein the heterologous nucleic acid is human codon optimized and encodes a polypeptide as set forth in SEQ ID NO:4. 26. The retrovirus of claim 1, wherein the 3′ LTR is derived from a gammaretrovirus. 27. The retrovirus of claim 26, wherein the 3′ LTR comprises a U3-R-U5 domain. 28. The retrovirus of claim 1, wherein the heterologous nucleic acid sequence encodes a biological response modifier or an immunopotentiating cytokine. 29. The retrovirus according to claim 28, wherein the immunopotentiating cytokine is selected from the group consisting of interleukins 1 through 15, interferon, tumor necrosis factor (TNF), and granulocyte-macrophage-colony stimulating factor (GM-CSF). 30. The retrovirus according to claim 28, wherein the immunopotentiating cytokine is interferon gamma. 31. The retrovirus according to claim 1, wherein the heterologous nucleic acid encodes a polypeptide that converts a nontoxic prodrug in to a toxic drug. 32. The retrovirus according to claim 31, wherein the polypeptide that converts a nontoxic prodrug in to a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP), or cytosine deaminase. 33. (canceled) 34. The retrovirus according to claim 1, wherein the heterologous nucleic acid sequence comprises an inhibitory polynucleotide. 35. The retrovirus according to claim 34, wherein the inhibitory polynucleotide comprises an miRNA, siRNA sequence or other RNAi construct. 36. The retrovirus of claim 35, wherein the therapeutic cassette comprises a mini-promoter operably linked to heterologous nucleic acid and a polIII promoter operably linked to the miRNA, miRNA, siRNA sequence or other RNAi coding domain. 37. A recombinant retroviral polynucleotide genome for producing a retrovirus of claim 1. 38. A method of delivering a therapeutic molecule to a subject comprising contact the subject with a retrovirus of claim 1. 39. A method of treating a cell proliferative disorder comprising contacting the subject with a retrovirus of claim 23 under conditions such that the polynucleotide is expressed and contacting the subject with 5-fluorocytosine. 40. The method of claim 39, wherein the cell proliferative disorder is glioblastoma multiforme. 41. The method of claim 39, wherein the cell proliferative disorder is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer. 42. A method of treating a cell proliferative disorder in a subject comprising contacting the subject with a retrovirus of claim 1, wherein the heterologous nucleic acid sequence encodes a therapeutic protein that inhibits proliferation of a neoplastic cell. 43. The method of claim 42, wherein the therapeutic protein comprises a polypeptide that converts a non-cytotoxic drug to a cytotoxic drug. 44. The method of claim 43, wherein the polypeptide has cytosine deaminase activity. 45. The method of claim 44, wherein the polypeptide comprises a sequence as set forth in SEQ ID NO:4, 12, 14, 16, or 18. 46. The method of claim 43, wherein the non-cytotoxic drug is 5-fluorocytosine. 47. A method of treating a cell proliferative disorder comprising administering a retrovirus of claim 1 to a subject having a cell proliferative disorder under conditions such that the retrovirus infects cells with the disorder and contacting the subject with an anti-cancer agent or chemotherapeutic agent. 48. The method of claim 47, wherein the anti-cancer agent is selected from the group consisting of bevacizumab, pegaptanib, ranibizumab, sorafenib, sunitinib, AE-941, VEGF Trap, pazopanib, vandetanib, vatalanib, cediranib, fenretinide, squalamine, INGN-241, oral tetrathiomolybdate, tetrathiomolybdate, Panzem NCD, 2-methoxyestradiol, AEE-788, AG-013958, bevasiranib sodium, AMG-706, axitinib, BIBF-1120, CDP-791, CP-547632, PI-88, SU-14813, SU-6668, XL-647, XL-999, IMC-1121B, ABT-869, BAY-57-9352, BAY-73-4506, BMS-582664, CEP-7055, CHIR-265, CT-322, CX-3542, E-7080, ENMD-1198, OSI-930, PTC-299, Sirna-027, TKI-258, Veglin, XL-184, and ZK-304709. 49. The method of claim 47, wherein the retrovirus is administered from about 103 to 107 TU/g brain weight. 50. The method of claim 49, wherein the retrovirus is administered from about 104 to 106 TU/g brain weight. 51. A recombinant retroviral replicating vector (RRV) comprising: a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′ end of the retroviral polynucleotide sequence, a promoter sequence at the 5′ end of the retroviral polynucleotide, said promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain; a therapeutic cassette comprising a mini-promoter cassette operably linked to a heterologous polynucleotide and a miRNA cassette comprising a polIII promoter linked to a primary precursor miRNA (pri-miRNA) for an miRNA or siRNA sequence; and cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell. 52. The RRV of claim 51, wherein the miRNA is selected from the group consisting of miR-142-3p, miR-181, miR-223, miR 128-1 and miR 128-2.

A rapid assay for detection of human cellular fibronectin (c-Fn) where ELISA-based assays have previously been developed for detecting and measuring cellular fibronectin in biological fluids, but these methods are too time-consuming for practical clinical diagnostic use. The assay of the present invention enables prediction of bleeding events on a rapid timescale. Described as well are high affinity human monoclonal antibodies, particularly those directed against isotopic determinants of cellular fibronectin (c-Fn), as well as direct equivalents and derivatives of these antibodies. These antibodies bind to their respective target with an affinity at least 100 fold greater than they do to plasma fibronectin, and enable assays to be created that detect c-Fn in less than 30 minutes in a variety of detection formats, and in some detection formats less than 15 minutes. These antibodies are useful for diagnostics, particularly prediction of bleeding events, prophylaxis and treatment of disease.

1-22. (canceled) 23. A rapid assay for the prediction of bleeding in a human test subject that can determine in 60 minutes or less the level of human cellular fibronectin (c-Fn) in a test sample taken from a subject; wherein said test sample is drawn from the group consisting of whole blood, serum, or a plasma sample. 24. The rapid assay according to claim 23 wherein said rapid assay consists essentially of an isolated human monoclonal antibody, or an antigen-binding portion thereof, or both an antibody and its antigen-binding portion, wherein the antibody binds to the EDA region of the c-Fn molecule, amino acid sequence 1631-1721; and a reference antibody consisting essentially of a synthetic polypeptide having a sequence, from left to right and in the direction from amino-terminus to carboxy-terminus, drawn from the group consisting of any of the sub-sequences (SEQ ID NO: 1) a) DGEEDTAELQGLRPGSEC, (SEQ ID NO: 2) b) ESPQGQVSRYRVTYSSPEDC, (SEQ ID NO: 3) c) HDDMESQPLIGTQSC, and the entire sequence (SEQ ID NO: 4) d) NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIH ELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTA. 25. The rapid assay according to claim 23 wherein said rapid assay do determines the level of human cellular fibronectin in 20 minutes or less. 26. A use of the rapid assay of claim 23 comprising: evaluating the level of human cellular fibronectin determined to be in the test sample taken from the subject as a predictor of future bleeding of the subject should the subject be treated for neurological disease. 27. A use of the rapid assay of claim 23 comprising: evaluating the level of human cellular fibronectin determined to be in the test sample taken from the subject as a predictor of future bleeding of the subject should the subject be treated for cardiovascular disease. 28. A use of the rapid assay of claim 23 comprising: evaluating the level of human cellular fibronectin determined to be in the test sample taken from the subject as a predictor of future bleeding of the subject should the subject be subject to bleeding following anti-coagulant therapy. 29. An isolated human monoclonal antibody, or an antigen-binding portion thereof, or both an antibody and its antigen-binding portion; wherein the antibody binds to the EDA region of the c-Fn molecule, amino acid sequence 1631-1721; and wherein a reference antibody consists essentially of a synthetic polypeptide having a sequence, from left to right and in the direction from amino-terminus to carboxy-terminus, drawn from the group consisting of any of the sub-sequences (SEQ ID NO: 1) a) DGEEDTAELQGLRPGSEC, (SEQ ID NO: 2) b) ESPQGQVSRYRVTYSSPEDC, (SEQ ID NO: 3) c) HDDMESQPLIGTQSC, and the entire sequence (SEQ ID NO: 4) d) NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIH ELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTA. 30. The antibody, or antigen-binding portion thereof, or both an antibody and its antigen binding portion according to claim 29 linked to a second functional moiety having a different binding specificity than either or both said antibody and said antigen binding portion thereof. 31. The antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion according to claim 30 further linked to a label. 32. The antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion according to claim 30 assembled as a diagnostic kit. 33. The antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion according to claim 29 further linked to a label. 34. The antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion according to claim 29 assembled as a diagnostic kit. 35. A method of using the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion, according to claim 29 comprising: contacting a blood or blood plasma or both a blood and blood plasma sample of the subject, which sample includes cellular fibronectin molecules, with the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion; determining the level of retention of the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion as the case may be, by the sample relative to a control sample of a control subject; wherein a higher or lower level of retention by the sample of the subject relative to the control sample indicates that the subject has a higher or lower level of cellular fibronectin molecules relative to that in the control subject; therein to determine by comparison the level of cellular fibronectin molecules in the subject. 36. A method of using the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion according to claim 29 comprising: contacting a blood or blood plasma or both a blood and blood plasma sample of the subject, which sample includes cellular fibronectin molecules, with the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion; and determining the level of retention of the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion, by the sample relative to a control sample of a control subject; wherein a higher or lower level of retention of the antibody by the sample of the subject relative to the control sample indicates that the subject has a disorder associated with an abnormal level of cellular fibronectin. 37. The method of claim 36, wherein the disorder associated with an abnormal level of cellular fibronectin is taken from the group consisting of neurological or cardiovascular bleeding, likelihood of a neurological or cardiovascular bleeding, and likelihood of a neurological or cardiovascular bleeding following administration to the subject of a therapeutic agent. 38. The method of claim 37 wherein the therapeutic agent is a thrombolytic. 39. The method of claim 37 wherein the therapeutic agent is a anti-platelet agent. 40. The method of claim 37 wherein the therapeutic agent is an anti-coagulation agent. 41. The method of claim 37 wherein the therapeutic agent is a Factor Xa inhibitor. 42. The method of claim 23 wherein the therapeutic agent is a therapeutic agent against Alzheimer's or Parkinson's disease. 43. The method of claim 36 using synthetic polypeptides that are useful as reference antibodies reacting with cellular fibronectin in a test for presence of cellular fibronectin extended to a test for prediction of bleeding in a human test subject, the bleeding prediction test comprising: combining (1) some one or ones of the synthetic polypeptides with (2) a sample drawn from the group consisting of any of whole blood and serum and a plasma sample, each from a human test subject; wherein the one or ones of the synthetic polypeptides react with the test sample so that essentially all components are completely reacted in 60 minutes or less. 44. Synthetic polypeptides useful as reference antibodies reacting with cellular fibronectin, and thus useful in a test for presence of cellular fibronectin, the synthetic polypeptides drawn from the group consisting of any of the sub-sequences, expressed from left to right and in the direction from amino-terminus to carboxy-terminus, (SEQ ID NO: 1) a) DGEEDTAELQGLRPGSEC, (SEQ ID NO: 2) b) ESPQGQVSRYRVTYSSPEDC, (SEQ ID NO: 3) c) HDDMESQPLIGTQSC, and the entire sequence (SEQ ID NO: 4) d) NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIH ELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTA.

A rapid assay for detection of human cellular fibronectin (c-Fn) where ELISA-based assays have previously been developed for detecting and measuring cellular fibronectin in biological fluids, but these methods are too time-consuming for practical clinical diagnostic use. The assay of the present invention enables prediction of bleeding events on a rapid timescale. Described as well are high affinity human monoclonal antibodies, particularly those directed against isotopic determinants of cellular fibronectin (c-Fn), as well as direct equivalents and derivatives of these antibodies. These antibodies bind to their respective target with an affinity at least 100 fold greater than they do to plasma fibronectin, and enable assays to be created that detect c-Fn in less than 30 minutes in a variety of detection formats, and in some detection formats less than 15 minutes. These antibodies are useful for diagnostics, particularly prediction of bleeding events, prophylaxis and treatment of disease.1-22. (canceled) 23. A rapid assay for the prediction of bleeding in a human test subject that can determine in 60 minutes or less the level of human cellular fibronectin (c-Fn) in a test sample taken from a subject; wherein said test sample is drawn from the group consisting of whole blood, serum, or a plasma sample. 24. The rapid assay according to claim 23 wherein said rapid assay consists essentially of an isolated human monoclonal antibody, or an antigen-binding portion thereof, or both an antibody and its antigen-binding portion, wherein the antibody binds to the EDA region of the c-Fn molecule, amino acid sequence 1631-1721; and a reference antibody consisting essentially of a synthetic polypeptide having a sequence, from left to right and in the direction from amino-terminus to carboxy-terminus, drawn from the group consisting of any of the sub-sequences (SEQ ID NO: 1) a) DGEEDTAELQGLRPGSEC, (SEQ ID NO: 2) b) ESPQGQVSRYRVTYSSPEDC, (SEQ ID NO: 3) c) HDDMESQPLIGTQSC, and the entire sequence (SEQ ID NO: 4) d) NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIH ELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTA. 25. The rapid assay according to claim 23 wherein said rapid assay do determines the level of human cellular fibronectin in 20 minutes or less. 26. A use of the rapid assay of claim 23 comprising: evaluating the level of human cellular fibronectin determined to be in the test sample taken from the subject as a predictor of future bleeding of the subject should the subject be treated for neurological disease. 27. A use of the rapid assay of claim 23 comprising: evaluating the level of human cellular fibronectin determined to be in the test sample taken from the subject as a predictor of future bleeding of the subject should the subject be treated for cardiovascular disease. 28. A use of the rapid assay of claim 23 comprising: evaluating the level of human cellular fibronectin determined to be in the test sample taken from the subject as a predictor of future bleeding of the subject should the subject be subject to bleeding following anti-coagulant therapy. 29. An isolated human monoclonal antibody, or an antigen-binding portion thereof, or both an antibody and its antigen-binding portion; wherein the antibody binds to the EDA region of the c-Fn molecule, amino acid sequence 1631-1721; and wherein a reference antibody consists essentially of a synthetic polypeptide having a sequence, from left to right and in the direction from amino-terminus to carboxy-terminus, drawn from the group consisting of any of the sub-sequences (SEQ ID NO: 1) a) DGEEDTAELQGLRPGSEC, (SEQ ID NO: 2) b) ESPQGQVSRYRVTYSSPEDC, (SEQ ID NO: 3) c) HDDMESQPLIGTQSC, and the entire sequence (SEQ ID NO: 4) d) NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIH ELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTA. 30. The antibody, or antigen-binding portion thereof, or both an antibody and its antigen binding portion according to claim 29 linked to a second functional moiety having a different binding specificity than either or both said antibody and said antigen binding portion thereof. 31. The antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion according to claim 30 further linked to a label. 32. The antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion according to claim 30 assembled as a diagnostic kit. 33. The antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion according to claim 29 further linked to a label. 34. The antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion according to claim 29 assembled as a diagnostic kit. 35. A method of using the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion, according to claim 29 comprising: contacting a blood or blood plasma or both a blood and blood plasma sample of the subject, which sample includes cellular fibronectin molecules, with the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion; determining the level of retention of the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion as the case may be, by the sample relative to a control sample of a control subject; wherein a higher or lower level of retention by the sample of the subject relative to the control sample indicates that the subject has a higher or lower level of cellular fibronectin molecules relative to that in the control subject; therein to determine by comparison the level of cellular fibronectin molecules in the subject. 36. A method of using the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion according to claim 29 comprising: contacting a blood or blood plasma or both a blood and blood plasma sample of the subject, which sample includes cellular fibronectin molecules, with the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion; and determining the level of retention of the antibody, or the antigen-binding portion thereof, or both the antibody and its antigen binding portion, by the sample relative to a control sample of a control subject; wherein a higher or lower level of retention of the antibody by the sample of the subject relative to the control sample indicates that the subject has a disorder associated with an abnormal level of cellular fibronectin. 37. The method of claim 36, wherein the disorder associated with an abnormal level of cellular fibronectin is taken from the group consisting of neurological or cardiovascular bleeding, likelihood of a neurological or cardiovascular bleeding, and likelihood of a neurological or cardiovascular bleeding following administration to the subject of a therapeutic agent. 38. The method of claim 37 wherein the therapeutic agent is a thrombolytic. 39. The method of claim 37 wherein the therapeutic agent is a anti-platelet agent. 40. The method of claim 37 wherein the therapeutic agent is an anti-coagulation agent. 41. The method of claim 37 wherein the therapeutic agent is a Factor Xa inhibitor. 42. The method of claim 23 wherein the therapeutic agent is a therapeutic agent against Alzheimer's or Parkinson's disease. 43. The method of claim 36 using synthetic polypeptides that are useful as reference antibodies reacting with cellular fibronectin in a test for presence of cellular fibronectin extended to a test for prediction of bleeding in a human test subject, the bleeding prediction test comprising: combining (1) some one or ones of the synthetic polypeptides with (2) a sample drawn from the group consisting of any of whole blood and serum and a plasma sample, each from a human test subject; wherein the one or ones of the synthetic polypeptides react with the test sample so that essentially all components are completely reacted in 60 minutes or less. 44. Synthetic polypeptides useful as reference antibodies reacting with cellular fibronectin, and thus useful in a test for presence of cellular fibronectin, the synthetic polypeptides drawn from the group consisting of any of the sub-sequences, expressed from left to right and in the direction from amino-terminus to carboxy-terminus, (SEQ ID NO: 1) a) DGEEDTAELQGLRPGSEC, (SEQ ID NO: 2) b) ESPQGQVSRYRVTYSSPEDC, (SEQ ID NO: 3) c) HDDMESQPLIGTQSC, and the entire sequence (SEQ ID NO: 4) d) NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIH ELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTA.

Disclosed are methods and systems for determining the three-dimensional structure of chromatin in eukaryotic cells. More specifically, disclosed are methods and systems for obtaining chromatin structural information by surface immobilization, i.e tethering crosslinked protein:DNA complexes and/or ligated DNA complexes to media such as beads, gels, and or matrices during the conformation capture assay. In general, the method includes contacting a cell with a cross-linking reagent to cross-link DNA and protein in the cell; lysing the cell, producing cross-linked protein:DNA complexes by cutting the chromatin using a chemical, physical or enzymatic method, substantially immobilizing the cross-linked protein:DNA complexes, ligating the cross-linked protein:DNA complexes intramolecularly such that the ligated protein:DNA complexes represent structural organization of the chromatin; characterizing the ligated DNA by sequencing or other methods; and identifying any structural organization of the chromatin. The structural organization preferably includes information relating to interacting loci of the chromatin.

1. A method of determining the three-dimensional arrangement of chromatin in a cell, comprising: Contacting a cell with a cross-linking reagent to cross-link DNA and protein in the cell such that the structural organization of the chromatin or other protein:DNA complexes is preserved; lysing the cell; producing cross-linked protein:DNA complexes by cutting the chromatin using a chemical, physical or enzymatic method; substantially immobilizing the cross-linked protein:DNA complexes; connecting the cross-linked protein:DNA complexes intramolecularly such that the connected protein:DNA complexes represent structural organization of the chromatin; characterizing the DNA of the protein:DNA complexes by sequencing or other methods; and identifying any structural organization of the chromatin. 2. The method of claim 1, further comprising, denaturing the chromatin. 3. The method of claim 1, wherein the protein:DNA complexes are cut by restriction digestion; 4. The method of claim 1, wherein the protein:DNA complexes are substantially immobilized by tethering the protein:DNA complexes to one or more media. 5. The method of claim 4, where the media is selected from the group consisting of beads, chip, colloids, matrix, and gel. 6. The method of claim 1, wherein the protein:DNA complexes are substantially immobilized by a covalent or non-covalent (streptavidin-biotin bonding is not covalent but is very strong) bond between the side-chains of the amino acids of the proteins of chromatin and a reactive chemical group on the surface or inside of one or more media selected from the group consisting of beads, chip, colloids, matrix, and gel. 7. The method of claim 1, wherein the protein:DNA complexes are substantially immobilized through modifying the proteins of the chromatin so to anchor the modified protein:DNA complexes to the surface or inside of one or more media selected from the group consisting of beads, colloids, matrix, and gel. 8. The method of claim 1, wherein the protein:DNA complexes are substantially immobilized by biotinylating the proteins of the chromatin so to anchor the biotinylated protein:DNA complexes to a biotin binding surface 9. The method of claim 8, wherein the protein:DNA complexes are substantially immobilized by biotinylating the thiol groups of the proteins of the chromatin so to anchor the biotinylated protein:DNA complexes to a biotin binding surface. 10. The method of claim 8, wherein the protein:DNA complexes are substantially immobilized by biotinylating the cysteine residues of the proteins of the chromatin so to anchor the biotinylated protein:DNA complexes to a biotin binding surface. 11. The method of claim 8, wherein the protein:DNA complexes are substantially immobilized by biotinylating the N-termini and lysine residues of the proteins of the chromatin so to anchor the biotinylated protein:DNA complexes to a biotin binding surface. 12. The method of claim 8, wherein the protein:DNA complexes are substantially immobilized by biotinylating the glutamate or aspartate residues of the proteins of the chromatin so to anchor the biotinylated protein:DNA complexes to a biotin binding surface. 13. The method of claim 9, wherein thiol groups are added to the proteins of chromatin through a chemical reagent. 14. The method of claim 12, wherein thiol groups are added to the proteins of chromatin through reacting the proteins of chromatin with an aminothiol and a crosslinking reagent. 15. The method of claim 14, wherein thiol groups are added to the lysines of the proteins of chromatin by reacting them with a cross-linking reagent and cysteamine. 16. The method of claim 14 wherein thiol groups are added to the lysines of the proteins of chromatin by reacting them with formaldehyde and cysteamine. 17. The method of claim 8, wherein the substrate is streptavidin-coated chips or magnetic beads. 18. The method of claim 1, wherein the cells are denatured with Sodium Dodecyl Sulfate. 19. The method of claim 4, wherein the chromatin is digested with a restriction enzyme that produces a 5′ overhang of at least two non-identical bases. 20. The method of claim 19, wherein the 5′ overhang is blunted. 21. The method of claim 20, wherein the connection of the protein:DNA complexes intramolecularly is done by blunt-ended ligation using DNA ligase. 22. The method of claim 20, wherein blunting is done with nucleotide analogues. 23. The method of claim 20, wherein a biotinylated nucleotide is used for blunting. 24. The method of claim 20, wherein a nuclease resistant nucleotide analogue is used in blunting. 25. The method of claim 20, wherein a 2-deoxy-nucleoside-5′-(alpha-thio)-triphosphate is used in blunting. 26. The method of claim 1, wherein after the connecting step, protein:DNA complexes that have not undergone connection are removed. 27. The method of claim 1, wherein the sequencing is massively parallel or ultrahigh-throughput sequencing. 28. The method of claim 1, wherein the structural organization is interacting loci in the nucleus of the cell. 29. An improved method for determination of the structural organization of chromatin having less noise and higher resolution, said improved method comprising: providing chromatin having DNA cross-linked to protein such that the structural organization of the chromatin is preserved; producing cross-linked protein:DNA complexes by cutting the chromatin with a restriction enzyme; substantially immobilizing the cross-linked protein:DNA complexes on a surface and removing non-crosslinked DNA generated by digesting the chromatin; connecting the cross-linked protein:DNA complexes intramolecularly and removing DNA molecules without a connection; 30. The method of claim 29, further comprising sequencing the DNA of the connected protein:DNA complexes. 31. The method of claim 29, wherein the chromatin is digested with a restriction enzyme that produces a 5′ overhang of at least two non-identical bases 32. The method of claim 29, wherein the protein:DNA complexes are substantially immobilized by tethering the protein:DNA complexes on the surface of one or more media selected from the group consisting of beads, matrix, and gel. (see claim 3) 33. The method of claim 29, wherein the non-crosslinked DNA generated by digesting the chromatin is removed by washing. 34. The method of claim 29, wherein the immobilizing the cross-linked protein:DNA complexes reduces the frequency of formation of inter-molecular connections. 35. The method of claim 29, wherein DNA molecules without a connection junction are removed by an exonuclease. 36. A kit for determining the three-dimensional arrangement of chromatin in a cell, comprising: A cross-linking reagent for cross-linking the DNA and proteins of the chromatin; a lysing reagent; a denaturing reagent; a restriction enzyme for producing cross-linked protein:DNA complexes; (or any other chemical, physical, or enzymatic method for cutting DNA), a substrate for substantially immobilizing the cross-linked protein:DNA complexes; and one or more connecting reagents for connecting the protein:DNA complexes intramolecularly. 37. The method of claim 1, wherein protein:DNA complexes are substantially immobilized by a covalent or non-covalent bond between the DNA of chromatin and a reactive chemical group on the surface or inside of one or more media selected from the group consisting of beads, chip, colloids, matrix, and gel. 38. The method of claim 1, wherein connection of the DNA of the crosslinked Protein:DNA complexes intramolecularly is done by ligation using DNA ligase. 39. The method of claim 1, wherein the protein:DNA complexes are substantially immobilized relative to each other.

Disclosed are methods and systems for determining the three-dimensional structure of chromatin in eukaryotic cells. More specifically, disclosed are methods and systems for obtaining chromatin structural information by surface immobilization, i.e tethering crosslinked protein:DNA complexes and/or ligated DNA complexes to media such as beads, gels, and or matrices during the conformation capture assay. In general, the method includes contacting a cell with a cross-linking reagent to cross-link DNA and protein in the cell; lysing the cell, producing cross-linked protein:DNA complexes by cutting the chromatin using a chemical, physical or enzymatic method, substantially immobilizing the cross-linked protein:DNA complexes, ligating the cross-linked protein:DNA complexes intramolecularly such that the ligated protein:DNA complexes represent structural organization of the chromatin; characterizing the ligated DNA by sequencing or other methods; and identifying any structural organization of the chromatin. The structural organization preferably includes information relating to interacting loci of the chromatin.1. A method of determining the three-dimensional arrangement of chromatin in a cell, comprising: Contacting a cell with a cross-linking reagent to cross-link DNA and protein in the cell such that the structural organization of the chromatin or other protein:DNA complexes is preserved; lysing the cell; producing cross-linked protein:DNA complexes by cutting the chromatin using a chemical, physical or enzymatic method; substantially immobilizing the cross-linked protein:DNA complexes; connecting the cross-linked protein:DNA complexes intramolecularly such that the connected protein:DNA complexes represent structural organization of the chromatin; characterizing the DNA of the protein:DNA complexes by sequencing or other methods; and identifying any structural organization of the chromatin. 2. The method of claim 1, further comprising, denaturing the chromatin. 3. The method of claim 1, wherein the protein:DNA complexes are cut by restriction digestion; 4. The method of claim 1, wherein the protein:DNA complexes are substantially immobilized by tethering the protein:DNA complexes to one or more media. 5. The method of claim 4, where the media is selected from the group consisting of beads, chip, colloids, matrix, and gel. 6. The method of claim 1, wherein the protein:DNA complexes are substantially immobilized by a covalent or non-covalent (streptavidin-biotin bonding is not covalent but is very strong) bond between the side-chains of the amino acids of the proteins of chromatin and a reactive chemical group on the surface or inside of one or more media selected from the group consisting of beads, chip, colloids, matrix, and gel. 7. The method of claim 1, wherein the protein:DNA complexes are substantially immobilized through modifying the proteins of the chromatin so to anchor the modified protein:DNA complexes to the surface or inside of one or more media selected from the group consisting of beads, colloids, matrix, and gel. 8. The method of claim 1, wherein the protein:DNA complexes are substantially immobilized by biotinylating the proteins of the chromatin so to anchor the biotinylated protein:DNA complexes to a biotin binding surface 9. The method of claim 8, wherein the protein:DNA complexes are substantially immobilized by biotinylating the thiol groups of the proteins of the chromatin so to anchor the biotinylated protein:DNA complexes to a biotin binding surface. 10. The method of claim 8, wherein the protein:DNA complexes are substantially immobilized by biotinylating the cysteine residues of the proteins of the chromatin so to anchor the biotinylated protein:DNA complexes to a biotin binding surface. 11. The method of claim 8, wherein the protein:DNA complexes are substantially immobilized by biotinylating the N-termini and lysine residues of the proteins of the chromatin so to anchor the biotinylated protein:DNA complexes to a biotin binding surface. 12. The method of claim 8, wherein the protein:DNA complexes are substantially immobilized by biotinylating the glutamate or aspartate residues of the proteins of the chromatin so to anchor the biotinylated protein:DNA complexes to a biotin binding surface. 13. The method of claim 9, wherein thiol groups are added to the proteins of chromatin through a chemical reagent. 14. The method of claim 12, wherein thiol groups are added to the proteins of chromatin through reacting the proteins of chromatin with an aminothiol and a crosslinking reagent. 15. The method of claim 14, wherein thiol groups are added to the lysines of the proteins of chromatin by reacting them with a cross-linking reagent and cysteamine. 16. The method of claim 14 wherein thiol groups are added to the lysines of the proteins of chromatin by reacting them with formaldehyde and cysteamine. 17. The method of claim 8, wherein the substrate is streptavidin-coated chips or magnetic beads. 18. The method of claim 1, wherein the cells are denatured with Sodium Dodecyl Sulfate. 19. The method of claim 4, wherein the chromatin is digested with a restriction enzyme that produces a 5′ overhang of at least two non-identical bases. 20. The method of claim 19, wherein the 5′ overhang is blunted. 21. The method of claim 20, wherein the connection of the protein:DNA complexes intramolecularly is done by blunt-ended ligation using DNA ligase. 22. The method of claim 20, wherein blunting is done with nucleotide analogues. 23. The method of claim 20, wherein a biotinylated nucleotide is used for blunting. 24. The method of claim 20, wherein a nuclease resistant nucleotide analogue is used in blunting. 25. The method of claim 20, wherein a 2-deoxy-nucleoside-5′-(alpha-thio)-triphosphate is used in blunting. 26. The method of claim 1, wherein after the connecting step, protein:DNA complexes that have not undergone connection are removed. 27. The method of claim 1, wherein the sequencing is massively parallel or ultrahigh-throughput sequencing. 28. The method of claim 1, wherein the structural organization is interacting loci in the nucleus of the cell. 29. An improved method for determination of the structural organization of chromatin having less noise and higher resolution, said improved method comprising: providing chromatin having DNA cross-linked to protein such that the structural organization of the chromatin is preserved; producing cross-linked protein:DNA complexes by cutting the chromatin with a restriction enzyme; substantially immobilizing the cross-linked protein:DNA complexes on a surface and removing non-crosslinked DNA generated by digesting the chromatin; connecting the cross-linked protein:DNA complexes intramolecularly and removing DNA molecules without a connection; 30. The method of claim 29, further comprising sequencing the DNA of the connected protein:DNA complexes. 31. The method of claim 29, wherein the chromatin is digested with a restriction enzyme that produces a 5′ overhang of at least two non-identical bases 32. The method of claim 29, wherein the protein:DNA complexes are substantially immobilized by tethering the protein:DNA complexes on the surface of one or more media selected from the group consisting of beads, matrix, and gel. (see claim 3) 33. The method of claim 29, wherein the non-crosslinked DNA generated by digesting the chromatin is removed by washing. 34. The method of claim 29, wherein the immobilizing the cross-linked protein:DNA complexes reduces the frequency of formation of inter-molecular connections. 35. The method of claim 29, wherein DNA molecules without a connection junction are removed by an exonuclease. 36. A kit for determining the three-dimensional arrangement of chromatin in a cell, comprising: A cross-linking reagent for cross-linking the DNA and proteins of the chromatin; a lysing reagent; a denaturing reagent; a restriction enzyme for producing cross-linked protein:DNA complexes; (or any other chemical, physical, or enzymatic method for cutting DNA), a substrate for substantially immobilizing the cross-linked protein:DNA complexes; and one or more connecting reagents for connecting the protein:DNA complexes intramolecularly. 37. The method of claim 1, wherein protein:DNA complexes are substantially immobilized by a covalent or non-covalent bond between the DNA of chromatin and a reactive chemical group on the surface or inside of one or more media selected from the group consisting of beads, chip, colloids, matrix, and gel. 38. The method of claim 1, wherein connection of the DNA of the crosslinked Protein:DNA complexes intramolecularly is done by ligation using DNA ligase. 39. The method of claim 1, wherein the protein:DNA complexes are substantially immobilized relative to each other.

The present invention provides: a method for detecting a nucleic acid, which efficiently eliminates non-specific detection of nucleic acids other than the nucleic acid as a detection target, so that detection specificity of the nucleic acid as a detection target can be further improved; or the like. This method for detecting a nucleic acid comprises: (a) a step wherein a gel, on which a probe is immobilized, is brought into contact with a reaction solution which contains a nucleic acid that serves as a template for nucleic acid amplification, a primer set for nucleic acid amplification, a nucleotide unit and a DNA extension enzyme; (b) a step wherein the gel and the reaction solution are subjected to a heat cycle for performing a nucleic acid amplification reaction; (c) a step wherein nucleic acid fragments having a specific base length are selected from among the amplified nucleic acid fragments; and (d) a step wherein the selected nucleic acid fragments are detected.

1. A method for detecting a nucleic acid, comprising: contacting a plurality of types of gels of different gel concentrations, on which a probe is immobilized, with a reaction solution comprising a nucleic acid that serves as a template for nucleic acid amplification, a primer set for nucleic acid amplification, a nucleotide unit and a DNA extension enzyme; subjecting the gels and the reaction solution to a heat cycle for performing a nucleic acid amplification reaction; selecting nucleic acid fragments having a specific base length from among the amplified nucleic acid fragments; and detecting the selected nucleic acid fragments. 2. (canceled) 3. The method according to claim 1, wherein a ratio (V/S) of a volume of the gel (V(μm3)) to a contact surface area of the gel on which the probe is immobilized and the reaction solution (S(μm2)) is 50 or more. 4. The method according to claim 1, wherein the gels on which the probe is immobilized are held in a well or through-hole in a substrate. 5. The method according to claim 4, wherein the gels on which the probe is immobilized comprises a substituted (meth)acrylamide derivative, an agarose derivative, or both. 6. The method according to claim 5, wherein a gel concentration of the gels on which the probe is immobilized is more than 2% by mass and less than 5% by mass. 7. A microarray, by which a plurality of types of gels with different gel concentrations are carried, and wherein a probe is immobilized on the gels. 8. A method for producing the microarray according to claim 7, comprising: three-dimensionally arranging a plurality of hollow fibers so that fiber axial directions of the hollow fibers become the same, and wherein the arrangement is fixed with a resin to produce a hollow fiber bundle; introducing a plurality of types of gel precursor solutions with different monomer concentrations comprising a probe into respective hollow portions of the hollow fibers of the hollow fiber bundle; reacting the gel precursor solutions introduced into the hollow portions to hold a gel-like product comprising the probe in the hollow portions of the hollow fibers; and slicing the hollow fiber bundle in a direction crossing a longitudinal direction of the fibers into thin sections.

The present invention provides: a method for detecting a nucleic acid, which efficiently eliminates non-specific detection of nucleic acids other than the nucleic acid as a detection target, so that detection specificity of the nucleic acid as a detection target can be further improved; or the like. This method for detecting a nucleic acid comprises: (a) a step wherein a gel, on which a probe is immobilized, is brought into contact with a reaction solution which contains a nucleic acid that serves as a template for nucleic acid amplification, a primer set for nucleic acid amplification, a nucleotide unit and a DNA extension enzyme; (b) a step wherein the gel and the reaction solution are subjected to a heat cycle for performing a nucleic acid amplification reaction; (c) a step wherein nucleic acid fragments having a specific base length are selected from among the amplified nucleic acid fragments; and (d) a step wherein the selected nucleic acid fragments are detected.1. A method for detecting a nucleic acid, comprising: contacting a plurality of types of gels of different gel concentrations, on which a probe is immobilized, with a reaction solution comprising a nucleic acid that serves as a template for nucleic acid amplification, a primer set for nucleic acid amplification, a nucleotide unit and a DNA extension enzyme; subjecting the gels and the reaction solution to a heat cycle for performing a nucleic acid amplification reaction; selecting nucleic acid fragments having a specific base length from among the amplified nucleic acid fragments; and detecting the selected nucleic acid fragments. 2. (canceled) 3. The method according to claim 1, wherein a ratio (V/S) of a volume of the gel (V(μm3)) to a contact surface area of the gel on which the probe is immobilized and the reaction solution (S(μm2)) is 50 or more. 4. The method according to claim 1, wherein the gels on which the probe is immobilized are held in a well or through-hole in a substrate. 5. The method according to claim 4, wherein the gels on which the probe is immobilized comprises a substituted (meth)acrylamide derivative, an agarose derivative, or both. 6. The method according to claim 5, wherein a gel concentration of the gels on which the probe is immobilized is more than 2% by mass and less than 5% by mass. 7. A microarray, by which a plurality of types of gels with different gel concentrations are carried, and wherein a probe is immobilized on the gels. 8. A method for producing the microarray according to claim 7, comprising: three-dimensionally arranging a plurality of hollow fibers so that fiber axial directions of the hollow fibers become the same, and wherein the arrangement is fixed with a resin to produce a hollow fiber bundle; introducing a plurality of types of gel precursor solutions with different monomer concentrations comprising a probe into respective hollow portions of the hollow fibers of the hollow fiber bundle; reacting the gel precursor solutions introduced into the hollow portions to hold a gel-like product comprising the probe in the hollow portions of the hollow fibers; and slicing the hollow fiber bundle in a direction crossing a longitudinal direction of the fibers into thin sections.

A composition having biocidal properties is disclosed. The composition includes a first biocide, optionally a second biocide, and a biocide enhancing agent. The first biocide may comprise an isothiazolin. The second biocide may comprise a pyrithione. The biocide enhancing agent may comprise an amine, an amine salt, an amine oxide, or mixtures thereof. The amine compound may have a carbon chain length of at least six carbon atoms, such as at least eight carbon atoms.

1. A composition having biocidal properties comprising: a first biocide comprising an isothiazolin; a second biocide comprising pyrithione; and a biocide enhancing agent comprising an amine, an amine salt, an amine oxide, or mixtures thereof, the amine, the amine salt, or the amine oxide having a carbon chain length of at least 6 carbon atoms. 2. A composition as defined in claim 1, wherein the first biocide and the second biocide are present in the composition at a weight ratio of from about 1:200 to about 10:1. 3. A composition as defined in claim 1, wherein the first biocide and the biocide enhancing agent are present in the composition at weight ratio of from about 1:600 to about 2:1. 4. A composition as defined in claim 1, wherein the composition contains at least two biocide enhancing agents, each of the biocide enhancing agents comprising an amine, an amine salt, or an amine oxide. 5. A composition as defined in claim 1, further comprising a zinc compound, the zinc compound comprising a zinc salt of an organic acid, a zinc salt of an inorganic acid, a zinc oxide, a zinc hydroxide, or mixtures thereof. 6. A composition as defined in claim 1, wherein the composition further comprises polyethylenimine. 7. A composition as defined in claim 1, wherein the first biocide comprises 1,2-benzisothiazolin-3-one; 5-chloro-2-methyl-2H-isothiazolin-3-one; 2-methyl-2H-isothiazolin-3-one; 2-Octyl-3(2H)-isothiazolone; dichloro-n-octyl-2H-isothiazolin-3-one; N-methyl-1,2-benzisothiazolin-3-one; 2,2-Dithiobis(N-N-butyl-1,2-benzisothiazolin-3-one, or mixtures thereof. 8. A composition as defined in claim 1, wherein the second biocide comprises sodium pyrithione, bismuth pyrithione, potassium pyrithione, lithium pyrithione, ammonium pyrithione, zinc pyrithione, copper pyrithione, calcium pyrithione, magnesium pyrithione, strontium pyrithione, silver pyrithione, gold pyrithione, manganese pyrithione, an organic amine pyrithione, or mixtures thereof. 9. A composition as defined in claim 1, wherein the biocide enhancing agent comprises 1-dodecylamine, cocodimethylamine oxide, 4-dodecylmorpholine, lauryl dimethyl amine, Bis(3-aminopropyl)dodecylamine, or mixtures thereof. 10. A composition as defined in claim 5, wherein the zinc compound comprises zinc acetate, zinc borate, zinc oxide, zinc carbonate, zinc chloride, zinc sulfate, zinc hydroxide, zinc citrate, zinc fluoride, zinc iodide, zinc lactate, zinc oleate, zinc oxalate, zinc phosphate, zinc propionate, zinc salicylate, zinc selenite, zinc silicate, zinc stearate, zinc sulfide, zinc tannate, zinc tartrate, zinc valerate, and mixtures thereof. 11. A composition as defined in claim 6, wherein the polyethylenimine has a branched structure. 12. A composition as defined in claim 1, wherein the biocide enhancing agent comprises: wherein: R1 is a linear, branched or cyclic C6-40 saturated or unsaturated group; and R2 and R3 are each independently of one another H or linear, branched or cyclic C1-40 saturated or unsaturated group or R2 and R3 are both taken together in combination with a nitrogen atom to which they are attached to form a cyclic or heterocyclic group, optionally substituted. 13. A product for coating a surface comprising: a base composition; and a preservative comprising a first biocide comprising an isothiazolin; a second biocide comprising pyrithione; and a biocide enhancing agent comprising an amine, an amine salt, an amine oxide, or mixtures thereof, the amine, the amine salt, or the amine oxide having a carbon chain length of at least 6 carbon atoms, and wherein the first biocide is present in the product at a concentration of up to about 50 ppm. 14. A product as defined in claim 13, wherein the first biocide and the second biocide are present in the composition at a weight ratio of from about 1:10 to about 1:4. 15. A product as defined in claim 13, wherein the first biocide and the biocide enhancing agent are present in the composition at weight ratio of from about 1:100 to about 1:2. 16. A product as defined in claim 13, wherein the composition contains at least two biocide enhancing agents, each of the biocide enhancing agents comprising an amine, an amine salt, or an amine oxide. 17. A product as defined in claim 13, further comprising a zinc compound, the zinc compound comprising a zinc salt of an organic acid, a zinc salt of an inorganic acid, a zinc oxide, a zinc hydroxide, or mixtures thereof. 18. A product as defined in claim 13, wherein the composition further comprises polyethylenimine. 19. A product as defined in claim 13, wherein the first biocide comprises 1,2-benzisothiazolin-3-one; 5-chloro-2-methyl-2H-isothiazolin-3-one; 2-methyl-2H-isothiazolin-3-one: 2-Octyl-3(2H)-isothiazolone; dichloro-n-octyl-2H-isothiazolin-3-one; N-methyl-1,2-benzisothiazolin-3-one; 2,2-Dithiobis(N-methylbenzamide; N-butyl-1,2-benzisothiazolin-3-one, and mixtures thereof and wherein the second biocide comprises sodium pyrithione, bismuth pyrithione, potassium pyrithione, lithium pyrithione, ammonium pyrithione, zinc pyrithione, copper pyrithione, calcium pyrithione, magnesium pyrithione, strontium pyrithione, silver pyrithione, gold pyrithione, manganese pyrithione, an organic amine pyrithione, and mixtures thereof, and wherein the biocide enhancing agent comprises 1-dodecylamine, cocodimethylamine oxide, 4-dodecylmorpholine, lauryl dimethyl amine, Bis(3-aminopropyl)dodecylamine, and mixtures thereof. 20. A product as defined in claim 13, wherein the biocide enhancing agent comprises: wherein: R1 is a linear, branched or cyclic C6-40 saturated or unsaturated group; and R2 and R3 are each independently of one another H or linear, branched or cyclic C1-40 saturated or unsaturated group or R2 and R3 are both taken together in combination with a nitrogen atom to which they are attached to form a cyclic or heterocyclic group, optionally substituted.

A composition having biocidal properties is disclosed. The composition includes a first biocide, optionally a second biocide, and a biocide enhancing agent. The first biocide may comprise an isothiazolin. The second biocide may comprise a pyrithione. The biocide enhancing agent may comprise an amine, an amine salt, an amine oxide, or mixtures thereof. The amine compound may have a carbon chain length of at least six carbon atoms, such as at least eight carbon atoms.1. A composition having biocidal properties comprising: a first biocide comprising an isothiazolin; a second biocide comprising pyrithione; and a biocide enhancing agent comprising an amine, an amine salt, an amine oxide, or mixtures thereof, the amine, the amine salt, or the amine oxide having a carbon chain length of at least 6 carbon atoms. 2. A composition as defined in claim 1, wherein the first biocide and the second biocide are present in the composition at a weight ratio of from about 1:200 to about 10:1. 3. A composition as defined in claim 1, wherein the first biocide and the biocide enhancing agent are present in the composition at weight ratio of from about 1:600 to about 2:1. 4. A composition as defined in claim 1, wherein the composition contains at least two biocide enhancing agents, each of the biocide enhancing agents comprising an amine, an amine salt, or an amine oxide. 5. A composition as defined in claim 1, further comprising a zinc compound, the zinc compound comprising a zinc salt of an organic acid, a zinc salt of an inorganic acid, a zinc oxide, a zinc hydroxide, or mixtures thereof. 6. A composition as defined in claim 1, wherein the composition further comprises polyethylenimine. 7. A composition as defined in claim 1, wherein the first biocide comprises 1,2-benzisothiazolin-3-one; 5-chloro-2-methyl-2H-isothiazolin-3-one; 2-methyl-2H-isothiazolin-3-one; 2-Octyl-3(2H)-isothiazolone; dichloro-n-octyl-2H-isothiazolin-3-one; N-methyl-1,2-benzisothiazolin-3-one; 2,2-Dithiobis(N-N-butyl-1,2-benzisothiazolin-3-one, or mixtures thereof. 8. A composition as defined in claim 1, wherein the second biocide comprises sodium pyrithione, bismuth pyrithione, potassium pyrithione, lithium pyrithione, ammonium pyrithione, zinc pyrithione, copper pyrithione, calcium pyrithione, magnesium pyrithione, strontium pyrithione, silver pyrithione, gold pyrithione, manganese pyrithione, an organic amine pyrithione, or mixtures thereof. 9. A composition as defined in claim 1, wherein the biocide enhancing agent comprises 1-dodecylamine, cocodimethylamine oxide, 4-dodecylmorpholine, lauryl dimethyl amine, Bis(3-aminopropyl)dodecylamine, or mixtures thereof. 10. A composition as defined in claim 5, wherein the zinc compound comprises zinc acetate, zinc borate, zinc oxide, zinc carbonate, zinc chloride, zinc sulfate, zinc hydroxide, zinc citrate, zinc fluoride, zinc iodide, zinc lactate, zinc oleate, zinc oxalate, zinc phosphate, zinc propionate, zinc salicylate, zinc selenite, zinc silicate, zinc stearate, zinc sulfide, zinc tannate, zinc tartrate, zinc valerate, and mixtures thereof. 11. A composition as defined in claim 6, wherein the polyethylenimine has a branched structure. 12. A composition as defined in claim 1, wherein the biocide enhancing agent comprises: wherein: R1 is a linear, branched or cyclic C6-40 saturated or unsaturated group; and R2 and R3 are each independently of one another H or linear, branched or cyclic C1-40 saturated or unsaturated group or R2 and R3 are both taken together in combination with a nitrogen atom to which they are attached to form a cyclic or heterocyclic group, optionally substituted. 13. A product for coating a surface comprising: a base composition; and a preservative comprising a first biocide comprising an isothiazolin; a second biocide comprising pyrithione; and a biocide enhancing agent comprising an amine, an amine salt, an amine oxide, or mixtures thereof, the amine, the amine salt, or the amine oxide having a carbon chain length of at least 6 carbon atoms, and wherein the first biocide is present in the product at a concentration of up to about 50 ppm. 14. A product as defined in claim 13, wherein the first biocide and the second biocide are present in the composition at a weight ratio of from about 1:10 to about 1:4. 15. A product as defined in claim 13, wherein the first biocide and the biocide enhancing agent are present in the composition at weight ratio of from about 1:100 to about 1:2. 16. A product as defined in claim 13, wherein the composition contains at least two biocide enhancing agents, each of the biocide enhancing agents comprising an amine, an amine salt, or an amine oxide. 17. A product as defined in claim 13, further comprising a zinc compound, the zinc compound comprising a zinc salt of an organic acid, a zinc salt of an inorganic acid, a zinc oxide, a zinc hydroxide, or mixtures thereof. 18. A product as defined in claim 13, wherein the composition further comprises polyethylenimine. 19. A product as defined in claim 13, wherein the first biocide comprises 1,2-benzisothiazolin-3-one; 5-chloro-2-methyl-2H-isothiazolin-3-one; 2-methyl-2H-isothiazolin-3-one: 2-Octyl-3(2H)-isothiazolone; dichloro-n-octyl-2H-isothiazolin-3-one; N-methyl-1,2-benzisothiazolin-3-one; 2,2-Dithiobis(N-methylbenzamide; N-butyl-1,2-benzisothiazolin-3-one, and mixtures thereof and wherein the second biocide comprises sodium pyrithione, bismuth pyrithione, potassium pyrithione, lithium pyrithione, ammonium pyrithione, zinc pyrithione, copper pyrithione, calcium pyrithione, magnesium pyrithione, strontium pyrithione, silver pyrithione, gold pyrithione, manganese pyrithione, an organic amine pyrithione, and mixtures thereof, and wherein the biocide enhancing agent comprises 1-dodecylamine, cocodimethylamine oxide, 4-dodecylmorpholine, lauryl dimethyl amine, Bis(3-aminopropyl)dodecylamine, and mixtures thereof. 20. A product as defined in claim 13, wherein the biocide enhancing agent comprises: wherein: R1 is a linear, branched or cyclic C6-40 saturated or unsaturated group; and R2 and R3 are each independently of one another H or linear, branched or cyclic C1-40 saturated or unsaturated group or R2 and R3 are both taken together in combination with a nitrogen atom to which they are attached to form a cyclic or heterocyclic group, optionally substituted.

Drug delivery involving hydrogels as used for various medical conditions, and includes hydrogels formed in an eye with extended drug release times. An embodiment of the invention is a method of delivering a therapeutic agent to a tissue comprising forming a hydrogel in situ in an eye with a therapeutic agent dispersed in the hydrogel, the agent having a low solubility in water. The agent may be essentially insoluble in water. The hydrogel may be made so that 50% to 100% w/w of the agent is released when the hydrogel is from 100% to 50% persistent, with the persistence being a measure of the dry weight of the hydrogel relative to an initial dry weight of the hydrogel.

1. A method of delivering a therapeutic agent to a tissue comprising forming a hydrogel in situ in an eye with a therapeutic agent dispersed in the hydrogel, the agent having a low solubility in water. 2. The method of claim 1 with the agent being suspended in the hydrogel. 3. The method of claim 1 with 50% to 100% w/w of the agent being released when the hydrogel is from 100% to 50% persistent, with the persistence being a measure of the dry weight of the hydrogel relative to an initial dry weight of the hydrogel. 4. The method of claim 3 with 90% w/w of the agent being released when the hydrogel is at least 90% persistent. 5. The method of claim 1 wherein the agent is released to provide an effective concentration of the agent in an eye over a period of time. 6. The method of claim 5 wherein the period of time is 2-36 months. 7. The method of claim 5 wherein, after the period of time, the hydrogel has released all of the agent and has a persistence of at least 80%. 8. The method of claim 5 wherein, after the period of time, the hydrogel releases a further amount of the agent that is non-toxic. 9. The method of claim 1 wherein the hydrogel is water-degradable, as measurable by the hydrogel being dissolvable in vitro in an excess of water by degradation of water-degradable groups. 10. The method of claim 1 wherein the hydrogel is formed at an intravitreal site. 11. The method of claim 1 wherein the agent is for treatment of a back of the eye disease. 12. The method of claim 11 wherein the back of the eye disease is age-related macular degeneration (AMD) cystoid macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy, or glaucoma. 13. The method of claim 1 wherein the agent comprises anti-VEGF, blocks VEGFR1, blocks VEGFR2, blocks VEGFR3, anti-PDGF, anti-PDGF-R blocks PDGFRβ, an anti-angiogenic agent, Sunitinib, E7080, Takeda-6d, Tivozanib, Regorafenib, Sorafenib, Pazopanib, Axitinib, Nintedanib, Cediranib, Vatalanib, Motesanib, macrolides, sirolimus, everolimus, tyrosine kinase inhibitors (TKIs), Imatinibn gefinitib, toceranib, Erlotinib, Lapatinib, Nilotinib, Bosutinib Neratinib, lapatinib, Vatalanib, comprises low-soluble prostaglandin analogues for glaucoma, nepafenac, macrolides, rapamycin, sirolimus, tacrolimus, or serves to block mTOR receptors for AMD/CNV. 14. The method of claim 1 wherein the hydrogel is formed by combining a first precursor and a second precursor that react with each other to form the hydrogel. 15. The method of claim 14 wherein the hydrogel is formed with covalent crosslinks between the first precursor and the second precursor. 16. The method of claim 15 wherein the first precursor and the second precursor are hydrophilic. 17. The method of claim 1 wherein the hydrogel is essentially spherical, essentially discoidal, or essentially cylindroid. 18. A hydrogel in an eye with a therapeutic agent dispersed in the hydrogel, the agent having a low solubility in water. 19. The hydrogel of claim 1 with 50% to 100% w/w of the agent being released when the hydrogel is from 100% to 50% persistent, with the persistence being a measure of the dry weight of the hydrogel relative to an initial dry weight of the hydrogel. 20. The hydrogel of claim 19 wherein the period of time is 2-36 months.

Drug delivery involving hydrogels as used for various medical conditions, and includes hydrogels formed in an eye with extended drug release times. An embodiment of the invention is a method of delivering a therapeutic agent to a tissue comprising forming a hydrogel in situ in an eye with a therapeutic agent dispersed in the hydrogel, the agent having a low solubility in water. The agent may be essentially insoluble in water. The hydrogel may be made so that 50% to 100% w/w of the agent is released when the hydrogel is from 100% to 50% persistent, with the persistence being a measure of the dry weight of the hydrogel relative to an initial dry weight of the hydrogel.1. A method of delivering a therapeutic agent to a tissue comprising forming a hydrogel in situ in an eye with a therapeutic agent dispersed in the hydrogel, the agent having a low solubility in water. 2. The method of claim 1 with the agent being suspended in the hydrogel. 3. The method of claim 1 with 50% to 100% w/w of the agent being released when the hydrogel is from 100% to 50% persistent, with the persistence being a measure of the dry weight of the hydrogel relative to an initial dry weight of the hydrogel. 4. The method of claim 3 with 90% w/w of the agent being released when the hydrogel is at least 90% persistent. 5. The method of claim 1 wherein the agent is released to provide an effective concentration of the agent in an eye over a period of time. 6. The method of claim 5 wherein the period of time is 2-36 months. 7. The method of claim 5 wherein, after the period of time, the hydrogel has released all of the agent and has a persistence of at least 80%. 8. The method of claim 5 wherein, after the period of time, the hydrogel releases a further amount of the agent that is non-toxic. 9. The method of claim 1 wherein the hydrogel is water-degradable, as measurable by the hydrogel being dissolvable in vitro in an excess of water by degradation of water-degradable groups. 10. The method of claim 1 wherein the hydrogel is formed at an intravitreal site. 11. The method of claim 1 wherein the agent is for treatment of a back of the eye disease. 12. The method of claim 11 wherein the back of the eye disease is age-related macular degeneration (AMD) cystoid macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy, or glaucoma. 13. The method of claim 1 wherein the agent comprises anti-VEGF, blocks VEGFR1, blocks VEGFR2, blocks VEGFR3, anti-PDGF, anti-PDGF-R blocks PDGFRβ, an anti-angiogenic agent, Sunitinib, E7080, Takeda-6d, Tivozanib, Regorafenib, Sorafenib, Pazopanib, Axitinib, Nintedanib, Cediranib, Vatalanib, Motesanib, macrolides, sirolimus, everolimus, tyrosine kinase inhibitors (TKIs), Imatinibn gefinitib, toceranib, Erlotinib, Lapatinib, Nilotinib, Bosutinib Neratinib, lapatinib, Vatalanib, comprises low-soluble prostaglandin analogues for glaucoma, nepafenac, macrolides, rapamycin, sirolimus, tacrolimus, or serves to block mTOR receptors for AMD/CNV. 14. The method of claim 1 wherein the hydrogel is formed by combining a first precursor and a second precursor that react with each other to form the hydrogel. 15. The method of claim 14 wherein the hydrogel is formed with covalent crosslinks between the first precursor and the second precursor. 16. The method of claim 15 wherein the first precursor and the second precursor are hydrophilic. 17. The method of claim 1 wherein the hydrogel is essentially spherical, essentially discoidal, or essentially cylindroid. 18. A hydrogel in an eye with a therapeutic agent dispersed in the hydrogel, the agent having a low solubility in water. 19. The hydrogel of claim 1 with 50% to 100% w/w of the agent being released when the hydrogel is from 100% to 50% persistent, with the persistence being a measure of the dry weight of the hydrogel relative to an initial dry weight of the hydrogel. 20. The hydrogel of claim 19 wherein the period of time is 2-36 months.

The present invention is generally directed to a hierarchical genome assembly process for producing high-quality de novo genome assemblies. The method utilizes a single, long-insert, shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT®) DNA sequencing, and obviates the need for additional sample preparation and sequencing data sets required for previously described hybrid assembly strategies. Efficient de novo assembly from genomic DNA to a finished genome sequence is demonstrated for several microorganisms using as little as three SMRT® cells, and for bacterial artificial chromosomes (BACs) using sequencing data from just one SMRT® Cell. Part of this new assembly workflow is a new consensus algorithm which takes advantage of SMRT® sequencing primary quality values, to produce a highly accurate de novo genome sequence, exceeding 99.999% (QV 50) accuracy. The methods are typically performed on a computer and comprise an algorithm that constructs sequence alignment graphs from pairwise alignment of sequence reads to a common reference.

1. A method of identifying a sequence of a nucleic acid, the method comprising: obtaining a set of reads for the nucleic acid sequence; forming a seed sequence dataset comprising one or more seed reads identified as reads in the set of reads having lengths greater than 1,000 base pairs; aligning each read in the set of reads to a seed read, thereby producing a sequence alignment for each seed read that comprises a subset of the set of reads; creating a pre-assembled read for each seed read, from each sequence alignment, thereby generating a set of pre-assembled reads, one for each seed read; using a sequence assembler to assemble the set of pre-assembled reads into a contig for the nucleic acid; and determining a consensus sequence of the nucleic acid. 2. The method of claim 1, wherein the creating further comprises optimizing each pre-assembled read to produce a set of refined pre-assembled reads prior to using the sequence assembler. 3. The method of claim 1, wherein the creating further comprises trimming alignments of the reads to the seed reads, thereby reducing chimeric reads and read noise in the set of pre-assembled reads prior to using the sequence assembler. 4. The method of claim 1, wherein the set of reads is produced by single-molecule real-time sequencing. 5. The method of claim 1, wherein the reads in the set of reads have an average accuracy of between 80 and 95%. 6. The method of claim 1, wherein the seed reads have a length greater than 2,000 bp. 7. The method of claim 1, wherein the seed read has length between 1,000 bp and 10,000 bp. 8. The method of claim 1, wherein the seed reads in the seeding sequence dataset comprise 15-20× coverage of the nucleic acid. 9. The method of claim 1, wherein the creating a pre-assembled read for each seed read comprises: a) using the sequence alignments from said aligning to construct a sequence alignment graph; and b) based upon the sequence alignment graph, determining a consensus sequence for the seed read. 10. The method of claim 9, further comprising subjecting each sequence alignment produced in said aligning to normalization prior to constructing the sequence alignment graph. 11. The method of claim 10, wherein the normalization comprises at least one of the group consisting of: changing mismatches to indels and moving gaps. 12. The method of claim 11, wherein said moving gaps comprises moving gaps to right-most equivalent positions. 13. The method of claim 9, wherein constructing the sequence alignment graph comprises constructing a multi-graph and merging nodes and edges in the multi-graph. 14. The method of claim 1, wherein the determining a consensus sequence of the nucleic acid comprises: a) aligning each sequence in the set of original reads to the assembled sequence contig; b) using the set of sequence alignments to find the reads coming from each region of the genome; c) for each region of the genome, choosing an initial estimate of the consensus of the reads in that window using a graph-based algorithm; d) computing a statistical likelihood that a sequence whose identity is the current consensus estimate was the sequence that generated the reads found to originate from this region of the genome, using a statistical model of the types of errors made in reading nucleic acid sequence; e) perturbing the current estimate by applying a single-base mutation to its sequence, such that the perturbation yields a statistical likelihood that is greater than before the perturbation; and f) repeating the steps (d) and (e) until the estimate is such that no perturbation yields a greater statistical likelihood. 15. The method of claim 1, wherein the consensus sequence is at least 99.99% accurate. 16. The method of claim 1, wherein the consensus sequence is at least 99.999% accurate. 17. A system for identifying a sequence of a nucleic acid, the system comprising: an input device configured to accept raw, single molecule, real-time sequence data; an output device; a memory; a processor, and an output device, wherein the processor is configured to execute instructions according to claim 1, and to direct the consensus sequence for the nucleic acid to the output device. 18. A computer readable medium, comprising non-transitory signals, the medium encoded with instructions to: process raw, single molecule, real-time sequence data according to the method of claim 1.

The present invention is generally directed to a hierarchical genome assembly process for producing high-quality de novo genome assemblies. The method utilizes a single, long-insert, shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT®) DNA sequencing, and obviates the need for additional sample preparation and sequencing data sets required for previously described hybrid assembly strategies. Efficient de novo assembly from genomic DNA to a finished genome sequence is demonstrated for several microorganisms using as little as three SMRT® cells, and for bacterial artificial chromosomes (BACs) using sequencing data from just one SMRT® Cell. Part of this new assembly workflow is a new consensus algorithm which takes advantage of SMRT® sequencing primary quality values, to produce a highly accurate de novo genome sequence, exceeding 99.999% (QV 50) accuracy. The methods are typically performed on a computer and comprise an algorithm that constructs sequence alignment graphs from pairwise alignment of sequence reads to a common reference.1. A method of identifying a sequence of a nucleic acid, the method comprising: obtaining a set of reads for the nucleic acid sequence; forming a seed sequence dataset comprising one or more seed reads identified as reads in the set of reads having lengths greater than 1,000 base pairs; aligning each read in the set of reads to a seed read, thereby producing a sequence alignment for each seed read that comprises a subset of the set of reads; creating a pre-assembled read for each seed read, from each sequence alignment, thereby generating a set of pre-assembled reads, one for each seed read; using a sequence assembler to assemble the set of pre-assembled reads into a contig for the nucleic acid; and determining a consensus sequence of the nucleic acid. 2. The method of claim 1, wherein the creating further comprises optimizing each pre-assembled read to produce a set of refined pre-assembled reads prior to using the sequence assembler. 3. The method of claim 1, wherein the creating further comprises trimming alignments of the reads to the seed reads, thereby reducing chimeric reads and read noise in the set of pre-assembled reads prior to using the sequence assembler. 4. The method of claim 1, wherein the set of reads is produced by single-molecule real-time sequencing. 5. The method of claim 1, wherein the reads in the set of reads have an average accuracy of between 80 and 95%. 6. The method of claim 1, wherein the seed reads have a length greater than 2,000 bp. 7. The method of claim 1, wherein the seed read has length between 1,000 bp and 10,000 bp. 8. The method of claim 1, wherein the seed reads in the seeding sequence dataset comprise 15-20× coverage of the nucleic acid. 9. The method of claim 1, wherein the creating a pre-assembled read for each seed read comprises: a) using the sequence alignments from said aligning to construct a sequence alignment graph; and b) based upon the sequence alignment graph, determining a consensus sequence for the seed read. 10. The method of claim 9, further comprising subjecting each sequence alignment produced in said aligning to normalization prior to constructing the sequence alignment graph. 11. The method of claim 10, wherein the normalization comprises at least one of the group consisting of: changing mismatches to indels and moving gaps. 12. The method of claim 11, wherein said moving gaps comprises moving gaps to right-most equivalent positions. 13. The method of claim 9, wherein constructing the sequence alignment graph comprises constructing a multi-graph and merging nodes and edges in the multi-graph. 14. The method of claim 1, wherein the determining a consensus sequence of the nucleic acid comprises: a) aligning each sequence in the set of original reads to the assembled sequence contig; b) using the set of sequence alignments to find the reads coming from each region of the genome; c) for each region of the genome, choosing an initial estimate of the consensus of the reads in that window using a graph-based algorithm; d) computing a statistical likelihood that a sequence whose identity is the current consensus estimate was the sequence that generated the reads found to originate from this region of the genome, using a statistical model of the types of errors made in reading nucleic acid sequence; e) perturbing the current estimate by applying a single-base mutation to its sequence, such that the perturbation yields a statistical likelihood that is greater than before the perturbation; and f) repeating the steps (d) and (e) until the estimate is such that no perturbation yields a greater statistical likelihood. 15. The method of claim 1, wherein the consensus sequence is at least 99.99% accurate. 16. The method of claim 1, wherein the consensus sequence is at least 99.999% accurate. 17. A system for identifying a sequence of a nucleic acid, the system comprising: an input device configured to accept raw, single molecule, real-time sequence data; an output device; a memory; a processor, and an output device, wherein the processor is configured to execute instructions according to claim 1, and to direct the consensus sequence for the nucleic acid to the output device. 18. A computer readable medium, comprising non-transitory signals, the medium encoded with instructions to: process raw, single molecule, real-time sequence data according to the method of claim 1.

The present invention provides a method for analysis of target nucleic acids which are present in low amounts. In particular, the method comprises the following steps: i. providing a sample wherein target nucleic acids are present in a low amount, ii. generating a reduced representation library of said target nucleic acids by a method comprising: fragmenting said target nucleic acids; ligating adaptors to said fragments; and selecting a subset of said adaptor-ligated fragments, iii. massively parallel sequencing said reduced representation library, and iv. identifying variants in said target nucleic acids by analyzing results obtained by said sequencing.

1. A method for analysis of target nucleic acids, the method comprising: i. providing a sample wherein target nucleic acids are present in a low amount, ii. generating a reduced representation library of said target nucleic acids by a method comprising: fragmenting said target nucleic acids; ligating adaptors to said fragments; and selecting a subset of said adaptor-ligated fragments, iii. massively parallel sequencing said reduced representation library, and iv. identifying variants in said target nucleic acids by analyzing results obtained by said sequencing. 2. The method of claim 1, wherein said low amount is an amount of 100 pg or less. 3. The method of claim 1, wherein said fragmenting is performed using one or more restriction enzymes. 4. The method of claim 1, wherein said selecting a subset is based on the size of said fragments. 5. The method of claim 1, wherein said selecting a subset is performed using PCR-amplification. 6. The method of claim 1, wherein said selecting a subset includes PCR amplification using a selective primer. 7. The method of claim 1, wherein generating a reduced representation library comprises amplifying a subset of fragments which, when combined, comprise only a part of the target nucleic acids. 8. The method of claim 1, further comprising v. constructing a genotype and/or haplotype based on identified variants in said target nucleic acid. 9. The method of claim 1, further comprising v. identifying a genetic aberration in said sample based on identified variants in said target nucleic acid. 10. The method of claim 1, wherein said target nucleic acids originate from an embryo or fetus. 11. The method of claim 1, wherein said target nucleic acids originate from a cancer or tumor cell. 12. The method of claim 1, wherein providing a sample comprises isolating one or a few target cells. 13. The method of claim 12, wherein providing a sample further comprises lysing said one or few target cells. 14. The method of claim 1, further comprising whole genome amplification (WGA) of said target nucleic acids. 15. The method of claim 1, wherein sequencing said reduced representation library assures that each variant position in said library is sampled with high redundancy. 16. A system for generating a sequencing library, said system being adapted to receive a sample wherein target nucleic acids are present in a low amount, said system comprising a control unit that controls the generation of a reduced representation sequencing library of said target nucleic acids by controlling the fragmentation of said target nucleic acids; the ligation of adaptors to said fragments; and the selection of a subset of said adaptor-ligated fragments. 17. A system for analysis of target nucleic acids, said system being adapted to receive a sample wherein target nucleic acids are present in a low amount, said system comprising one or more control units that control: fragmenting said target nucleic acids, ligating adaptors to said fragments, selecting a subset of said adaptor-ligated fragments, and sequencing said subset. 18. A combination of devices comprising: a cell isolation device adapted to isolate one to twenty cells; a sample processing device adapted to generate a reduced representation library by a method comprising fragmenting said target nucleic acids, ligating adaptors to said fragments, and selecting a subset of said adaptor-ligated fragments; and a massive parallel sequencing device.

The present invention provides a method for analysis of target nucleic acids which are present in low amounts. In particular, the method comprises the following steps: i. providing a sample wherein target nucleic acids are present in a low amount, ii. generating a reduced representation library of said target nucleic acids by a method comprising: fragmenting said target nucleic acids; ligating adaptors to said fragments; and selecting a subset of said adaptor-ligated fragments, iii. massively parallel sequencing said reduced representation library, and iv. identifying variants in said target nucleic acids by analyzing results obtained by said sequencing.1. A method for analysis of target nucleic acids, the method comprising: i. providing a sample wherein target nucleic acids are present in a low amount, ii. generating a reduced representation library of said target nucleic acids by a method comprising: fragmenting said target nucleic acids; ligating adaptors to said fragments; and selecting a subset of said adaptor-ligated fragments, iii. massively parallel sequencing said reduced representation library, and iv. identifying variants in said target nucleic acids by analyzing results obtained by said sequencing. 2. The method of claim 1, wherein said low amount is an amount of 100 pg or less. 3. The method of claim 1, wherein said fragmenting is performed using one or more restriction enzymes. 4. The method of claim 1, wherein said selecting a subset is based on the size of said fragments. 5. The method of claim 1, wherein said selecting a subset is performed using PCR-amplification. 6. The method of claim 1, wherein said selecting a subset includes PCR amplification using a selective primer. 7. The method of claim 1, wherein generating a reduced representation library comprises amplifying a subset of fragments which, when combined, comprise only a part of the target nucleic acids. 8. The method of claim 1, further comprising v. constructing a genotype and/or haplotype based on identified variants in said target nucleic acid. 9. The method of claim 1, further comprising v. identifying a genetic aberration in said sample based on identified variants in said target nucleic acid. 10. The method of claim 1, wherein said target nucleic acids originate from an embryo or fetus. 11. The method of claim 1, wherein said target nucleic acids originate from a cancer or tumor cell. 12. The method of claim 1, wherein providing a sample comprises isolating one or a few target cells. 13. The method of claim 12, wherein providing a sample further comprises lysing said one or few target cells. 14. The method of claim 1, further comprising whole genome amplification (WGA) of said target nucleic acids. 15. The method of claim 1, wherein sequencing said reduced representation library assures that each variant position in said library is sampled with high redundancy. 16. A system for generating a sequencing library, said system being adapted to receive a sample wherein target nucleic acids are present in a low amount, said system comprising a control unit that controls the generation of a reduced representation sequencing library of said target nucleic acids by controlling the fragmentation of said target nucleic acids; the ligation of adaptors to said fragments; and the selection of a subset of said adaptor-ligated fragments. 17. A system for analysis of target nucleic acids, said system being adapted to receive a sample wherein target nucleic acids are present in a low amount, said system comprising one or more control units that control: fragmenting said target nucleic acids, ligating adaptors to said fragments, selecting a subset of said adaptor-ligated fragments, and sequencing said subset. 18. A combination of devices comprising: a cell isolation device adapted to isolate one to twenty cells; a sample processing device adapted to generate a reduced representation library by a method comprising fragmenting said target nucleic acids, ligating adaptors to said fragments, and selecting a subset of said adaptor-ligated fragments; and a massive parallel sequencing device.

Formulations and methods for transdermal drug delivery compositions that include anastrozole are disclosed. Transdermal anastrozole compositions of the present disclosure may be indicated for treating testosterone deficiency. Disclosed transdermal anastrozole compositions may include permeation enhancers that may improve penetration of anastrozole in human skin. Permeation enhancers within transdermal anastrozole compositions may include oils from Amazon rainforest such as Pracaxi oil, Plukenetia volubilis seed oil, Inaja oil, and Patauá oil, which includes behenic and oleic fatty acids that may provide penetration power. Transdermal anastrozole may include organic solvents as transdermal penetration enhancers. Additionally, transdermal anastrozole compositions may include physiological lipids, phospholipids, and one or more butters rich in linoleic acid and linolenic acid that may also provide penetration power with restorative benefits to the skin.

1. A pharmaceutical composition, comprising: anastrozole and at least one permeation enhancer. 2. The pharmaceutical composition of claim 1, wherein the anastrozole comprises about 0.01% to about 0.1% by weight of the pharmaceutical composition. 3. The pharmaceutical composition of claim 1, wherein the permeation enhancer comprises about 5% to about 50% by weight of the pharmaceutical composition. 4. The pharmaceutical composition of claim 1, wherein the permeation enhancer comprises about 10% to about 20% by weight of the pharmaceutical composition. 5. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated as an ointment, cream, gel, lotion, solution, or paste. 6. A method of increasing hormone levels comprising: administering to a patient a pharmaceutical composition comprising anastrozole and at least one permeation enhancer. 7. The method according to claim 6, wherein the anastrozole is applied at about 0.1 mg/day to about 1 mg/day. 8. The method according to claim 6, wherein the administration of the pharmaceutical composition is to the skin. 9. The method according to claim 6, wherein the permeation enhancer is selected from the group comprising pracaxi oil, Plukenetia volubilis seed oil, Inaja oil, and Patauá oil, and combinations thereof. 10. The method according to claim 6, wherein the permeation enhancer is selected from the group comprising behenic acid, oleic acid, and combinations thereof. 11. The method according to claim 6, wherein the permeation enhancer further comprises water, one or more skin lipids, one or more butters having linoleic acid or linolenic acid, and one or more phospholipids, and combinations thereof. 12. The method according to claim 6, wherein the permeation enhancer comprises at least one medium chain triglyceride, ethyl alcohol, ethoxy diglycol, dimethyl sulfoxide, glycerin, ispropryl myristate, isoproply palmitate, propylene glycol, and combinations thereof. 13. The method according to claim 12, wherein the at least one medium chain triglyceride comprises at least one from the group consisting of caprylic triglyceride, capric triglyceride, and combinations thereof. 14. The method according to claim 6, wherein the anastrozole comprises about 0.01% to about 0.1% by weight of the pharmaceutical composition. 15. The method according to claim 6, wherein the permeation enhancer comprises about 5% to about 50% by weight of the pharmaceutical composition. 16. The method according to claim 6, wherein the permeation enhancer comprises about 10% to about 20% by weight of the pharmaceutical composition. 17. The method according to claim 6, wherein the pharmaceutical composition is formulated as an ointment, cream, gel, lotion, solution, or paste.

Formulations and methods for transdermal drug delivery compositions that include anastrozole are disclosed. Transdermal anastrozole compositions of the present disclosure may be indicated for treating testosterone deficiency. Disclosed transdermal anastrozole compositions may include permeation enhancers that may improve penetration of anastrozole in human skin. Permeation enhancers within transdermal anastrozole compositions may include oils from Amazon rainforest such as Pracaxi oil, Plukenetia volubilis seed oil, Inaja oil, and Patauá oil, which includes behenic and oleic fatty acids that may provide penetration power. Transdermal anastrozole may include organic solvents as transdermal penetration enhancers. Additionally, transdermal anastrozole compositions may include physiological lipids, phospholipids, and one or more butters rich in linoleic acid and linolenic acid that may also provide penetration power with restorative benefits to the skin.1. A pharmaceutical composition, comprising: anastrozole and at least one permeation enhancer. 2. The pharmaceutical composition of claim 1, wherein the anastrozole comprises about 0.01% to about 0.1% by weight of the pharmaceutical composition. 3. The pharmaceutical composition of claim 1, wherein the permeation enhancer comprises about 5% to about 50% by weight of the pharmaceutical composition. 4. The pharmaceutical composition of claim 1, wherein the permeation enhancer comprises about 10% to about 20% by weight of the pharmaceutical composition. 5. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated as an ointment, cream, gel, lotion, solution, or paste. 6. A method of increasing hormone levels comprising: administering to a patient a pharmaceutical composition comprising anastrozole and at least one permeation enhancer. 7. The method according to claim 6, wherein the anastrozole is applied at about 0.1 mg/day to about 1 mg/day. 8. The method according to claim 6, wherein the administration of the pharmaceutical composition is to the skin. 9. The method according to claim 6, wherein the permeation enhancer is selected from the group comprising pracaxi oil, Plukenetia volubilis seed oil, Inaja oil, and Patauá oil, and combinations thereof. 10. The method according to claim 6, wherein the permeation enhancer is selected from the group comprising behenic acid, oleic acid, and combinations thereof. 11. The method according to claim 6, wherein the permeation enhancer further comprises water, one or more skin lipids, one or more butters having linoleic acid or linolenic acid, and one or more phospholipids, and combinations thereof. 12. The method according to claim 6, wherein the permeation enhancer comprises at least one medium chain triglyceride, ethyl alcohol, ethoxy diglycol, dimethyl sulfoxide, glycerin, ispropryl myristate, isoproply palmitate, propylene glycol, and combinations thereof. 13. The method according to claim 12, wherein the at least one medium chain triglyceride comprises at least one from the group consisting of caprylic triglyceride, capric triglyceride, and combinations thereof. 14. The method according to claim 6, wherein the anastrozole comprises about 0.01% to about 0.1% by weight of the pharmaceutical composition. 15. The method according to claim 6, wherein the permeation enhancer comprises about 5% to about 50% by weight of the pharmaceutical composition. 16. The method according to claim 6, wherein the permeation enhancer comprises about 10% to about 20% by weight of the pharmaceutical composition. 17. The method according to claim 6, wherein the pharmaceutical composition is formulated as an ointment, cream, gel, lotion, solution, or paste.

The present invention generally relates to the field of nutrition and health. In particular, the present invention provides a composition that allows it to treat, limit or prevent muscle atrophy. Embodiments of the present invention are directed at GLP-2 containing compositions and to compositions that stimulate the secretion of GLP-2 in a body to treat or prevent muscle atrophy.

1. A method for treating, limiting and/or preventing muscle atrophy comprising the steps of administering a composition comprising glucagon-like peptide 2 (GLP 2) having a caloric density in the range of 0.8-2.0 kcal/ml with at least 10% of the calories resulting from fat and at least 25% of the calories resulting from carbohydrates to a patient in need of same. 2. Method in accordance with claim 1, wherein the muscle atrophy is skeletal muscle atrophy. 3. Method in accordance of with claim 1 for use in the treatment, limitation and/or prevention of muscle atrophy via supporting the recovery of the intestine. 4. Method in accordance with claim 1, wherein the composition comprises intestinal trophic factors. 5. Composition in accordance with claim 1, wherein the GLP-2 has an amino acid sequence selected from the group consisting of HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ-ID No. 1), HGDGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ-ID No. 2), HADGSFSDEMNTILDNLATRDFINWLIQTKITD (SEQ-ID No. 3), HGDGSFSDEMNTILDNLATRDFINWLIQTKITD (SEQ-ID No. 4), and combinations thereof. 6. Method in accordance with claim 1 for use in the improvement of skeletal muscle recovery. 7. Method in accordance with claim 1 for use in the improvement of small intestine and muscle mass recovery. 8. Method in accordance with claim 1 for use in increasing liver protein mass regain and improving muscle mass recovery. 9. Method in accordance with claim 1, wherein the composition is administered during or after a period of malnutrition. 10. Method in accordance with claim 1, wherein the composition is selected from the group consisting of a food product, a nutraceutical, a drink, a food additive and a medicament. 11. Method in accordance with claim 1, wherein the composition is a nutritional formula. 12. Method in accordance with claim 1, wherein the composition is administered through a route selected from the group consisting of oral, enteral and parenteral administration. 13. Method in accordance with claim 1, wherein the composition comprises a carbohydrate source in an amount of 7-19 g/100 kcal of the composition. 14. Method in accordance with claim 1, wherein the composition comprises a lipid source in an amount of 1.5-5.0 g/100 kcal of the composition. 15. Method in accordance with claim 1, wherein the composition comprises a protein source in an amount of 2.0-8.5 g/100 kcal of the composition. 16. Method for use in treating, limiting and/or preventing muscle atrophy comprising administering a composition comprising a formulation that stimulates the secretion of GLP-2 in the body, having a caloric density in the range of 0.8-2.0 kcal/ml with at least 10% of the calories resulting from fat and/of at least 25% of the calories resulting from carbohydrates to a patient in need of same. 17. Method in accordance with claim 16, wherein the muscle atrophy is skeletal muscle atrophy. 18. Method in accordance of with claim 16 for use in the treatment, limitation and/or prevention of muscle atrophy via supporting the recovery of the intestine. 19. Method in accordance with claim 16, wherein the composition comprises intestinal trophic factors. 20. Composition in accordance with claim 16, wherein the GLP-2 has an amino acid sequence selected from the group consisting of HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ-ID No. 1), HGDGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ-ID No. 2), HADGSFSDEMNTILDNLATRDFINWLIQTKITD (SEQ-ID No. 3), HGDGSFSDEMNTILDNLATRDFINWLIQTKITD (SEQ-ID No. 4), and combinations thereof. 21. Method in accordance with claim 16 for use in the improvement of skeletal muscle recovery. 22. Method in accordance with claim 16 for use in the improvement of small intestine and muscle mass recovery. 23. Method in accordance with claim 16 for use in increasing liver protein mass regain and improving muscle mass recovery. 24. Method in accordance with claim 16, wherein the composition is administered during or after a period of malnutrition. 25. Method in accordance with claim 16, wherein the composition is selected from the group consisting of a food product, a nutraceutical, a drink, a food additive and a medicament. 26. Method in accordance with claim 16, wherein the composition is a nutritional formula. 27. Method in accordance with claim 16, wherein the composition is administered through a route selected from the group consisting of oral, enteral and parenteral administration. 28. Method in accordance with claim 16, wherein the composition comprises a carbohydrate source in an amount of 7-19 g/100 kcal of the composition. 29. Method in accordance with claim 16, wherein the composition comprises a lipid source in an amount of 1.5-5.0 g/100 kcal of the composition. 30. Method in accordance with claim 16, wherein the composition comprises a protein source in an amount of 2.0-8.5 g/100 kcal of the composition.

The present invention generally relates to the field of nutrition and health. In particular, the present invention provides a composition that allows it to treat, limit or prevent muscle atrophy. Embodiments of the present invention are directed at GLP-2 containing compositions and to compositions that stimulate the secretion of GLP-2 in a body to treat or prevent muscle atrophy.1. A method for treating, limiting and/or preventing muscle atrophy comprising the steps of administering a composition comprising glucagon-like peptide 2 (GLP 2) having a caloric density in the range of 0.8-2.0 kcal/ml with at least 10% of the calories resulting from fat and at least 25% of the calories resulting from carbohydrates to a patient in need of same. 2. Method in accordance with claim 1, wherein the muscle atrophy is skeletal muscle atrophy. 3. Method in accordance of with claim 1 for use in the treatment, limitation and/or prevention of muscle atrophy via supporting the recovery of the intestine. 4. Method in accordance with claim 1, wherein the composition comprises intestinal trophic factors. 5. Composition in accordance with claim 1, wherein the GLP-2 has an amino acid sequence selected from the group consisting of HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ-ID No. 1), HGDGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ-ID No. 2), HADGSFSDEMNTILDNLATRDFINWLIQTKITD (SEQ-ID No. 3), HGDGSFSDEMNTILDNLATRDFINWLIQTKITD (SEQ-ID No. 4), and combinations thereof. 6. Method in accordance with claim 1 for use in the improvement of skeletal muscle recovery. 7. Method in accordance with claim 1 for use in the improvement of small intestine and muscle mass recovery. 8. Method in accordance with claim 1 for use in increasing liver protein mass regain and improving muscle mass recovery. 9. Method in accordance with claim 1, wherein the composition is administered during or after a period of malnutrition. 10. Method in accordance with claim 1, wherein the composition is selected from the group consisting of a food product, a nutraceutical, a drink, a food additive and a medicament. 11. Method in accordance with claim 1, wherein the composition is a nutritional formula. 12. Method in accordance with claim 1, wherein the composition is administered through a route selected from the group consisting of oral, enteral and parenteral administration. 13. Method in accordance with claim 1, wherein the composition comprises a carbohydrate source in an amount of 7-19 g/100 kcal of the composition. 14. Method in accordance with claim 1, wherein the composition comprises a lipid source in an amount of 1.5-5.0 g/100 kcal of the composition. 15. Method in accordance with claim 1, wherein the composition comprises a protein source in an amount of 2.0-8.5 g/100 kcal of the composition. 16. Method for use in treating, limiting and/or preventing muscle atrophy comprising administering a composition comprising a formulation that stimulates the secretion of GLP-2 in the body, having a caloric density in the range of 0.8-2.0 kcal/ml with at least 10% of the calories resulting from fat and/of at least 25% of the calories resulting from carbohydrates to a patient in need of same. 17. Method in accordance with claim 16, wherein the muscle atrophy is skeletal muscle atrophy. 18. Method in accordance of with claim 16 for use in the treatment, limitation and/or prevention of muscle atrophy via supporting the recovery of the intestine. 19. Method in accordance with claim 16, wherein the composition comprises intestinal trophic factors. 20. Composition in accordance with claim 16, wherein the GLP-2 has an amino acid sequence selected from the group consisting of HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ-ID No. 1), HGDGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ-ID No. 2), HADGSFSDEMNTILDNLATRDFINWLIQTKITD (SEQ-ID No. 3), HGDGSFSDEMNTILDNLATRDFINWLIQTKITD (SEQ-ID No. 4), and combinations thereof. 21. Method in accordance with claim 16 for use in the improvement of skeletal muscle recovery. 22. Method in accordance with claim 16 for use in the improvement of small intestine and muscle mass recovery. 23. Method in accordance with claim 16 for use in increasing liver protein mass regain and improving muscle mass recovery. 24. Method in accordance with claim 16, wherein the composition is administered during or after a period of malnutrition. 25. Method in accordance with claim 16, wherein the composition is selected from the group consisting of a food product, a nutraceutical, a drink, a food additive and a medicament. 26. Method in accordance with claim 16, wherein the composition is a nutritional formula. 27. Method in accordance with claim 16, wherein the composition is administered through a route selected from the group consisting of oral, enteral and parenteral administration. 28. Method in accordance with claim 16, wherein the composition comprises a carbohydrate source in an amount of 7-19 g/100 kcal of the composition. 29. Method in accordance with claim 16, wherein the composition comprises a lipid source in an amount of 1.5-5.0 g/100 kcal of the composition. 30. Method in accordance with claim 16, wherein the composition comprises a protein source in an amount of 2.0-8.5 g/100 kcal of the composition.

The present invention generally relates to nitric oxide releasing pharmaceutical compositions and methods of using the same.

1. A pharmaceutical composition for topical delivery of a moisture activated active pharmaceutical ingredient, the composition comprising: (a) a hydrophobic base; and (b) an amphiphilic compound. 2. The pharmaceutical composition of claim 1, wherein the hydrophobic base is present in the composition at a concentration from about 35% to about 90%; and the amphiphilic compound is present in the composition at a concentration from about 1% to about 30%. 3. The pharmaceutical composition of claim 1, wherein the amphiphilic compound has a hydrophilic-lipophilic balance (HLB) value of 12 to 20. 4. The pharmaceutical composition of claim 1, wherein the amphiphilic compound comprises a fatty acid ester. 5-6. (canceled) 7. The pharmaceutical composition of claim 1, wherein the amphiphilic compound absorbs moisture and does not substantially absorb vaporous moisture. 8. The pharmaceutical composition of claim 1, wherein the hydrophobic base comprises at least one of mineral oil and a hydrophobic polymer. 9. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a cosolvent and the cosolvent is present in the composition at a concentration from about 1% to about 30%. 10. The pharmaceutical composition of claim 9, wherein the cosolvent comprises a fatty acid ester. 11. (canceled) 12. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a humectant and the humectant is present in the composition at a concentration from about 1% to about 25%. 13. The pharmaceutical composition of claim 12, wherein the humectant comprises a polyhydric alcohol. 14. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a moisture activated active pharmaceutical ingredient and the moisture activated active pharmaceutical ingredient is present in the composition at a concentration from about 0.1% to about 70%. 15. The pharmaceutical composition of claim 14, wherein the moisture activated active pharmaceutical ingredient comprises a nitric oxide releasing compound. 16. The pharmaceutical composition of claim 15, wherein the nitric oxide releasing compound comprises a diazeniumdiolate functional group. 17. The pharmaceutical composition of claim 15, wherein the nitric oxide releasing compound comprises a NO-releasing co-condensed silica particle. 18. The pharmaceutical composition of claim 17, wherein the co-condensed silica particle has a mean particle size of less than about 10 μm. 19. A pharmaceutical composition for topical delivery of a moisture activated active pharmaceutical ingredient, the composition comprising: a moisture activated active pharmaceutical ingredient present in the composition at a concentration from about 0.1% to about 35%; a hydrophobic polymer present in the composition at a concentration from about 30% to about 60%; a mineral oil present in the composition at a concentration from about 1% to about 30%; an amphiphilic compound present in the composition at a concentration from about 1% to about 20%; a cosolvent present in the composition at a concentration from about 1% to about 25%; and a humectant present in the composition at a concentration from about 1% to about 25%. 20. The pharmaceutical composition of claim 1, wherein the composition comprises an ointment. 21. The pharmaceutical composition of claim 1, wherein water is present in the composition at a concentration of less than about 2%. 22. A method of treating the skin of a subject, the method comprising topically administering the pharmaceutical composition of claim 1 in an amount effective to treat the skin of a subject. 23. The method of claim 22, further comprising contacting water to the pharmaceutical composition before, after, and/or during the step of topically administering the composition.

The present invention generally relates to nitric oxide releasing pharmaceutical compositions and methods of using the same.1. A pharmaceutical composition for topical delivery of a moisture activated active pharmaceutical ingredient, the composition comprising: (a) a hydrophobic base; and (b) an amphiphilic compound. 2. The pharmaceutical composition of claim 1, wherein the hydrophobic base is present in the composition at a concentration from about 35% to about 90%; and the amphiphilic compound is present in the composition at a concentration from about 1% to about 30%. 3. The pharmaceutical composition of claim 1, wherein the amphiphilic compound has a hydrophilic-lipophilic balance (HLB) value of 12 to 20. 4. The pharmaceutical composition of claim 1, wherein the amphiphilic compound comprises a fatty acid ester. 5-6. (canceled) 7. The pharmaceutical composition of claim 1, wherein the amphiphilic compound absorbs moisture and does not substantially absorb vaporous moisture. 8. The pharmaceutical composition of claim 1, wherein the hydrophobic base comprises at least one of mineral oil and a hydrophobic polymer. 9. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a cosolvent and the cosolvent is present in the composition at a concentration from about 1% to about 30%. 10. The pharmaceutical composition of claim 9, wherein the cosolvent comprises a fatty acid ester. 11. (canceled) 12. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a humectant and the humectant is present in the composition at a concentration from about 1% to about 25%. 13. The pharmaceutical composition of claim 12, wherein the humectant comprises a polyhydric alcohol. 14. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a moisture activated active pharmaceutical ingredient and the moisture activated active pharmaceutical ingredient is present in the composition at a concentration from about 0.1% to about 70%. 15. The pharmaceutical composition of claim 14, wherein the moisture activated active pharmaceutical ingredient comprises a nitric oxide releasing compound. 16. The pharmaceutical composition of claim 15, wherein the nitric oxide releasing compound comprises a diazeniumdiolate functional group. 17. The pharmaceutical composition of claim 15, wherein the nitric oxide releasing compound comprises a NO-releasing co-condensed silica particle. 18. The pharmaceutical composition of claim 17, wherein the co-condensed silica particle has a mean particle size of less than about 10 μm. 19. A pharmaceutical composition for topical delivery of a moisture activated active pharmaceutical ingredient, the composition comprising: a moisture activated active pharmaceutical ingredient present in the composition at a concentration from about 0.1% to about 35%; a hydrophobic polymer present in the composition at a concentration from about 30% to about 60%; a mineral oil present in the composition at a concentration from about 1% to about 30%; an amphiphilic compound present in the composition at a concentration from about 1% to about 20%; a cosolvent present in the composition at a concentration from about 1% to about 25%; and a humectant present in the composition at a concentration from about 1% to about 25%. 20. The pharmaceutical composition of claim 1, wherein the composition comprises an ointment. 21. The pharmaceutical composition of claim 1, wherein water is present in the composition at a concentration of less than about 2%. 22. A method of treating the skin of a subject, the method comprising topically administering the pharmaceutical composition of claim 1 in an amount effective to treat the skin of a subject. 23. The method of claim 22, further comprising contacting water to the pharmaceutical composition before, after, and/or during the step of topically administering the composition.

The present invention relates to a transmucosal administration system to administer quinones, benzoquinones, and especially 1,4-benzoquinones, via the oromucosal route.

1. A transmuccosal administration system for a pharmaceutical active ingredient comprising 0.01-80% by weight of an active ingredient of the structural formula (I) wherein R1 is a C1-4 lower alkyl group; R2 is a hydrogen atom or an optional substituted alkyl or an optional substituted alkenyl group; R3 and R4 each represents an optional substituted C1-6 lower alkyl or C1-3 lower alkoxy group or, R3 and R4 taken together a butadienylene group and 20-99.99% by weight of a carrier material. 2. The administration system as claimed in claim 1, wherein R1 is a C1-4 lower alkyl group, R2 is linear or branched, saturated or unsaturated, aliphatic hydrocarbon or hydroxyalkyl chain from 5 to 50 carbon atoms and R3 and R4 is a C1-4 alkyl group or a C1-3 alkoxy group. 3. The administration system as claimed in claim 1, wherein R1 is a C1-4 alkyl; R2 is (a) hydrogen, (b) a C1-22 alkyl which may be substituted by 1 to 10 substituents selected from the group consisting of (i) C1-4 alkyl, (ii) hydroxy, (iii) oxo, (iv) amino, (v) mono-C1-6 alkylamino, (vi) di-C1-6 alkylamino, (vii) carboxy, (viii) C1-4 alkoxy-carbonyl, (ix) C6-14 aryl which may be substituted by 1 or 2 substituents selected from the group consisting of C1-4, alkyl, hydroxy, carboxy and C1-6 alkoxy-carbonyl, (x) 5 or 6-membered heterocyclic group which may be substituted by 1 or 2 substituents selected from the group consisting of C1-4 alkyl, hydroxy, carboxy and C1-6 alkoxy-carbonyl, and (xi) halogen, or (c) a C2-15 alkenyl which may be substituted by 1 to 10 substituents selected from the group consisting of (i) C1-4 alkyl, (ii) hydroxy, (iii) oxo, (iv) amino, (v) mono-C1-6 alkylamino, (vi) di-C1-6 alkylamino, (vii) carboxy, (viii) C1-4 alkoxy-carbonyl, (ix) C6-14 aryl which may be substituted by 1 or 2 substituents selected from the group consisting of C1-4 alkyl, hydroxy, carboxy and C1-6 alkoxy-carbonyl, (x) 5- or 6 membered heterocyclic group which may be substituted by 1 or 2 substituents selected from the group consisting of C1-4 alkyl, hydroxy, carboxy and C1-6 alkoxy-carbonyl, and (xi) halogen; R3 and R4 each is a C1-6 alkyl which may be substituted by 1 to 3 substituents selected from the group consisting of hydroxy, halogen, nitro. C1-3 alkyl which may be halogenated, carboxy, C1-6 alkoxy-carbonyl, 3-pyridyl, 1-imidazolyl and 5-thiazolyl or a C1-3 alkoxy; or R3 and R4 form, taken together with the respective adjacent carbon atoms, a benzene ring which may be substituted by 1 to 3 substituents selected from the group consisting of C1-3 alkyl, C1-3 alkoxy, hydroxy, nitro and halogen. 4. The administration system as claimed in claim 1, wherein R1 is a C1-3 alkyl, R2 is a C6-14 alky optionally substituted by hydroxy and R3 and R4 each is C1-3 alkoxy. 5. The administration system as claimed in claim 1, wherein said active ingredient is selected from the group consisting of idebenone, idebenone analouges, decylubiquinone, ubiquinone and ubiquinone analouges. 6. The administration system as claimed in claim 1, wherein the carrier material is selected from the group consisting of cellulose, cellulose derivatives, polyvinyl-alcohol, poly-N-vinylpyrrolidones, vinylpyrrolidone-vinyl acetate copolymers, starch, starch derivatives, gelatine, gelatine derivatives, Soluplus. Kollicoat, and combinations thereof. 7. The administration system as claimed in claim 6, wherein the cellulose derivate is methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose or combinations thereof. 8. The administration system as claimed in claim 1, wherein said system comprises 50% by weight of a 1,4-benzoquinone as active ingredient selected from the group consisting of idebenone, ubiquinone or ubiquinone analouges and 40% by weight polyvinyl alcohol and 10% by weight of sodium carboxymethyl-cellulose as carrier material. 9. The administration system as claimed in claim 1, wherein said system comprises 10% by weight of a 1,4-benzoquinone as active ingredient selected from the group consisting of idebenone, ubiquinone or ubiquinone analogues and 90% by weight of hydroxypropylmethylcellulose as carrier material. 10. The administration system as claimed in claim 1, wherein the system is a mucoadhesive film which dissolves in the mouth. 11. The administration system as claimed in claim 10, wherein the film dissolves completely in a period of less than 30 minutes in the mouth. 12. The administration system as claimed in claim 10, wherein the film dissolves completely in a period of less than 15 minutes in the mouth. 13. The administration system as claimed in claim 1, wherein a maximum concentration of the active ingredient in the blood is reached in a period of less than 240 minutes after application. 14. The administration system as claimed in claim 13, wherein a maximum concentration of the active ingredient in the blood is reached in a period of less than 60 minutes after application. 15. The administration system as claimed in claim 13, wherein a maximum concentration of the active ingredient in the blood is reached in a period of between 5 and 30 min after application. 16. The administration system as claimed in claim 1, wherein the system additionally comprises at least one excipient from the group of flavorings, colorants, sweeteners, fillers, plasticizers, surface-active substances, solubilizers, liquid excipient, pH stabilizers, disintegrants, solubility enhancers and absorption enhancers. 17. The administration system as claimed in claim 1, wherein the active ingredient is present dissolved in the carrier material and/or a liquid excipient. 18. The administration system as claimed in claim 17, wherein the liquid excipient is a liquid lipophilic excipient. 19. The administration system as claimed in claim 1, wherein the system has an area of between 1 and 10 cm2. 20. The administration system as claimed in claim 1, wherein the system has an area of between 2 and 8 cm2. 21. The administration system as claimed in claim 1, wherein the system has an area of between 5 and 7 cm2. 22. The administration system as claimed in claim 1, wherein system has a weight per unit area of between 50 and 250 g/m2. 23. The administration system as claimed in claim 1, wherein system has a weight per unit area of between 100 and 150 g/m2. 24. The administration system as claimed in claim 1, wherein the system has a thickness of between 40 and 300 μm. 25. The administration system as claimed in claim 1, wherein the system has a thickness of between 50 and 100 μm. 26. The administration system as claimed in claim 1, wherein the system contains a suspension comprising 30-60% by weight of the active ingredient and 40-70% by weight of a polyalcohol and/or a cellulose derivate as carrier material. 27. The administration system as claimed in claim 1, wherein the system contains a suspension comprising 40-50% by weight of the active ingredient and 40-70% by weight of a polyalcohol and/or a cellulose derivate as carrier material. 28. The administration system as claimed in claim 1, wherein the active ingredient is micronized. 29. The administration system as claimed in claim 1, wherein the system contains an amorphous presentation comprising 3-20% by weight of the active ingredient and 80-97% by weight of a water soluble polymer as carrier material. 30. The administration system as claimed in claim 29, wherein the system contains an amorphous presentation comprising 5-10% by weight of the active ingredient and 80-97% by weight of a water soluble polymer as carrier material. 31. The administration as claimed in claim 29, wherein the carrier material is a substituted carbohydrate. 32. The administration system as claimed in claim 1, wherein the system contains an emulsion comprising 3-50% by weight of the active ingredient and 50-97% by weight of a cellulose derivate as carrier material. 33. The administration system as claimed in claim 1, wherein the system contains an emulsion comprising 5-30% by weight of the active ingredient and 50-97% by weight of a cellulose derivate as carrier material. 34. The administration system as claimed in claim 1, wherein the system has a monolayer or double-layer construction. 35. A treatment method comprising administering to a patient suffering from a disease and/or condition a dose of the system as claimed in claim 1 in an amount of equal to or less than 60 mg/kg/day. 36. The treatment method as claimed in claim 35, wherein the disease is disease caused by mitochondrial dysfunction, neurological diseases and/or neuromuscular diseases. 37. The treatment method as claimed in claim 35, wherein the disease and/or condition is Friedreich's Ataxia (FRDA), Leber's Hereditary Optic Neuropathy (LHON), mitochondrial myopathy, encephalopathy, lactic acidosis with stroke-like episodes (MELAS) and/or mitochondrial myopathies. 38. The treatment method as claimed in claim 35, wherein the disease and/or condition is Duchenne Muscular Dystrophy and/or Becker Muscular Dystrophy. 39. The treatment method as claimed in claim 35, wherein the disease and/or condition is Alzheimer's disease and/or Parkinson's disease. 40. The treatment method as claimed in claim 35, wherein the pharmaceutical active ingredient is idebenone and the idebenone is administered in a dosage from 0.01 mg/kg/day to 10 mg/kg/day. 41. The treatment method as claimed in claim 35, wherein the pharmaceutical active ingredient is idebenone and the system further comprises a polymer matrix wherein the idebenone is incorporated within the matrix as a suspension, a suspension after micronization, an emulsion, a micro- or nano-emulsion, or in solubilised and/or molecularly dispersed form. 42. The treatment method as claimed in claim 35, wherein the administering step comprising admininstering a dosage via the oral mucosa on or under the tongue in the buccal cavity or any other location in the oral cavity. 43. The treatment method as claimed in claim 35, wherein said method further comprises administering a second therapeutic agent.

The present invention relates to a transmucosal administration system to administer quinones, benzoquinones, and especially 1,4-benzoquinones, via the oromucosal route.1. A transmuccosal administration system for a pharmaceutical active ingredient comprising 0.01-80% by weight of an active ingredient of the structural formula (I) wherein R1 is a C1-4 lower alkyl group; R2 is a hydrogen atom or an optional substituted alkyl or an optional substituted alkenyl group; R3 and R4 each represents an optional substituted C1-6 lower alkyl or C1-3 lower alkoxy group or, R3 and R4 taken together a butadienylene group and 20-99.99% by weight of a carrier material. 2. The administration system as claimed in claim 1, wherein R1 is a C1-4 lower alkyl group, R2 is linear or branched, saturated or unsaturated, aliphatic hydrocarbon or hydroxyalkyl chain from 5 to 50 carbon atoms and R3 and R4 is a C1-4 alkyl group or a C1-3 alkoxy group. 3. The administration system as claimed in claim 1, wherein R1 is a C1-4 alkyl; R2 is (a) hydrogen, (b) a C1-22 alkyl which may be substituted by 1 to 10 substituents selected from the group consisting of (i) C1-4 alkyl, (ii) hydroxy, (iii) oxo, (iv) amino, (v) mono-C1-6 alkylamino, (vi) di-C1-6 alkylamino, (vii) carboxy, (viii) C1-4 alkoxy-carbonyl, (ix) C6-14 aryl which may be substituted by 1 or 2 substituents selected from the group consisting of C1-4, alkyl, hydroxy, carboxy and C1-6 alkoxy-carbonyl, (x) 5 or 6-membered heterocyclic group which may be substituted by 1 or 2 substituents selected from the group consisting of C1-4 alkyl, hydroxy, carboxy and C1-6 alkoxy-carbonyl, and (xi) halogen, or (c) a C2-15 alkenyl which may be substituted by 1 to 10 substituents selected from the group consisting of (i) C1-4 alkyl, (ii) hydroxy, (iii) oxo, (iv) amino, (v) mono-C1-6 alkylamino, (vi) di-C1-6 alkylamino, (vii) carboxy, (viii) C1-4 alkoxy-carbonyl, (ix) C6-14 aryl which may be substituted by 1 or 2 substituents selected from the group consisting of C1-4 alkyl, hydroxy, carboxy and C1-6 alkoxy-carbonyl, (x) 5- or 6 membered heterocyclic group which may be substituted by 1 or 2 substituents selected from the group consisting of C1-4 alkyl, hydroxy, carboxy and C1-6 alkoxy-carbonyl, and (xi) halogen; R3 and R4 each is a C1-6 alkyl which may be substituted by 1 to 3 substituents selected from the group consisting of hydroxy, halogen, nitro. C1-3 alkyl which may be halogenated, carboxy, C1-6 alkoxy-carbonyl, 3-pyridyl, 1-imidazolyl and 5-thiazolyl or a C1-3 alkoxy; or R3 and R4 form, taken together with the respective adjacent carbon atoms, a benzene ring which may be substituted by 1 to 3 substituents selected from the group consisting of C1-3 alkyl, C1-3 alkoxy, hydroxy, nitro and halogen. 4. The administration system as claimed in claim 1, wherein R1 is a C1-3 alkyl, R2 is a C6-14 alky optionally substituted by hydroxy and R3 and R4 each is C1-3 alkoxy. 5. The administration system as claimed in claim 1, wherein said active ingredient is selected from the group consisting of idebenone, idebenone analouges, decylubiquinone, ubiquinone and ubiquinone analouges. 6. The administration system as claimed in claim 1, wherein the carrier material is selected from the group consisting of cellulose, cellulose derivatives, polyvinyl-alcohol, poly-N-vinylpyrrolidones, vinylpyrrolidone-vinyl acetate copolymers, starch, starch derivatives, gelatine, gelatine derivatives, Soluplus. Kollicoat, and combinations thereof. 7. The administration system as claimed in claim 6, wherein the cellulose derivate is methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose or combinations thereof. 8. The administration system as claimed in claim 1, wherein said system comprises 50% by weight of a 1,4-benzoquinone as active ingredient selected from the group consisting of idebenone, ubiquinone or ubiquinone analouges and 40% by weight polyvinyl alcohol and 10% by weight of sodium carboxymethyl-cellulose as carrier material. 9. The administration system as claimed in claim 1, wherein said system comprises 10% by weight of a 1,4-benzoquinone as active ingredient selected from the group consisting of idebenone, ubiquinone or ubiquinone analogues and 90% by weight of hydroxypropylmethylcellulose as carrier material. 10. The administration system as claimed in claim 1, wherein the system is a mucoadhesive film which dissolves in the mouth. 11. The administration system as claimed in claim 10, wherein the film dissolves completely in a period of less than 30 minutes in the mouth. 12. The administration system as claimed in claim 10, wherein the film dissolves completely in a period of less than 15 minutes in the mouth. 13. The administration system as claimed in claim 1, wherein a maximum concentration of the active ingredient in the blood is reached in a period of less than 240 minutes after application. 14. The administration system as claimed in claim 13, wherein a maximum concentration of the active ingredient in the blood is reached in a period of less than 60 minutes after application. 15. The administration system as claimed in claim 13, wherein a maximum concentration of the active ingredient in the blood is reached in a period of between 5 and 30 min after application. 16. The administration system as claimed in claim 1, wherein the system additionally comprises at least one excipient from the group of flavorings, colorants, sweeteners, fillers, plasticizers, surface-active substances, solubilizers, liquid excipient, pH stabilizers, disintegrants, solubility enhancers and absorption enhancers. 17. The administration system as claimed in claim 1, wherein the active ingredient is present dissolved in the carrier material and/or a liquid excipient. 18. The administration system as claimed in claim 17, wherein the liquid excipient is a liquid lipophilic excipient. 19. The administration system as claimed in claim 1, wherein the system has an area of between 1 and 10 cm2. 20. The administration system as claimed in claim 1, wherein the system has an area of between 2 and 8 cm2. 21. The administration system as claimed in claim 1, wherein the system has an area of between 5 and 7 cm2. 22. The administration system as claimed in claim 1, wherein system has a weight per unit area of between 50 and 250 g/m2. 23. The administration system as claimed in claim 1, wherein system has a weight per unit area of between 100 and 150 g/m2. 24. The administration system as claimed in claim 1, wherein the system has a thickness of between 40 and 300 μm. 25. The administration system as claimed in claim 1, wherein the system has a thickness of between 50 and 100 μm. 26. The administration system as claimed in claim 1, wherein the system contains a suspension comprising 30-60% by weight of the active ingredient and 40-70% by weight of a polyalcohol and/or a cellulose derivate as carrier material. 27. The administration system as claimed in claim 1, wherein the system contains a suspension comprising 40-50% by weight of the active ingredient and 40-70% by weight of a polyalcohol and/or a cellulose derivate as carrier material. 28. The administration system as claimed in claim 1, wherein the active ingredient is micronized. 29. The administration system as claimed in claim 1, wherein the system contains an amorphous presentation comprising 3-20% by weight of the active ingredient and 80-97% by weight of a water soluble polymer as carrier material. 30. The administration system as claimed in claim 29, wherein the system contains an amorphous presentation comprising 5-10% by weight of the active ingredient and 80-97% by weight of a water soluble polymer as carrier material. 31. The administration as claimed in claim 29, wherein the carrier material is a substituted carbohydrate. 32. The administration system as claimed in claim 1, wherein the system contains an emulsion comprising 3-50% by weight of the active ingredient and 50-97% by weight of a cellulose derivate as carrier material. 33. The administration system as claimed in claim 1, wherein the system contains an emulsion comprising 5-30% by weight of the active ingredient and 50-97% by weight of a cellulose derivate as carrier material. 34. The administration system as claimed in claim 1, wherein the system has a monolayer or double-layer construction. 35. A treatment method comprising administering to a patient suffering from a disease and/or condition a dose of the system as claimed in claim 1 in an amount of equal to or less than 60 mg/kg/day. 36. The treatment method as claimed in claim 35, wherein the disease is disease caused by mitochondrial dysfunction, neurological diseases and/or neuromuscular diseases. 37. The treatment method as claimed in claim 35, wherein the disease and/or condition is Friedreich's Ataxia (FRDA), Leber's Hereditary Optic Neuropathy (LHON), mitochondrial myopathy, encephalopathy, lactic acidosis with stroke-like episodes (MELAS) and/or mitochondrial myopathies. 38. The treatment method as claimed in claim 35, wherein the disease and/or condition is Duchenne Muscular Dystrophy and/or Becker Muscular Dystrophy. 39. The treatment method as claimed in claim 35, wherein the disease and/or condition is Alzheimer's disease and/or Parkinson's disease. 40. The treatment method as claimed in claim 35, wherein the pharmaceutical active ingredient is idebenone and the idebenone is administered in a dosage from 0.01 mg/kg/day to 10 mg/kg/day. 41. The treatment method as claimed in claim 35, wherein the pharmaceutical active ingredient is idebenone and the system further comprises a polymer matrix wherein the idebenone is incorporated within the matrix as a suspension, a suspension after micronization, an emulsion, a micro- or nano-emulsion, or in solubilised and/or molecularly dispersed form. 42. The treatment method as claimed in claim 35, wherein the administering step comprising admininstering a dosage via the oral mucosa on or under the tongue in the buccal cavity or any other location in the oral cavity. 43. The treatment method as claimed in claim 35, wherein said method further comprises administering a second therapeutic agent.

The present invention relates to an enriched or purified population of dopaminergic neuronal progenitor cells and an enriched or purified population of dopaminergic neurons. These enriched or purified populations are derived from a population of embryonic stem cells by inducing production of dopaminergic neuronal progenitor cells. A promoter or enhancer which functions only in dopaminergic neuronal progenitor cells is selected and a nucleic acid molecule encoding a marker protein under control of said promoter or enhancer is introduced into the induced population of embryonic stem cells. The dopaminergic neuronal progenitor cells are allowed to express the marker protein, and the cells expressing the marker protein are separated from the induced population of embryonic stem cells. As a result, an enriched or purified population of dopaminergic neuronal progenitor cells is isolated. Alternatively, the nucleic acid molecule encoding the marker protein under control of the promoter or enhancer is introduced into the population of human embryonic stem cells followed by induction of the population of embryonic stem cells.

1. A method of isolating an enriched or purified population of dopaminergic neuronal progenitor cells from a population of embryonic stem cells comprising: providing a population of embryonic stem cells; inducing production of dopaminergic neuronal progenitor cells from the population of embryonic stem cells; selecting a promoter or enhancer which functions only in dopaminergic neuronal progenitor cells; introducing a nucleic acid molecule encoding a marker protein under control of said promoter or enhancer into the induced population of embryonic stem cells; allowing the dopaminergic neuronal progenitor cells to express the marker protein; and separating the cells expressing the marker protein from the induced population of embryonic stem cells, whereby an enriched or purified population of dopaminergic neuronal progenitor cells is isolated. 2. The method of claim 1, wherein said introducing comprises viral mediated transduction of the induced population of embryonic stem cells. 3. The method of claim 2, wherein said viral mediated transduction comprises adenovirus-mediated transduction, retrovirus-mediated transduction, lentivirus-mediated transduction, or adeno-associated virus-mediated transduction. 4. The method of claim 1, wherein said introducing comprises electroporation. 5. The method of claim 1, wherein said introducing comprises biolistic transformation. 6. The method of claim 1, wherein said introducing comprises liposomal mediated transformation. 7. The method of claim 1, wherein the marker protein is a fluorescent protein and said separating comprises fluorescence activated cell sorting. 8. The method of claim 1, wherein the marker protein is either lacZ/beta-galactosidase or alkaline phosphatase. 9. The method of claim 1, wherein said promoter or enhancer is for neurogenin-2. 10. The method of claim 1, wherein said promoter or enhancer is a promoter or enhancer for genes in the dopamine synthesis pathway, a promoter or enhancer for dopamine transport proteins, or a promoter or enhancer for genes expressed differentially in the ventral midbrain. 11. The method of claim 1, wherein the population of embryonic stem cells is in a cell culture. 12. The method of claim 11, wherein the cell culture further comprises astrocytes. 13. The method of claim 12, wherein the astrocytes are human mid-brain astrocytes. 14. The method of claim 12, wherein the astrocytes are immortalized. 15. The method of claim 1, wherein the embryonic stem cells are of human origin. 16. The method of claim 1 further comprising: transplanting the separated cells into a subject. 17. The method of claim 16, wherein the subject has a dopamine-depletion disease. 18. The method of claim 17, dopamine-depletion disease is Parkinson's Disease. 19. The method of claim 1, wherein said inducing is carried out by administering an inducer selected from the group consisting of FGF2, FGF8, FGF20, SHH, and mixtures thereof. 20. The method of claim 1 further comprising: differentiating the enriched or purified population of dopaminergic neuronal progenitor cells into dopamineric neurons. 21. An enriched or purified population of dopamineric neurons produced by the method of claim 20. 22. The enriched or purified population of dopaminergic neuron according to claim 21, wherein the dopaminergic neurons are of human origin. 23. An enriched or purified population of dopaminergic neuronal progenitor cells produced by the method of claim 1. 24. The enriched or purified population of dopaminergic neuronal progenitor cells according to claim 23, wherein the dopaminergic neuronal progenitor cells are of human origin. 25. A method of producing an enriched or purified population of dopaminergic neuronal progenitor cells from a population of embryonic stem cells comprising: selecting a promoter or enhancer which functions only in said dopaminergic neuronal progenitor cells; introducing a nucleic acid molecule encoding a marker protein under control of said promoter or enhancer into the population of human embryonic stem cells; inducing the population of embryonic stem cells to produce a mixed population of cells comprising dopaminergic neuronal progenitor cells; allowing the dopaminergic neuronal progenitor cells to express the marker protein; and separating the cells expressing the marker protein from the mixed population of cells, whereby an enriched or purified population of dopaminergic neuronal progenitor cells is isolated. 26. The method of claim 25, wherein said introducing comprises viral mediated transduction of the population of embryonic stem cells. 27. The method of claim 26, wherein said viral mediated transduction comprises adenovirus-mediated transduction, retrovirus-mediated transduction, lentivirus-mediated transduction, or adeno-associated virus-mediated transduction. 28. The method of claim 25, wherein said introducing comprises electroporation. 29. The method of claim 25, wherein said introducing comprises biolistic transformation. 30. The method of claim 25, wherein said introducing comprises liposomal mediated transformation. 31. The method of claim 25, wherein the marker protein is a fluorescent protein and said separating comprises fluorescence activated cell sorting. 32. The method of claim 25, wherein the marker protein is either lacZ/beta-galactosidase or alkaline phosphatase. 33. The method of claim 25, wherein said promoter or enhancer is for neurogenin-2. 34. The method of claim 25, wherein said promoter or enhancer is a promoter or enhancer for genes in the dopamine synthesis pathway, a promoter or enhancer for dopamine transport proteins, or a promoter or enhancer for genes expressed differentially in the ventral midbrain. 35. The method of claim 25, wherein the population of human embryonic stem cells is in a cell culture. 36. The method of claim 35, wherein the cell culture further comprises astrocytes. 37. The method of claim 36, wherein the astrocytes are human mid-brain astrocytes 38. The method of claim 36, wherein the astrocytes are immortalized. 39. The method of claim 25, wherein the embryonic stem cells are of human origin. 40. The method of claim 25 further comprising: transplanting the separated cells into a subject. 41. The method of claim 40, wherein the subject has a dopamine-depletion disease. 42. The method of claim 41, wherein the dopamine-depletion disease is Parkinson's Disease. 43. The method of claim 25, wherein said inducing is carried out by administering an inducer selected from the group consisting of FGF2, FGF8, FGF20, SHH, and mixtures thereof. 44. The method of claim 25 further comprising: differentiating the enriched or purified population of dopaminergic neuronal progenitor cells into dopamineric neurons. 45. An enriched or purified population of dopaminergic neurons produced by the method of claim 44. 46. The enriched or purified population of dopaninergic neurons of claim 41, wherein the dopaminergic neurons are of human origin. 47. An enriched or purified population of dopaminergic neuronal progenitor cells produced by the method of claim 25. 48. The enriched or purified population of dopaminergic neuronal progenitor cells of claim 47, wherein the dopaminergic neurons are of human origin. 49. An enriched or purified preparation of isolated dopaminergic neurons derived from embryonic stem cells. 50. The enriched or purified preparation of isolated dopaminergic neurons of claim 49, wherein the dopaminergic neurons are of human origin. 51. The enriched or purified preparation of isolated dopaminergic neurons of claim 50, wherein a neurogenin-2 promoter or enhancer functions in all cells of the enriched or purified preparation. 52. The enriched or purified preparation of dopaminergic neurons of claim 49, wherein the enriched or purified preparation of dopaminergic neurons are generated from the embryonic stem cells without contacting non-human cells. 53. The enriched or purified preparation of dopaminergic neurons of claim 49, wherein the preparation comprises at least 90% of the isolated dopaminergic neurons. 54. The enriched or purified preparation of dopaminergic neurons of claim 49, wherein the preparation comprises at least 99% of the isolated dopaminergic neurons. 55. An enriched or purified preparation of isolated dopaminergic neuronal progenitor cells derived from embryonic stem cells. 56. The enriched or purified preparation of isolated dopaminergic neuronal progenitor cells of claim 55, wherein the dopaminergic neuronal progenitor cells are of human origin. 57. The enriched or purified preparation of isolated dopaminergic neuronal progenitor cells of claim 55, wherein a neurogenin-2 promoter or enhancer functions in all cells of the enriched or purified preparation. 58. The enriched or purified preparation of isolated dopaminergic neuronal progenitor cells of claim 55, wherein the enriched or purified preparation of dopaminergic neuronal progenitor cells are generated from embryonic stem cells without contacting non-human cells. 59. The enriched or purified preparation of isolated dopaminergic neuronal progenitor cells of claim 55, wherein the preparation comprises at least 90% of the isolated dopaminergic neuronal progenitor cells. 60. The enriched or purified preparation of isolated dopaminergic neuronal progenitor cells of claim 55, wherein the preparation comprises at least 99% of the isolated dopaminergic neuronal progenitor cells. 61. A cell line of immortalized human mid-brain astrocytes. 62. The cell line of claim 61, wherein the astrocytes are fetal-derived. 63. The cell line of claim 61, wherein the astrocytes are adult-derived. 64. The cell line of claim 61, wherein the astrocytes are TERT-immortalized.

The present invention relates to an enriched or purified population of dopaminergic neuronal progenitor cells and an enriched or purified population of dopaminergic neurons. These enriched or purified populations are derived from a population of embryonic stem cells by inducing production of dopaminergic neuronal progenitor cells. A promoter or enhancer which functions only in dopaminergic neuronal progenitor cells is selected and a nucleic acid molecule encoding a marker protein under control of said promoter or enhancer is introduced into the induced population of embryonic stem cells. The dopaminergic neuronal progenitor cells are allowed to express the marker protein, and the cells expressing the marker protein are separated from the induced population of embryonic stem cells. As a result, an enriched or purified population of dopaminergic neuronal progenitor cells is isolated. Alternatively, the nucleic acid molecule encoding the marker protein under control of the promoter or enhancer is introduced into the population of human embryonic stem cells followed by induction of the population of embryonic stem cells.1. A method of isolating an enriched or purified population of dopaminergic neuronal progenitor cells from a population of embryonic stem cells comprising: providing a population of embryonic stem cells; inducing production of dopaminergic neuronal progenitor cells from the population of embryonic stem cells; selecting a promoter or enhancer which functions only in dopaminergic neuronal progenitor cells; introducing a nucleic acid molecule encoding a marker protein under control of said promoter or enhancer into the induced population of embryonic stem cells; allowing the dopaminergic neuronal progenitor cells to express the marker protein; and separating the cells expressing the marker protein from the induced population of embryonic stem cells, whereby an enriched or purified population of dopaminergic neuronal progenitor cells is isolated. 2. The method of claim 1, wherein said introducing comprises viral mediated transduction of the induced population of embryonic stem cells. 3. The method of claim 2, wherein said viral mediated transduction comprises adenovirus-mediated transduction, retrovirus-mediated transduction, lentivirus-mediated transduction, or adeno-associated virus-mediated transduction. 4. The method of claim 1, wherein said introducing comprises electroporation. 5. The method of claim 1, wherein said introducing comprises biolistic transformation. 6. The method of claim 1, wherein said introducing comprises liposomal mediated transformation. 7. The method of claim 1, wherein the marker protein is a fluorescent protein and said separating comprises fluorescence activated cell sorting. 8. The method of claim 1, wherein the marker protein is either lacZ/beta-galactosidase or alkaline phosphatase. 9. The method of claim 1, wherein said promoter or enhancer is for neurogenin-2. 10. The method of claim 1, wherein said promoter or enhancer is a promoter or enhancer for genes in the dopamine synthesis pathway, a promoter or enhancer for dopamine transport proteins, or a promoter or enhancer for genes expressed differentially in the ventral midbrain. 11. The method of claim 1, wherein the population of embryonic stem cells is in a cell culture. 12. The method of claim 11, wherein the cell culture further comprises astrocytes. 13. The method of claim 12, wherein the astrocytes are human mid-brain astrocytes. 14. The method of claim 12, wherein the astrocytes are immortalized. 15. The method of claim 1, wherein the embryonic stem cells are of human origin. 16. The method of claim 1 further comprising: transplanting the separated cells into a subject. 17. The method of claim 16, wherein the subject has a dopamine-depletion disease. 18. The method of claim 17, dopamine-depletion disease is Parkinson's Disease. 19. The method of claim 1, wherein said inducing is carried out by administering an inducer selected from the group consisting of FGF2, FGF8, FGF20, SHH, and mixtures thereof. 20. The method of claim 1 further comprising: differentiating the enriched or purified population of dopaminergic neuronal progenitor cells into dopamineric neurons. 21. An enriched or purified population of dopamineric neurons produced by the method of claim 20. 22. The enriched or purified population of dopaminergic neuron according to claim 21, wherein the dopaminergic neurons are of human origin. 23. An enriched or purified population of dopaminergic neuronal progenitor cells produced by the method of claim 1. 24. The enriched or purified population of dopaminergic neuronal progenitor cells according to claim 23, wherein the dopaminergic neuronal progenitor cells are of human origin. 25. A method of producing an enriched or purified population of dopaminergic neuronal progenitor cells from a population of embryonic stem cells comprising: selecting a promoter or enhancer which functions only in said dopaminergic neuronal progenitor cells; introducing a nucleic acid molecule encoding a marker protein under control of said promoter or enhancer into the population of human embryonic stem cells; inducing the population of embryonic stem cells to produce a mixed population of cells comprising dopaminergic neuronal progenitor cells; allowing the dopaminergic neuronal progenitor cells to express the marker protein; and separating the cells expressing the marker protein from the mixed population of cells, whereby an enriched or purified population of dopaminergic neuronal progenitor cells is isolated. 26. The method of claim 25, wherein said introducing comprises viral mediated transduction of the population of embryonic stem cells. 27. The method of claim 26, wherein said viral mediated transduction comprises adenovirus-mediated transduction, retrovirus-mediated transduction, lentivirus-mediated transduction, or adeno-associated virus-mediated transduction. 28. The method of claim 25, wherein said introducing comprises electroporation. 29. The method of claim 25, wherein said introducing comprises biolistic transformation. 30. The method of claim 25, wherein said introducing comprises liposomal mediated transformation. 31. The method of claim 25, wherein the marker protein is a fluorescent protein and said separating comprises fluorescence activated cell sorting. 32. The method of claim 25, wherein the marker protein is either lacZ/beta-galactosidase or alkaline phosphatase. 33. The method of claim 25, wherein said promoter or enhancer is for neurogenin-2. 34. The method of claim 25, wherein said promoter or enhancer is a promoter or enhancer for genes in the dopamine synthesis pathway, a promoter or enhancer for dopamine transport proteins, or a promoter or enhancer for genes expressed differentially in the ventral midbrain. 35. The method of claim 25, wherein the population of human embryonic stem cells is in a cell culture. 36. The method of claim 35, wherein the cell culture further comprises astrocytes. 37. The method of claim 36, wherein the astrocytes are human mid-brain astrocytes 38. The method of claim 36, wherein the astrocytes are immortalized. 39. The method of claim 25, wherein the embryonic stem cells are of human origin. 40. The method of claim 25 further comprising: transplanting the separated cells into a subject. 41. The method of claim 40, wherein the subject has a dopamine-depletion disease. 42. The method of claim 41, wherein the dopamine-depletion disease is Parkinson's Disease. 43. The method of claim 25, wherein said inducing is carried out by administering an inducer selected from the group consisting of FGF2, FGF8, FGF20, SHH, and mixtures thereof. 44. The method of claim 25 further comprising: differentiating the enriched or purified population of dopaminergic neuronal progenitor cells into dopamineric neurons. 45. An enriched or purified population of dopaminergic neurons produced by the method of claim 44. 46. The enriched or purified population of dopaninergic neurons of claim 41, wherein the dopaminergic neurons are of human origin. 47. An enriched or purified population of dopaminergic neuronal progenitor cells produced by the method of claim 25. 48. The enriched or purified population of dopaminergic neuronal progenitor cells of claim 47, wherein the dopaminergic neurons are of human origin. 49. An enriched or purified preparation of isolated dopaminergic neurons derived from embryonic stem cells. 50. The enriched or purified preparation of isolated dopaminergic neurons of claim 49, wherein the dopaminergic neurons are of human origin. 51. The enriched or purified preparation of isolated dopaminergic neurons of claim 50, wherein a neurogenin-2 promoter or enhancer functions in all cells of the enriched or purified preparation. 52. The enriched or purified preparation of dopaminergic neurons of claim 49, wherein the enriched or purified preparation of dopaminergic neurons are generated from the embryonic stem cells without contacting non-human cells. 53. The enriched or purified preparation of dopaminergic neurons of claim 49, wherein the preparation comprises at least 90% of the isolated dopaminergic neurons. 54. The enriched or purified preparation of dopaminergic neurons of claim 49, wherein the preparation comprises at least 99% of the isolated dopaminergic neurons. 55. An enriched or purified preparation of isolated dopaminergic neuronal progenitor cells derived from embryonic stem cells. 56. The enriched or purified preparation of isolated dopaminergic neuronal progenitor cells of claim 55, wherein the dopaminergic neuronal progenitor cells are of human origin. 57. The enriched or purified preparation of isolated dopaminergic neuronal progenitor cells of claim 55, wherein a neurogenin-2 promoter or enhancer functions in all cells of the enriched or purified preparation. 58. The enriched or purified preparation of isolated dopaminergic neuronal progenitor cells of claim 55, wherein the enriched or purified preparation of dopaminergic neuronal progenitor cells are generated from embryonic stem cells without contacting non-human cells. 59. The enriched or purified preparation of isolated dopaminergic neuronal progenitor cells of claim 55, wherein the preparation comprises at least 90% of the isolated dopaminergic neuronal progenitor cells. 60. The enriched or purified preparation of isolated dopaminergic neuronal progenitor cells of claim 55, wherein the preparation comprises at least 99% of the isolated dopaminergic neuronal progenitor cells. 61. A cell line of immortalized human mid-brain astrocytes. 62. The cell line of claim 61, wherein the astrocytes are fetal-derived. 63. The cell line of claim 61, wherein the astrocytes are adult-derived. 64. The cell line of claim 61, wherein the astrocytes are TERT-immortalized.

The invention relates to the use of osmolytes, for example, ectoine, hydroxyectoine, firoin, firoin-A, diglycerol phosphate, cyclic diphosphoglycerate, 1,3-dimannosyl-di-myo-inositol-phosphate (DMIP) and/or diinositol phosphate and their equally effective derivatives and/or pharmacologically acceptable salts thereof, for the combating of pulmonary diseases caused by the effect of airborne particles and/or causally related cardiovascular diseases. The invention further relates to a dosing device suitable for inhalable medicaments and filled with active agent, the atomizable content of which consisting of an active agent composition comprising at least one osmolyte or its derivatives and/or pharmacologically acceptable salts thereof.

1.-10. (canceled) 11. A method of treatment of diseases caused by the effects of suspended particulate on lung tissue and/or the cardiovascular diseases related thereto comprising administering an effective amount of at least one osmolyte selected from ectoine, hydroxyectoine or a pharmacologically compatible salt thereof to a patient. 12. A method for the production of inhalable pharmaceutical preparations aimed at combating of diseases caused by the effects of suspended particulate on lung tissue and/or the cardiovascular diseases related thereto comprising using an effective amount of at least one osmolyte selected from ectoine hydroxyectoine or a pharmacologically compatible salt thereof. 13. A method according to claim 11, characterized in that further active agents are added to the preparation. 14. A method according to claim 12, characterized in that further active agents are added to the preparation. 15. The method of claim 11, wherein the diseases are inflammatory diseases. 16. The method of claim 12, wherein the diseases are inflammatory diseases. 17. A method for the production of inhalable pharmaceutical preparations aimed at combating diseases caused by the effect of suspended particulate on lung tissue and/or cardiovascular diseases related thereto, comprising providing in an inhalation device an effective amount of at least one osmolyte selected from ectoine, hydroxyectoine or a pharmacologically compatible salt thereof as an active agent in form of an atomazible solid or liquid. 18. The method of claim 17, wherein the diseases are inflammatory diseases.

The invention relates to the use of osmolytes, for example, ectoine, hydroxyectoine, firoin, firoin-A, diglycerol phosphate, cyclic diphosphoglycerate, 1,3-dimannosyl-di-myo-inositol-phosphate (DMIP) and/or diinositol phosphate and their equally effective derivatives and/or pharmacologically acceptable salts thereof, for the combating of pulmonary diseases caused by the effect of airborne particles and/or causally related cardiovascular diseases. The invention further relates to a dosing device suitable for inhalable medicaments and filled with active agent, the atomizable content of which consisting of an active agent composition comprising at least one osmolyte or its derivatives and/or pharmacologically acceptable salts thereof.1.-10. (canceled) 11. A method of treatment of diseases caused by the effects of suspended particulate on lung tissue and/or the cardiovascular diseases related thereto comprising administering an effective amount of at least one osmolyte selected from ectoine, hydroxyectoine or a pharmacologically compatible salt thereof to a patient. 12. A method for the production of inhalable pharmaceutical preparations aimed at combating of diseases caused by the effects of suspended particulate on lung tissue and/or the cardiovascular diseases related thereto comprising using an effective amount of at least one osmolyte selected from ectoine hydroxyectoine or a pharmacologically compatible salt thereof. 13. A method according to claim 11, characterized in that further active agents are added to the preparation. 14. A method according to claim 12, characterized in that further active agents are added to the preparation. 15. The method of claim 11, wherein the diseases are inflammatory diseases. 16. The method of claim 12, wherein the diseases are inflammatory diseases. 17. A method for the production of inhalable pharmaceutical preparations aimed at combating diseases caused by the effect of suspended particulate on lung tissue and/or cardiovascular diseases related thereto, comprising providing in an inhalation device an effective amount of at least one osmolyte selected from ectoine, hydroxyectoine or a pharmacologically compatible salt thereof as an active agent in form of an atomazible solid or liquid. 18. The method of claim 17, wherein the diseases are inflammatory diseases.

Disclosed are formulations comprising multivesicular liposomes and one or more non-steroidal anti-inflammatory drugs which minimize the side effects of unencapsulated non-steroidal anti-inflammatory drugs while maintaining or improving efficacy. Methods of making and administering the formulations comprising multivesicular liposomes and one or more non-steroidal anti-inflammatory drugs and their use as medicaments are also provided.

1. A formulation of one or more non-steroidal anti-inflammatory drugs, comprising: one or more non-steroidal anti-inflammatory drugs; and multivesicular liposomes, wherein the one or more non-steroidal anti-inflammatory drugs are encapsulated in the multivesicular liposomes. 2. The formulation of claim 1, wherein said non-steroidal anti-inflammatory drug is chosen from the group consisting of indomethacin, sulindac, etodolac, mefenamic acid, meclofenamic acid, meclofenamate sodium, flufenamic acid, tolmetin, ketorolac, diclofenac, diclofenac sodium, ibuprofen, naproxen, naproxen sodium, fenoprofen, ketoprofen, flurbiprofen, oxaprozin piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam, lornoxicam, cinnoxicam, sudoxicam, and tenoxicam. 3. The formulation of claim 1, wherein said non-steroidal anti-inflammatory drug is diclofenac. 4. The formulation of claim 1, wherein said non-steroidal anti-inflammatory drug is piroxicam. 5. The formulation of claim 1, wherein said non-steroidal anti-inflammatory drug is meloxicam. 6. The formula of claim 1, wherein said non-steroidal anti-inflammatory drug is ketorolac. 7. The formulation as in claim 1, wherein the multivesicular liposomes further comprise cholesterol, one or more phospholipids, including salts of the phospholipids, and one or more triglycerides. 8. The formulation as in claim 7, wherein the phospholipid is a phosphatidyl choline, a phosphatidyl glycerol and salts thereof, or a combination thereof. 9. The formulation as in claim 8, wherein the phosphatidyl glycerol is DPPG. 10. The formulation as in claim 8, wherein the phosphatidyl choline is DEPC. 11. The formulation as in claim 7, wherein the triglyceride is triolein, tricaprylin, or a combination of the two. 12. The formulation as in claim 1, wherein the multivesicular liposomes further comprise a pH modifier. 13. The formulation as in claim 12, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 14. The formulation as in claim 12, wherein the pH modifier is an inorganic acid. 15. The formulation as in claim 12, wherein the pH modifier is an organic base. 16. The formulation as in claim 12, wherein the pH modifier is an inorganic base. 17. The formulation as in claim 1, wherein the multivesicular liposomes further comprise a cyclodextrin. 18. The formulation of claim 17, wherein the cyclodextrin is in a concentration of from about 10 mg/ml to about 400 mg/ml complexed with the non-steroidal anti-inflammatory drug within the multivesicular liposomes. 19. The formulation of claim 17, wherein said cyclodextrin is selected from the group consisting of (2,6-Di-O—)ethyl-β-cyclodextrin, (2-Carboxyethyl)-β-cyclodextrin sodium salt, (2-hydroxyethyl)-β-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, sulfobutylether-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, 6-monodeoxy-6-monoamino-β-cyclodextrin, 6-O-α-maltosyl-β-cyclodextrin, butyl-β-cyclodextrin, butyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin, methyl-β-cyclodextrin, succinyl-α-cyclodextrin, succinyl-β-cyclodextrin, triacetyl-β-cyclodextrin, α-cyclodextrin β-cyclodextrin, and γ-cyclodextrin. 20. A method of treating pain and inflammation, comprising administering a formulation of claim 1 to a subject in need thereof. 21. The method of claim 20, wherein said non-steroidal anti-inflammatory drug is chosen from the group consisting of indomethacin, sulindac, etodolac, mefenamic acid, meclofenamic acid, meclofenamate sodium, flufenamic acid, tolmetin, ketorolac, diclofenac, diclofenac sodium, ibuprofen, naproxen, naproxen sodium, fenoprofen, ketoprofen, flurbiprofen, oxaprozin piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam, lornoxicam, cinnoxicam, sudoxicam, and tenoxicam. 22. The method of claim 21, further comprising a pharmaceutically acceptable carrier for injection. 23. The method of claim 20, wherein the multivesicular liposomes further comprise cholesterol, one or more phospholipids, including salts of the phospholipid, and one or more triglycerides. 24. The method of claim 20, wherein the multivesicular liposomes further comprise DPPG, DEPC, and tricaprylin. 25. The method of claim 20, wherein the multivesicular liposomes further comprise lysine. 26. The method of claim 20, wherein the multivesicular liposomes further comprise glutamic acid. 27. The method of claim 20, wherein said administration is subcutaneous injection. 28. The method of claim 20, wherein said administration is intramuscular injection. 29. The method of claim 20, wherein said administration is intraarticular injection. 30. The method of claim 20, wherein said administration is wound infiltration by local injection into a wound margin, or instillation into an incision wound, or a combination thereof. 31. The method of claim 20, wherein said administration is topical. 32. The method of claim 31, wherein said topical administration is ocular. 33. The method of claim 31, wherein said topical administration is nasal. 34. The method of claim 31, wherein said topical administration is otic. 35. The method of claim 20, wherein said administration is intraocular. 36. The method of claim 20, wherein administration is every 1 to 7 days. 37. A process for preparing multivesicular liposomal formulations, the process comprising: providing a first emulsion by mixing a first aqueous phase and a volatile water-immiscible solvent phase, said solvent phase comprising at least one amphipathic lipid and at least one neutral lipid; mixing and emulsifying said first emulsion and a second aqueous phase to provide a second emulsion, said second emulsion comprising a continuous aqueous phase; removing the volatile water-immiscible solvent from the second emulsion to form a composition of blank multivesicular liposomal particles; and remote loading one or more non-steroidal anti-inflammatory drugs into said multivesicular liposomes, wherein a gradient of low pH outside the multivesicular liposomes to high pH inside the multivesicular liposomes is present to drive the one or more non-steroidal anti-inflammatory drugs into the multivesicular liposomes. 38. The process as in claim 37, wherein the multivesicular liposomes further comprise a pH modifier. 39. The process as in claim 38, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 40. The process as in claim 38, wherein the pH modifier is an inorganic acid. 41. The process as in claim 38, wherein the pH modifier is an organic base. 42. The process as in claim 38, wherein the pH modifier is an inorganic base. 43. The process of claim 39, wherein the glutamic acid is adjusted to a pH from about 4.7 to about 9.2. 44. The process of claim 37, wherein the non-steroidal anti-inflammatory drug is diclofenac. 45. The process of claim 37, wherein the non-steroidal anti-inflammatory drug is piroxicam. 46. The process of claim 37, wherein the non-steroidal anti-inflammatory drug is meloxicam. 47. The process of claim 37, wherein the non-steroidal anti-inflammatory drug is ketorolac. 48. The process of claim 37, wherein the gradient is from about 1 to about 2 pH units. 49. A process for preparing multivesicular liposomal formulations, the process comprising: providing a first emulsion by mixing at least one non-steroidal anti-inflammatory drug, a first aqueous phase and a volatile water-immiscible solvent phase, said solvent phase comprising at least one amphipathic lipid and at least one neutral lipid; mixing and emulsifying said first emulsion and a second aqueous phase to provide a second emulsion, said second emulsion comprising a continuous aqueous phase; and removing the volatile water-immiscible solvent from the second emulsion to form a composition of blank multivesicular liposomal particles. 50. The process as in claim 49, wherein the non-steroidal anti-inflammatory drug is added to the first aqueous solution prior to mixing. 51. The process as in claim 49, wherein the non-steroidal anti-inflammatory drug is added to the volatile water-immiscible solvent phase prior to mixing. 52. The process as in claim 49, wherein the non-steroidal anti-inflammatory drug is added to both the first aqueous solution and volatile water-immiscible solvent phase prior to mixing. 53. The process as in claim 49, wherein the multivesicular liposomes further comprise a pH modifier. 54. The process as in claim 53, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 55. The process as in claim 53, wherein the pH modifier is an inorganic acid. 56. The process as in claim 53, wherein the pH modifier is an organic base. 57. The process as in claim 53, wherein the pH modifier is an inorganic base. 58. The process of claim 49, wherein the non-steroidal anti-inflammatory drug is diclofenac. 59. The process of claim 49, wherein the non-steroidal anti-inflammatory drug is piroxicam. 60. The process of claim 49, wherein the non-steroidal anti-inflammatory drug is meloxicam. 61. The process of claim 49, wherein the non-steroidal anti-inflammatory drug is ketorolac. 62. The formulation of claim 1 prepared by a process comprising: providing a volume of first emulsion by mixing a first aqueous phase and a volatile water-immiscible solvent phase, said solvent phase comprising at least one amphipathic lipid and at least one neutral lipid; providing a volume of second emulsion comprising a continuous aqueous phase by mixing and emulsifying said first emulsion and a second aqueous phase; removing the volatile water-immiscible solvent from the second emulsion to form a composition of multivesicular liposomal particles; and remote loading one or more non-steroidal anti-inflammatory drugs into said multivesicular liposomes, wherein a gradient of low pH outside the multivesicular liposomes to high pH inside the multivesicular liposomes is present to drive the one or more non-steroidal anti-inflammatory drugs into the multivesicular liposomes. 63. The formulation as in claim 62, wherein the multivesicular liposomes further comprise a pH modifier. 64. The formulation as in claim 63, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 65. The formulation as in claim 63, wherein the pH modifier is an inorganic acid. 66. The formulation as in claim 63, wherein the pH modifier is an organic base. 67. The formulation as in claim 63, wherein the pH modifier is an inorganic base. 68. The formulation of claim 64, wherein the glutamic acid is adjusted to a pH from about pH from about 4.7 to about 9.2. 69. The formulation of claim 62, wherein the non-steroidal anti-inflammatory drug is diclofenac. 70. The formulation of claim 62, wherein the non-steroidal anti-inflammatory drug is piroxicam. 71. The formulation of claim 62, wherein the non-steroidal anti-inflammatory drug is meloxicam. 72. The formulation of claim 62, wherein the non-steroidal anti-inflammatory drug is ketorolac. 73. The formulation of claim 62, wherein the gradient is from about 1 to about 2 pH units. 74. The formulation of claim 1 prepared by a process comprising: providing a volume of first emulsion by mixing at least one non-steroidal anti-inflammatory drug, a first aqueous phase and a volatile water-immiscible solvent phase, said solvent phase comprising at least one amphipathic lipid and at least one neutral lipid; providing a volume of second emulsion comprising a continuous aqueous phase by mixing and emulsifying said first emulsion and a second aqueous phase; and removing the volatile water-immiscible solvent from the second emulsion to form a composition of multivesicular liposomal particles. 75. The formulation as in claim 74, wherein the non-steroidal anti-inflammatory drug is added to the first aqueous solution prior to mixing. 76. The formulation as in claim 74, wherein the non-steroidal anti-inflammatory drug is added to the volatile water-immiscible solvent phase prior to mixing. 77. The formulation as in claim 74, wherein the non-steroidal anti-inflammatory drug is added to both the first aqueous solution and volatile water-immiscible solvent phase prior to mixing. 78. The formulation as in claim 74, wherein the multivesicular liposomes further comprise a pH modifier. 79. The formulation as in claim 78, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 80. The formulation as in claim 78, wherein the pH modifier is an inorganic acid. 81. The formulation as in claim 78, wherein the pH modifier is an organic base. 82. The formulation as in claim 78, wherein the pH modifier is an inorganic base. 83. The formulation of claim 74, wherein the non-steroidal anti-inflammatory drug is diclofenac. 84. The formulation of claim 74, wherein the non-steroidal anti-inflammatory drug is piroxicam. 85. The formulation of claim 74, wherein the non-steroidal anti-inflammatory drug is meloxicam. 86. The formulation of claim 74, wherein the non-steroidal anti-inflammatory drug is ketorolac. 87. A method of treating pain and inflammation for an extended period of time by wound infiltration, comprising administering a multivesicular liposome (MVL) formulation by local injection into a wound margin, or instillation into an incision wound, or a combination thereof, wherein the formulation comprises: one or more non-steroidal anti-inflammatory drugs; and multivesicular liposomes; wherein said one or more non-steroidal anti-inflammatory drugs are encapsulated in the multivesicular liposomes. 88. The method of claim 87, wherein said non-steroidal anti-inflammatory drug is chosen from the group consisting of indomethacin, sulindac, etodolac, mefenamic acid, meclofenamic acid, meclofenamate sodium, flufenamic acid, tolmetin, ketorolac, diclofenac, diclofenac sodium, ibuprofen, naproxen, naproxen sodium, fenoprofen, ketoprofen, flurbiprofen, oxaprozin piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam, lornoxicam, cinnoxicam, sudoxicam, and tenoxicam. 89. The method of claim 87, wherein said non-steroidal anti-inflammatory drug is diclofenac. 90. The method of claim 87, wherein said non-steroidal anti-inflammatory drug is piroxicam. 91. The method of claim 87, wherein said non-steroidal anti-inflammatory drug is meloxicam. 92. The method of claim 87, wherein said non-steroidal anti-inflammatory drug is ketorolac. 93. The method of claim 87, wherein the multivesicular liposomes further comprise cholesterol, one or more phospholipids, including salts of the phospholipids, and one or more triglycerides. 94. The method of claim 93, wherein the phospholipid is a phosphatidyl choline, a phosphatidyl glycerol and salts thereof, or a combination thereof. 95. The method of claim 94, wherein the phosphatidyl glycerol is DPPG. 96. The method of claim 94, wherein the phosphatidyl choline is DEPC. 97. The method of claim 93, wherein the triglyceride is triolein, tricaprylin, or a combination of the two. 98. The method of claim 87, wherein the multivesicular liposomes further comprise a pH modifier. 99. The method of claim 98, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 100. The method of claim 98, wherein the pH modifier is an inorganic acid. 101. The method of claim 98, wherein the pH modifier is an organic base. 102. The method of claim 98, wherein the pH modifier is an inorganic base. 103. The method of claim 87, wherein the multivesicular liposomes further comprise a cyclodextrin. 104. The method of claim 103, wherein the cyclodextrin is in a concentration of from about 10 mg/ml to about 400 mg/ml complexed with the non-steroidal anti-inflammatory drug within the multivesicular liposomes. 105. The method of claim 103, wherein said cyclodextrin is selected from the group consisting of (2,6-Di-O-)ethyl-f3-cyclodextrin, (2-Carboxyethyl)-β-cyclodextrin sodium salt, (2-hydroxyethyl)-β-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, sulfobutylether-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, 6-monodeoxy-6-monoamino-β-cyclodextrin, 6-O-α-maltosyl-β-cyclodextrin, butyl-β-cyclodextrin, butyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin, methyl-β- cyclodextrin, succinyl-α-cyclodextrin, succinyl-β-cyclodextrin, triacetyl-β-cyclodextrin, α-cyclodextrin β-cyclodextrin, and γ-cyclodextrin.

Disclosed are formulations comprising multivesicular liposomes and one or more non-steroidal anti-inflammatory drugs which minimize the side effects of unencapsulated non-steroidal anti-inflammatory drugs while maintaining or improving efficacy. Methods of making and administering the formulations comprising multivesicular liposomes and one or more non-steroidal anti-inflammatory drugs and their use as medicaments are also provided.1. A formulation of one or more non-steroidal anti-inflammatory drugs, comprising: one or more non-steroidal anti-inflammatory drugs; and multivesicular liposomes, wherein the one or more non-steroidal anti-inflammatory drugs are encapsulated in the multivesicular liposomes. 2. The formulation of claim 1, wherein said non-steroidal anti-inflammatory drug is chosen from the group consisting of indomethacin, sulindac, etodolac, mefenamic acid, meclofenamic acid, meclofenamate sodium, flufenamic acid, tolmetin, ketorolac, diclofenac, diclofenac sodium, ibuprofen, naproxen, naproxen sodium, fenoprofen, ketoprofen, flurbiprofen, oxaprozin piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam, lornoxicam, cinnoxicam, sudoxicam, and tenoxicam. 3. The formulation of claim 1, wherein said non-steroidal anti-inflammatory drug is diclofenac. 4. The formulation of claim 1, wherein said non-steroidal anti-inflammatory drug is piroxicam. 5. The formulation of claim 1, wherein said non-steroidal anti-inflammatory drug is meloxicam. 6. The formula of claim 1, wherein said non-steroidal anti-inflammatory drug is ketorolac. 7. The formulation as in claim 1, wherein the multivesicular liposomes further comprise cholesterol, one or more phospholipids, including salts of the phospholipids, and one or more triglycerides. 8. The formulation as in claim 7, wherein the phospholipid is a phosphatidyl choline, a phosphatidyl glycerol and salts thereof, or a combination thereof. 9. The formulation as in claim 8, wherein the phosphatidyl glycerol is DPPG. 10. The formulation as in claim 8, wherein the phosphatidyl choline is DEPC. 11. The formulation as in claim 7, wherein the triglyceride is triolein, tricaprylin, or a combination of the two. 12. The formulation as in claim 1, wherein the multivesicular liposomes further comprise a pH modifier. 13. The formulation as in claim 12, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 14. The formulation as in claim 12, wherein the pH modifier is an inorganic acid. 15. The formulation as in claim 12, wherein the pH modifier is an organic base. 16. The formulation as in claim 12, wherein the pH modifier is an inorganic base. 17. The formulation as in claim 1, wherein the multivesicular liposomes further comprise a cyclodextrin. 18. The formulation of claim 17, wherein the cyclodextrin is in a concentration of from about 10 mg/ml to about 400 mg/ml complexed with the non-steroidal anti-inflammatory drug within the multivesicular liposomes. 19. The formulation of claim 17, wherein said cyclodextrin is selected from the group consisting of (2,6-Di-O—)ethyl-β-cyclodextrin, (2-Carboxyethyl)-β-cyclodextrin sodium salt, (2-hydroxyethyl)-β-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, sulfobutylether-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, 6-monodeoxy-6-monoamino-β-cyclodextrin, 6-O-α-maltosyl-β-cyclodextrin, butyl-β-cyclodextrin, butyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin, methyl-β-cyclodextrin, succinyl-α-cyclodextrin, succinyl-β-cyclodextrin, triacetyl-β-cyclodextrin, α-cyclodextrin β-cyclodextrin, and γ-cyclodextrin. 20. A method of treating pain and inflammation, comprising administering a formulation of claim 1 to a subject in need thereof. 21. The method of claim 20, wherein said non-steroidal anti-inflammatory drug is chosen from the group consisting of indomethacin, sulindac, etodolac, mefenamic acid, meclofenamic acid, meclofenamate sodium, flufenamic acid, tolmetin, ketorolac, diclofenac, diclofenac sodium, ibuprofen, naproxen, naproxen sodium, fenoprofen, ketoprofen, flurbiprofen, oxaprozin piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam, lornoxicam, cinnoxicam, sudoxicam, and tenoxicam. 22. The method of claim 21, further comprising a pharmaceutically acceptable carrier for injection. 23. The method of claim 20, wherein the multivesicular liposomes further comprise cholesterol, one or more phospholipids, including salts of the phospholipid, and one or more triglycerides. 24. The method of claim 20, wherein the multivesicular liposomes further comprise DPPG, DEPC, and tricaprylin. 25. The method of claim 20, wherein the multivesicular liposomes further comprise lysine. 26. The method of claim 20, wherein the multivesicular liposomes further comprise glutamic acid. 27. The method of claim 20, wherein said administration is subcutaneous injection. 28. The method of claim 20, wherein said administration is intramuscular injection. 29. The method of claim 20, wherein said administration is intraarticular injection. 30. The method of claim 20, wherein said administration is wound infiltration by local injection into a wound margin, or instillation into an incision wound, or a combination thereof. 31. The method of claim 20, wherein said administration is topical. 32. The method of claim 31, wherein said topical administration is ocular. 33. The method of claim 31, wherein said topical administration is nasal. 34. The method of claim 31, wherein said topical administration is otic. 35. The method of claim 20, wherein said administration is intraocular. 36. The method of claim 20, wherein administration is every 1 to 7 days. 37. A process for preparing multivesicular liposomal formulations, the process comprising: providing a first emulsion by mixing a first aqueous phase and a volatile water-immiscible solvent phase, said solvent phase comprising at least one amphipathic lipid and at least one neutral lipid; mixing and emulsifying said first emulsion and a second aqueous phase to provide a second emulsion, said second emulsion comprising a continuous aqueous phase; removing the volatile water-immiscible solvent from the second emulsion to form a composition of blank multivesicular liposomal particles; and remote loading one or more non-steroidal anti-inflammatory drugs into said multivesicular liposomes, wherein a gradient of low pH outside the multivesicular liposomes to high pH inside the multivesicular liposomes is present to drive the one or more non-steroidal anti-inflammatory drugs into the multivesicular liposomes. 38. The process as in claim 37, wherein the multivesicular liposomes further comprise a pH modifier. 39. The process as in claim 38, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 40. The process as in claim 38, wherein the pH modifier is an inorganic acid. 41. The process as in claim 38, wherein the pH modifier is an organic base. 42. The process as in claim 38, wherein the pH modifier is an inorganic base. 43. The process of claim 39, wherein the glutamic acid is adjusted to a pH from about 4.7 to about 9.2. 44. The process of claim 37, wherein the non-steroidal anti-inflammatory drug is diclofenac. 45. The process of claim 37, wherein the non-steroidal anti-inflammatory drug is piroxicam. 46. The process of claim 37, wherein the non-steroidal anti-inflammatory drug is meloxicam. 47. The process of claim 37, wherein the non-steroidal anti-inflammatory drug is ketorolac. 48. The process of claim 37, wherein the gradient is from about 1 to about 2 pH units. 49. A process for preparing multivesicular liposomal formulations, the process comprising: providing a first emulsion by mixing at least one non-steroidal anti-inflammatory drug, a first aqueous phase and a volatile water-immiscible solvent phase, said solvent phase comprising at least one amphipathic lipid and at least one neutral lipid; mixing and emulsifying said first emulsion and a second aqueous phase to provide a second emulsion, said second emulsion comprising a continuous aqueous phase; and removing the volatile water-immiscible solvent from the second emulsion to form a composition of blank multivesicular liposomal particles. 50. The process as in claim 49, wherein the non-steroidal anti-inflammatory drug is added to the first aqueous solution prior to mixing. 51. The process as in claim 49, wherein the non-steroidal anti-inflammatory drug is added to the volatile water-immiscible solvent phase prior to mixing. 52. The process as in claim 49, wherein the non-steroidal anti-inflammatory drug is added to both the first aqueous solution and volatile water-immiscible solvent phase prior to mixing. 53. The process as in claim 49, wherein the multivesicular liposomes further comprise a pH modifier. 54. The process as in claim 53, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 55. The process as in claim 53, wherein the pH modifier is an inorganic acid. 56. The process as in claim 53, wherein the pH modifier is an organic base. 57. The process as in claim 53, wherein the pH modifier is an inorganic base. 58. The process of claim 49, wherein the non-steroidal anti-inflammatory drug is diclofenac. 59. The process of claim 49, wherein the non-steroidal anti-inflammatory drug is piroxicam. 60. The process of claim 49, wherein the non-steroidal anti-inflammatory drug is meloxicam. 61. The process of claim 49, wherein the non-steroidal anti-inflammatory drug is ketorolac. 62. The formulation of claim 1 prepared by a process comprising: providing a volume of first emulsion by mixing a first aqueous phase and a volatile water-immiscible solvent phase, said solvent phase comprising at least one amphipathic lipid and at least one neutral lipid; providing a volume of second emulsion comprising a continuous aqueous phase by mixing and emulsifying said first emulsion and a second aqueous phase; removing the volatile water-immiscible solvent from the second emulsion to form a composition of multivesicular liposomal particles; and remote loading one or more non-steroidal anti-inflammatory drugs into said multivesicular liposomes, wherein a gradient of low pH outside the multivesicular liposomes to high pH inside the multivesicular liposomes is present to drive the one or more non-steroidal anti-inflammatory drugs into the multivesicular liposomes. 63. The formulation as in claim 62, wherein the multivesicular liposomes further comprise a pH modifier. 64. The formulation as in claim 63, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 65. The formulation as in claim 63, wherein the pH modifier is an inorganic acid. 66. The formulation as in claim 63, wherein the pH modifier is an organic base. 67. The formulation as in claim 63, wherein the pH modifier is an inorganic base. 68. The formulation of claim 64, wherein the glutamic acid is adjusted to a pH from about pH from about 4.7 to about 9.2. 69. The formulation of claim 62, wherein the non-steroidal anti-inflammatory drug is diclofenac. 70. The formulation of claim 62, wherein the non-steroidal anti-inflammatory drug is piroxicam. 71. The formulation of claim 62, wherein the non-steroidal anti-inflammatory drug is meloxicam. 72. The formulation of claim 62, wherein the non-steroidal anti-inflammatory drug is ketorolac. 73. The formulation of claim 62, wherein the gradient is from about 1 to about 2 pH units. 74. The formulation of claim 1 prepared by a process comprising: providing a volume of first emulsion by mixing at least one non-steroidal anti-inflammatory drug, a first aqueous phase and a volatile water-immiscible solvent phase, said solvent phase comprising at least one amphipathic lipid and at least one neutral lipid; providing a volume of second emulsion comprising a continuous aqueous phase by mixing and emulsifying said first emulsion and a second aqueous phase; and removing the volatile water-immiscible solvent from the second emulsion to form a composition of multivesicular liposomal particles. 75. The formulation as in claim 74, wherein the non-steroidal anti-inflammatory drug is added to the first aqueous solution prior to mixing. 76. The formulation as in claim 74, wherein the non-steroidal anti-inflammatory drug is added to the volatile water-immiscible solvent phase prior to mixing. 77. The formulation as in claim 74, wherein the non-steroidal anti-inflammatory drug is added to both the first aqueous solution and volatile water-immiscible solvent phase prior to mixing. 78. The formulation as in claim 74, wherein the multivesicular liposomes further comprise a pH modifier. 79. The formulation as in claim 78, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 80. The formulation as in claim 78, wherein the pH modifier is an inorganic acid. 81. The formulation as in claim 78, wherein the pH modifier is an organic base. 82. The formulation as in claim 78, wherein the pH modifier is an inorganic base. 83. The formulation of claim 74, wherein the non-steroidal anti-inflammatory drug is diclofenac. 84. The formulation of claim 74, wherein the non-steroidal anti-inflammatory drug is piroxicam. 85. The formulation of claim 74, wherein the non-steroidal anti-inflammatory drug is meloxicam. 86. The formulation of claim 74, wherein the non-steroidal anti-inflammatory drug is ketorolac. 87. A method of treating pain and inflammation for an extended period of time by wound infiltration, comprising administering a multivesicular liposome (MVL) formulation by local injection into a wound margin, or instillation into an incision wound, or a combination thereof, wherein the formulation comprises: one or more non-steroidal anti-inflammatory drugs; and multivesicular liposomes; wherein said one or more non-steroidal anti-inflammatory drugs are encapsulated in the multivesicular liposomes. 88. The method of claim 87, wherein said non-steroidal anti-inflammatory drug is chosen from the group consisting of indomethacin, sulindac, etodolac, mefenamic acid, meclofenamic acid, meclofenamate sodium, flufenamic acid, tolmetin, ketorolac, diclofenac, diclofenac sodium, ibuprofen, naproxen, naproxen sodium, fenoprofen, ketoprofen, flurbiprofen, oxaprozin piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam, lornoxicam, cinnoxicam, sudoxicam, and tenoxicam. 89. The method of claim 87, wherein said non-steroidal anti-inflammatory drug is diclofenac. 90. The method of claim 87, wherein said non-steroidal anti-inflammatory drug is piroxicam. 91. The method of claim 87, wherein said non-steroidal anti-inflammatory drug is meloxicam. 92. The method of claim 87, wherein said non-steroidal anti-inflammatory drug is ketorolac. 93. The method of claim 87, wherein the multivesicular liposomes further comprise cholesterol, one or more phospholipids, including salts of the phospholipids, and one or more triglycerides. 94. The method of claim 93, wherein the phospholipid is a phosphatidyl choline, a phosphatidyl glycerol and salts thereof, or a combination thereof. 95. The method of claim 94, wherein the phosphatidyl glycerol is DPPG. 96. The method of claim 94, wherein the phosphatidyl choline is DEPC. 97. The method of claim 93, wherein the triglyceride is triolein, tricaprylin, or a combination of the two. 98. The method of claim 87, wherein the multivesicular liposomes further comprise a pH modifier. 99. The method of claim 98, wherein the pH modifier is lysine or glutamic acid, or a combination thereof. 100. The method of claim 98, wherein the pH modifier is an inorganic acid. 101. The method of claim 98, wherein the pH modifier is an organic base. 102. The method of claim 98, wherein the pH modifier is an inorganic base. 103. The method of claim 87, wherein the multivesicular liposomes further comprise a cyclodextrin. 104. The method of claim 103, wherein the cyclodextrin is in a concentration of from about 10 mg/ml to about 400 mg/ml complexed with the non-steroidal anti-inflammatory drug within the multivesicular liposomes. 105. The method of claim 103, wherein said cyclodextrin is selected from the group consisting of (2,6-Di-O-)ethyl-f3-cyclodextrin, (2-Carboxyethyl)-β-cyclodextrin sodium salt, (2-hydroxyethyl)-β-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, sulfobutylether-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, 6-monodeoxy-6-monoamino-β-cyclodextrin, 6-O-α-maltosyl-β-cyclodextrin, butyl-β-cyclodextrin, butyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin, methyl-β- cyclodextrin, succinyl-α-cyclodextrin, succinyl-β-cyclodextrin, triacetyl-β-cyclodextrin, α-cyclodextrin β-cyclodextrin, and γ-cyclodextrin.

The present invention provides a composition containing a pyrithione compound and a pyranone compound and a method of reducing discoloration of compositions containing pyrithione compounds. The composition may be a soap composition.

1. A method for reducing discoloration of a pyrithione-containing composition due to the presence of an iron ion, said method comprising: adding a pyranone compound to the pyrithione-containing composition in an amount effective to complex iron ions introduced to the composition from impurities present in raw materials used to make the composition or from the processing equipment. 2. The method according to claim 1, wherein the pyranone compound comprises 3-acetyl-6-methyl-2H-pyranone or a salt thereof. 3. The method according to claim 2, wherein the salt of 3-acetyl-6-methyl-2H-pyranone comprises a sodium or a zinc salt. 4. The method according to claim 2, wherein the 3-acetyl-6-methyl-2H-pyranone is added to the pyrithione-containing composition as a solid or as an aqueous slurry. 5. The method according to claim 1, wherein the pyrithione-containing composition is a soap, a shampoo, a hand sanitizer, a deodorant, a metal working fluid, a wood preservative, a paint, a coating, or a plastic precursor. 6. The method according to claim 1, wherein the pyranone is added in a range of 0.001% to 5% by weight, based on the total weight of the composition. 7. The method according to claim 1, further comprising adding an iron co-chelator to the pyrithione-containing composition. 8. The method according to claim 7, said iron co-chelator comprising a compound selected from the group consisting of hydroxyethylidene diphosphonic acid, ethylene diamine tetraacetic acid, salts of each of the proceeding compounds and mixtures thereof. 9. The method according to claim 7, wherein the weight ratio of the pyranone compound to the iron co-chelator is in the range of about 0.01:1 to about 1:0.01. 10. The method according to claim 1, said pyrithione containing-composition comprising sodium pyrithione, zinc pyrithione or a mixture thereof. 11. The method according to claim 1, said pyrithione containing composition comprising a pyrithione in an amount between about 0.001% to about 5.0% by weight of the composition. 12. A biocidal composition comprising: a pyrithione compound or a pyrithione salt in an amount effective to provided biocidal properties to the composition; a pyranone compound; and an iron co-chelator, wherein each of the pyranone compound and the iron co-chelator are present in an amount effective to reduce or eliminate discoloration of the biocidal composition due to the presence of an iron ion. 13. The composition according to claim 12, said pyrithione compound comprising sodium pyrithione, zinc pyrithione or a mixture thereof. 14. The composition according to claim 12, said pyranone compound comprising 3-acetyl-6-methyl-2H-pyranone or a salt thereof. 15. The composition according to claim 14, said salt of 3-acetyl-6-methyl-2H-pyranone comprising a sodium or a zinc salt. 16. The composition according to claim 12, said iron chelator comprising a compound selected from the group consisting of hydroxyethylidene diphosphonic acid, ethylene diamine tetraacetic acid and its analogs, salts of each of the proceeding compounds and mixtures thereof. 17. The composition according to claim 16, wherein the weight ratio of the pyranone compound to the iron co-chelator is in the range of about 0.01:1 to about 1:0.01. 18. The composition according to claim 17, said pyrithione comprising between about 0.001% to about 5.0% by weight of the composition, said pyranone compound comprising between about 0.001% to about 5.0% by weight of the composition and said iron co-chelator comprising about 0.001% to about 5.0% by weight of the composition. 19. The composition according to claim 18, said composition comprising a balance of components which give the composition its properties for its intended purpose and the composition is a soap, a shampoo, a hand sanitizer, a deodorant, a metal working fluid, a wood preservative, a paint, a coating, or a plastic precursor. 20. The composition according to claim 19, wherein the composition is a soap composition.

The present invention provides a composition containing a pyrithione compound and a pyranone compound and a method of reducing discoloration of compositions containing pyrithione compounds. The composition may be a soap composition.1. A method for reducing discoloration of a pyrithione-containing composition due to the presence of an iron ion, said method comprising: adding a pyranone compound to the pyrithione-containing composition in an amount effective to complex iron ions introduced to the composition from impurities present in raw materials used to make the composition or from the processing equipment. 2. The method according to claim 1, wherein the pyranone compound comprises 3-acetyl-6-methyl-2H-pyranone or a salt thereof. 3. The method according to claim 2, wherein the salt of 3-acetyl-6-methyl-2H-pyranone comprises a sodium or a zinc salt. 4. The method according to claim 2, wherein the 3-acetyl-6-methyl-2H-pyranone is added to the pyrithione-containing composition as a solid or as an aqueous slurry. 5. The method according to claim 1, wherein the pyrithione-containing composition is a soap, a shampoo, a hand sanitizer, a deodorant, a metal working fluid, a wood preservative, a paint, a coating, or a plastic precursor. 6. The method according to claim 1, wherein the pyranone is added in a range of 0.001% to 5% by weight, based on the total weight of the composition. 7. The method according to claim 1, further comprising adding an iron co-chelator to the pyrithione-containing composition. 8. The method according to claim 7, said iron co-chelator comprising a compound selected from the group consisting of hydroxyethylidene diphosphonic acid, ethylene diamine tetraacetic acid, salts of each of the proceeding compounds and mixtures thereof. 9. The method according to claim 7, wherein the weight ratio of the pyranone compound to the iron co-chelator is in the range of about 0.01:1 to about 1:0.01. 10. The method according to claim 1, said pyrithione containing-composition comprising sodium pyrithione, zinc pyrithione or a mixture thereof. 11. The method according to claim 1, said pyrithione containing composition comprising a pyrithione in an amount between about 0.001% to about 5.0% by weight of the composition. 12. A biocidal composition comprising: a pyrithione compound or a pyrithione salt in an amount effective to provided biocidal properties to the composition; a pyranone compound; and an iron co-chelator, wherein each of the pyranone compound and the iron co-chelator are present in an amount effective to reduce or eliminate discoloration of the biocidal composition due to the presence of an iron ion. 13. The composition according to claim 12, said pyrithione compound comprising sodium pyrithione, zinc pyrithione or a mixture thereof. 14. The composition according to claim 12, said pyranone compound comprising 3-acetyl-6-methyl-2H-pyranone or a salt thereof. 15. The composition according to claim 14, said salt of 3-acetyl-6-methyl-2H-pyranone comprising a sodium or a zinc salt. 16. The composition according to claim 12, said iron chelator comprising a compound selected from the group consisting of hydroxyethylidene diphosphonic acid, ethylene diamine tetraacetic acid and its analogs, salts of each of the proceeding compounds and mixtures thereof. 17. The composition according to claim 16, wherein the weight ratio of the pyranone compound to the iron co-chelator is in the range of about 0.01:1 to about 1:0.01. 18. The composition according to claim 17, said pyrithione comprising between about 0.001% to about 5.0% by weight of the composition, said pyranone compound comprising between about 0.001% to about 5.0% by weight of the composition and said iron co-chelator comprising about 0.001% to about 5.0% by weight of the composition. 19. The composition according to claim 18, said composition comprising a balance of components which give the composition its properties for its intended purpose and the composition is a soap, a shampoo, a hand sanitizer, a deodorant, a metal working fluid, a wood preservative, a paint, a coating, or a plastic precursor. 20. The composition according to claim 19, wherein the composition is a soap composition.

This invention relates to the storage on a solid matrix of genetic material, in particular DNA that has been purified prior to the application to the solid matrix. More specifically, the invention relates to a solid matrix for the storage of purified DNA, which matrix has been treated with a solution comprising plant polysaccharide inulin. One advantage of the invention is that an increased amount of DNA can be stored in the solid matrix of the present invention.”

1. A solid matrix suitable for the storage of purified DNA which matrix has been treated with a solution comprising inulin. 2. The solid matrix of claim 1, which has also been treated with PEG. 3. The solid matrix of claim 1, wherein the inulin treatment is at a concentration of up to 20%. 4. The solid matrix of claim 1, which also contains PEG at up to 10%. 5. A method of increasing of the yield of purified DNA eluted from a solid matrix by treating the solid matrix with a solution comprising inulin prior to the addition of said nucleic acid. 6. The method of claim 5, wherein the solid matrix has also been treated with PEG or the DNA is applied in a buffer comprising PEG.

This invention relates to the storage on a solid matrix of genetic material, in particular DNA that has been purified prior to the application to the solid matrix. More specifically, the invention relates to a solid matrix for the storage of purified DNA, which matrix has been treated with a solution comprising plant polysaccharide inulin. One advantage of the invention is that an increased amount of DNA can be stored in the solid matrix of the present invention.”1. A solid matrix suitable for the storage of purified DNA which matrix has been treated with a solution comprising inulin. 2. The solid matrix of claim 1, which has also been treated with PEG. 3. The solid matrix of claim 1, wherein the inulin treatment is at a concentration of up to 20%. 4. The solid matrix of claim 1, which also contains PEG at up to 10%. 5. A method of increasing of the yield of purified DNA eluted from a solid matrix by treating the solid matrix with a solution comprising inulin prior to the addition of said nucleic acid. 6. The method of claim 5, wherein the solid matrix has also been treated with PEG or the DNA is applied in a buffer comprising PEG.

Described herein are oral care compositions comprising a deoxy sugar antimetabolite and methods of inhibiting microbial biofilm formation and/or degrading a microbial biofilm in a subject.

1. A method of inhibiting microbial biofilm formation and/or degrading a microbial biofilm in the oral cavity of a subject comprising administering to the subject an oral care composition comprising a deoxy sugar antimetabolite with no additional antibacterial agent and an orally acceptable carrier. 2. The method according to claim 1, wherein the deoxy sugar antimetabolite is a compound of formula (I): wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R4 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 3. The method of claim 2, wherein the deoxy sugar antimetabolite is the compound of formula (I) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH. 4. The method of claim 3 wherein, the deoxy sugar antimetabolite is the compound of formula (I) wherein: R1 is H, OH, or F; R2 is H, OH, or F; R3 is H, OH, or F; R4 is H, OH, or F; and at least three of R1, R2, R3 and R4 is OH. 5. The method of claim 1 wherein, the deoxy sugar antimetabolite comprises a compound of formula (II)-(V). wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R4 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 6. The method of claim 1 wherein, the deoxy sugar antimetabolite comprises a compound of formula (II) wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R4 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 7. The compound of claim 6 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH. 8. The compound of claim 7 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH, with the proviso that the deoxy sugar antimetabolite is not 2-deoxy-D-glucose or 2-amino-2-deoxy-D-glucose. 9. The compound of claim 8 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, or F; R2 is H, OH, or F; R3 is H, OH, or F; R4 is H, OH, or F; and at least three of R1, R2, R3 and R4 is OH, with the proviso that the deoxy sugar antimetabolite is not 2-deoxy-D-glucose. 10. The method of claim 9, wherein the deoxy sugar antimetabolite is selected from the group consisting of 6-deoxy-D-glucose, 2-fluoro-2-deoxy-D-glucose, and mixtures thereof. 11. The method of claim 8, wherein the deoxy sugar antimetabolite is 2-amino-2-deoxy-D-mannosamine. 12. The method of claim 1, wherein bacteria is decreased by less than 5% and biofilm is inhibited by at least 25%. 13. The method of claim 1, wherein the biofilm efficiency is at least 1000%. 14. The method according to claim 1, wherein the method comprises prevents or treats a disease condition of the oral cavity by inhibiting microbial biofilm formation and/or degrading a microbial biofilm in the oral cavity which is selected from the group consisting of dental plaque, tooth decay, periodontal disease and gingivitis. 15. The method according to claim 1, wherein the biofilm is formed from one or more species of bacteria selected from Actinomyces viscosus, Actinomyces naeslundii, Lactobacillus casei, Streptococcus oralis, Fusobacterium nucleatum and Veillonella parvula, and Porphyromonas gingivalis. 16. An oral care composition for inhibiting microbial biofilm formation and/or degrading a microbial biofilm in the oral cavity of a subject comprising a deoxy sugar antimetabolite wherein the deoxy sugar antimetabolite is present in the composition at a concentration of less than about 1 wt. % based on the total weight of the composition; an orally acceptable carrier for a toothpaste, a dental cream, a mouthwash, a chewing gum or a denture adhesive; one or more of agents selected from the group consisting of an anti-plaque agent, a whitening agent, cleaning agent, a flavoring agent, a sweetening agent, adhesion agents, surfactants, foam modulators, abrasives, pH modifying agents, humectants, mouth feel agents, colorants, abrasive, tartar control (anticalculus) agent, fluoride ion source, saliva stimulating agents, an antisensitivity agent, an antioxidant agent, nutrients, viscosity modifiers, diluents, opacifiers, breath freshening agents and zinc salts and combinations thereof; and does not contain an additional antibacterial agent. 17. The oral care composition according to claim 16, wherein the deoxy sugar antimetabolite is a compound of formula (I): wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R4 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 18. The oral care composition of claim 17, wherein the deoxy sugar antimetabolite is the compound of formula (I) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH. 19. The oral care composition of claim 18 wherein, the deoxy sugar antimetabolite is the compound of formula (I) wherein: R1 is H, OH, or F; R2 is H, OH, or F; R3 is H, OH, or F; R4 is H, OH, or F; and at least three of R1, R2, R3 and R4 is OH. 20. The oral care composition of claim 16 wherein, the deoxy sugar antimetabolite comprises a compound of formula (II)-(V). wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R3 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 21. The oral care composition of claim 20 wherein, the deoxy sugar antimetabolite comprises a compound of formula (II) wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R4 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 22. The oral care composition of claim 21 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH. 23. The oral care composition of claim 22 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH, with the proviso that the deoxy sugar antimetabolite is not 2-deoxy-D-glucose or 2-amino-2-deoxy-D-glucose. 24. The oral care composition of claim 23 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, or F; R2 is H, OH, or F; R3 is H, OH, or F; R4 is H, OH, or F; and at least three of R1, R2, R3 and R4 is OH, with the proviso that the deoxy sugar antimetabolite is not 2-deoxy-D-glucose. 25. The oral care composition of claim 24, wherein the deoxy sugar antimetabolite is selected from the group consisting of 6-deoxy-D-glucose, 2-fluoro-2-deoxy-D-glucose and mixtures thereof. 26. The method of claim 23, wherein the deoxy sugar antimetabolite is 2-amino-2-deoxy-D-mannosamine. 27. The oral care composition of claim 16, wherein the bacteria is decreased by less than 5% and biofilm formation is inhibited by at least 25%. 28. The oral care composition of claim 16, wherein the biofilm efficiency is at least 1000%.

Described herein are oral care compositions comprising a deoxy sugar antimetabolite and methods of inhibiting microbial biofilm formation and/or degrading a microbial biofilm in a subject.1. A method of inhibiting microbial biofilm formation and/or degrading a microbial biofilm in the oral cavity of a subject comprising administering to the subject an oral care composition comprising a deoxy sugar antimetabolite with no additional antibacterial agent and an orally acceptable carrier. 2. The method according to claim 1, wherein the deoxy sugar antimetabolite is a compound of formula (I): wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R4 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 3. The method of claim 2, wherein the deoxy sugar antimetabolite is the compound of formula (I) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH. 4. The method of claim 3 wherein, the deoxy sugar antimetabolite is the compound of formula (I) wherein: R1 is H, OH, or F; R2 is H, OH, or F; R3 is H, OH, or F; R4 is H, OH, or F; and at least three of R1, R2, R3 and R4 is OH. 5. The method of claim 1 wherein, the deoxy sugar antimetabolite comprises a compound of formula (II)-(V). wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R4 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 6. The method of claim 1 wherein, the deoxy sugar antimetabolite comprises a compound of formula (II) wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R4 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 7. The compound of claim 6 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH. 8. The compound of claim 7 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH, with the proviso that the deoxy sugar antimetabolite is not 2-deoxy-D-glucose or 2-amino-2-deoxy-D-glucose. 9. The compound of claim 8 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, or F; R2 is H, OH, or F; R3 is H, OH, or F; R4 is H, OH, or F; and at least three of R1, R2, R3 and R4 is OH, with the proviso that the deoxy sugar antimetabolite is not 2-deoxy-D-glucose. 10. The method of claim 9, wherein the deoxy sugar antimetabolite is selected from the group consisting of 6-deoxy-D-glucose, 2-fluoro-2-deoxy-D-glucose, and mixtures thereof. 11. The method of claim 8, wherein the deoxy sugar antimetabolite is 2-amino-2-deoxy-D-mannosamine. 12. The method of claim 1, wherein bacteria is decreased by less than 5% and biofilm is inhibited by at least 25%. 13. The method of claim 1, wherein the biofilm efficiency is at least 1000%. 14. The method according to claim 1, wherein the method comprises prevents or treats a disease condition of the oral cavity by inhibiting microbial biofilm formation and/or degrading a microbial biofilm in the oral cavity which is selected from the group consisting of dental plaque, tooth decay, periodontal disease and gingivitis. 15. The method according to claim 1, wherein the biofilm is formed from one or more species of bacteria selected from Actinomyces viscosus, Actinomyces naeslundii, Lactobacillus casei, Streptococcus oralis, Fusobacterium nucleatum and Veillonella parvula, and Porphyromonas gingivalis. 16. An oral care composition for inhibiting microbial biofilm formation and/or degrading a microbial biofilm in the oral cavity of a subject comprising a deoxy sugar antimetabolite wherein the deoxy sugar antimetabolite is present in the composition at a concentration of less than about 1 wt. % based on the total weight of the composition; an orally acceptable carrier for a toothpaste, a dental cream, a mouthwash, a chewing gum or a denture adhesive; one or more of agents selected from the group consisting of an anti-plaque agent, a whitening agent, cleaning agent, a flavoring agent, a sweetening agent, adhesion agents, surfactants, foam modulators, abrasives, pH modifying agents, humectants, mouth feel agents, colorants, abrasive, tartar control (anticalculus) agent, fluoride ion source, saliva stimulating agents, an antisensitivity agent, an antioxidant agent, nutrients, viscosity modifiers, diluents, opacifiers, breath freshening agents and zinc salts and combinations thereof; and does not contain an additional antibacterial agent. 17. The oral care composition according to claim 16, wherein the deoxy sugar antimetabolite is a compound of formula (I): wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R4 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 18. The oral care composition of claim 17, wherein the deoxy sugar antimetabolite is the compound of formula (I) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH. 19. The oral care composition of claim 18 wherein, the deoxy sugar antimetabolite is the compound of formula (I) wherein: R1 is H, OH, or F; R2 is H, OH, or F; R3 is H, OH, or F; R4 is H, OH, or F; and at least three of R1, R2, R3 and R4 is OH. 20. The oral care composition of claim 16 wherein, the deoxy sugar antimetabolite comprises a compound of formula (II)-(V). wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R3 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 21. The oral care composition of claim 20 wherein, the deoxy sugar antimetabolite comprises a compound of formula (II) wherein: R1 is H, OH, CH3, NH2 or halogen; R2 is H, OH, CH3, NH2 or halogen; R3 is H, OH, CH3, NH2 or halogen; R4 is H, OH, NH2 or halogen; and at least three of R1, R2, R3 and R4 is OH. 22. The oral care composition of claim 21 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH. 23. The oral care composition of claim 22 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, CH3, NH2, F or Cl; R2 is H, OH, CH3, NH2, F or Cl; R3 is H, OH, CH3, NH2, F or Cl; R4 is H, OH, NH2, F or Cl; and at least three of R1, R2, R3 and R4 is OH, with the proviso that the deoxy sugar antimetabolite is not 2-deoxy-D-glucose or 2-amino-2-deoxy-D-glucose. 24. The oral care composition of claim 23 wherein, the deoxy sugar antimetabolite is the compound of formula (II) wherein: R1 is H, OH, or F; R2 is H, OH, or F; R3 is H, OH, or F; R4 is H, OH, or F; and at least three of R1, R2, R3 and R4 is OH, with the proviso that the deoxy sugar antimetabolite is not 2-deoxy-D-glucose. 25. The oral care composition of claim 24, wherein the deoxy sugar antimetabolite is selected from the group consisting of 6-deoxy-D-glucose, 2-fluoro-2-deoxy-D-glucose and mixtures thereof. 26. The method of claim 23, wherein the deoxy sugar antimetabolite is 2-amino-2-deoxy-D-mannosamine. 27. The oral care composition of claim 16, wherein the bacteria is decreased by less than 5% and biofilm formation is inhibited by at least 25%. 28. The oral care composition of claim 16, wherein the biofilm efficiency is at least 1000%.

Described herein are methods and compositions for the treatment and monitoring the progress of autoimmune diseases. In some embodiments, the methods include the stimulation of regulatory T cells specific to autoantigens associated with the autoimmune disease. A specific embodiment relates to diabetes mellitus, and the prevention or delay of loss of residual β-cell mass, providing a longer remission period and delaying the onset of diabetes related, progressive, complications through immunotherapeutic induction of regulatory T cells specific for human insulin B chain. In addition, the methods described herein can be used to predict whether a subject, e.g., a subject with ongoing anti-insulin autoimmunity, will progress to T1DM, and to evaluate a subject's response to a therapeutic intervention.

1. A method of obtaining a population of insulin autoantigen-specific regulatory T cells, the method comprising: administering to a subject a composition comprising an effective amount of an insulin autoantigen and an oil-in-water adjuvant; obtaining a sample comprising peripheral blood mononuclear cells (PBMCs) from the subject; detecting the presence of insulin autoantigen-specific regulatory T cells in the sample; isolating at least one insulin autoantigen-specific regulatory T cell from the sample; and expanding the cell in culture until a predetermined number of cells is present in the culture, thereby obtaining a population of insulin autoantigen-specific regulatory T cells. 2. The method of claim 1, wherein the insulin autoantigen is insulin B chain peptide, and the insulin autoantigen-specific regulatory T cells are insulin B chain-specific regulatory T cells. 3. The method of claim 2, wherein the autoantigen-specific regulatory T cells are insulin B chain-specific regulatory T cells. 4. The method of claim 1, wherein the insulin autoantigen-specific regulatory T cells are CD4+. 5. The method of claim 1, wherein the insulin autoantigen-specific regulatory T cells are CD25+ and FoxP3+. 6. The method of claim 1, wherein the cells are administered as part of an islet transplantation protocol. 7. A method of treating the progression or development of autoimmune diabetes in a subject, the method comprising: administering to the subject a composition comprising an effective amount of an insulin autoantigen and an oil-and-water adjuvant; obtaining a sample comprising PBMCs from the subject; isolating at least one insulin autoantigen-specific regulatory T cell from the sample; and administering said autoantigen-specific regulatory T cell to a subject, thereby treating or preventing the progression of autoimmune diabetes in the subject. 8. The method of claim 7, further comprising expanding the autoantigen-specific regulatory T cell in culture until a predetermined number of autoantigen-specific regulatory T cells is present in the culture; and administering a therapeutically effective number of said autoantigen-specific regulatory T cells to the subject. 9. The method of claim 7, wherein the subject has ongoing insulin autoimmunity. 10. The method of claim 7, wherein the subject has a population of functional, insulin producing beta cells. 11. The method of claim 7, wherein the cells are administered to the same subject from which they were isolated. 12. A method of treating the development or progression of autoimmune diabetes in a subject, the method comprising: administering to the subject a composition comprising an effective amount of an insulin autoantigen and an oil-and-water adjuvant, wherein the effective amount is an amount sufficient to generate a selected level of autoantigen-specific regulatory T cells in the subject; determining a level of circulating insulin autoantigen-specific regulatory T cells in the subject; administering one or more subsequent doses of the composition comprising an effective amount of an insulin autoantigen and an oil-and-water adjuvant, in an amount and on a schedule sufficient to maintain the selected level of autoantigen-specific regulatory T cells in the subject; thereby treating or preventing the progression of autoimmune diabetes in the subject. 13. The method of claim 12, wherein the insulin autoantigen is insulin B chain peptide, and the insulin autoantigen-specific regulatory T cells are insulin B chain-specific regulatory T cells. 14. The method of claim 12, wherein the autoantigen-specific regulatory T cells are insulin B chain-specific regulatory T cells. 15. A method of evaluating the efficacy of a treatment for diabetes, the method comprising: administering to a subject a treatment for diabetes; obtaining a test sample comprising PBMCs from the subject; and detecting the presence or absence of insulin autoantigen-specific regulatory T cells in the test sample; wherein the presence or absence of insulin autoantigen-specific regulatory T cells in the test sample indicates whether the treatment is effective. 16. The method of claim 15, further comprising quantifying the insulin autoantigen-specific regulatory T cells in the test sample, wherein the quantity of insulin autoantigen specific regulatory T cells in the test sample indicates whether the treatment is effective. 17. The method of claim 15, further comprising, prior to administering the treatment intervention, obtaining a baseline sample comprising PBMCs from the subject; detecting the presence of insulin autoantigen-specific regulatory T cells in the baseline sample; and quantifying the insulin autoantigen-specific regulatory T cells in the baseline sample; wherein a significant increase in the quantity of insulin autoantigen-specific regulatory T cells from the baseline sample to the test sample indicates that the therapeutic intervention is effective. 18. A method of determining a subject's risk of developing type 1 diabetes mellitus (T1DM), the method comprising: obtaining a sample comprising PBMCs from the subject; and detecting the presence of insulin autoantigen-specific regulatory T cells in the sample; wherein the presence of insulin autoantigen-specific regulatory T cells in the sample indicates that the subject has a low risk of developing T1DM. 19. A method of treating an autoimmune disease in a mammalian subject, the method comprising: detecting the presence of autoantibodies to autoantigens associated with the autoimmune disease in the subject; administering to the subject an effective amount of a therapeutic composition comprising at least one autoantigen associated with the autoimmune disease in a water-in-oil emulsion; determining the level of autoantigen-specific regulatory T cells stimulated in the subject in response to the administered therapeutic composition, and administering a booster amount of the therapeutic composition to the subject if the level of the autoantigen-specific regulatory T cells declines. 20. The method of claim 19, wherein the autoimmune disease is diabetes, and the autoantigen is insulin B chain. 21. The method of claim 19, wherein the water-in-oil emulsion comprises IFA or MAS-1. 22. The method of claim 19, wherein the regulatory T cells are CD4+, CD25+ and FoxP3+. 23. The method of claim 19, wherein the regulatory T cells secrete IL-10, TGF-Beta, or both IL-10 and TGF-Beta.

Described herein are methods and compositions for the treatment and monitoring the progress of autoimmune diseases. In some embodiments, the methods include the stimulation of regulatory T cells specific to autoantigens associated with the autoimmune disease. A specific embodiment relates to diabetes mellitus, and the prevention or delay of loss of residual β-cell mass, providing a longer remission period and delaying the onset of diabetes related, progressive, complications through immunotherapeutic induction of regulatory T cells specific for human insulin B chain. In addition, the methods described herein can be used to predict whether a subject, e.g., a subject with ongoing anti-insulin autoimmunity, will progress to T1DM, and to evaluate a subject's response to a therapeutic intervention.1. A method of obtaining a population of insulin autoantigen-specific regulatory T cells, the method comprising: administering to a subject a composition comprising an effective amount of an insulin autoantigen and an oil-in-water adjuvant; obtaining a sample comprising peripheral blood mononuclear cells (PBMCs) from the subject; detecting the presence of insulin autoantigen-specific regulatory T cells in the sample; isolating at least one insulin autoantigen-specific regulatory T cell from the sample; and expanding the cell in culture until a predetermined number of cells is present in the culture, thereby obtaining a population of insulin autoantigen-specific regulatory T cells. 2. The method of claim 1, wherein the insulin autoantigen is insulin B chain peptide, and the insulin autoantigen-specific regulatory T cells are insulin B chain-specific regulatory T cells. 3. The method of claim 2, wherein the autoantigen-specific regulatory T cells are insulin B chain-specific regulatory T cells. 4. The method of claim 1, wherein the insulin autoantigen-specific regulatory T cells are CD4+. 5. The method of claim 1, wherein the insulin autoantigen-specific regulatory T cells are CD25+ and FoxP3+. 6. The method of claim 1, wherein the cells are administered as part of an islet transplantation protocol. 7. A method of treating the progression or development of autoimmune diabetes in a subject, the method comprising: administering to the subject a composition comprising an effective amount of an insulin autoantigen and an oil-and-water adjuvant; obtaining a sample comprising PBMCs from the subject; isolating at least one insulin autoantigen-specific regulatory T cell from the sample; and administering said autoantigen-specific regulatory T cell to a subject, thereby treating or preventing the progression of autoimmune diabetes in the subject. 8. The method of claim 7, further comprising expanding the autoantigen-specific regulatory T cell in culture until a predetermined number of autoantigen-specific regulatory T cells is present in the culture; and administering a therapeutically effective number of said autoantigen-specific regulatory T cells to the subject. 9. The method of claim 7, wherein the subject has ongoing insulin autoimmunity. 10. The method of claim 7, wherein the subject has a population of functional, insulin producing beta cells. 11. The method of claim 7, wherein the cells are administered to the same subject from which they were isolated. 12. A method of treating the development or progression of autoimmune diabetes in a subject, the method comprising: administering to the subject a composition comprising an effective amount of an insulin autoantigen and an oil-and-water adjuvant, wherein the effective amount is an amount sufficient to generate a selected level of autoantigen-specific regulatory T cells in the subject; determining a level of circulating insulin autoantigen-specific regulatory T cells in the subject; administering one or more subsequent doses of the composition comprising an effective amount of an insulin autoantigen and an oil-and-water adjuvant, in an amount and on a schedule sufficient to maintain the selected level of autoantigen-specific regulatory T cells in the subject; thereby treating or preventing the progression of autoimmune diabetes in the subject. 13. The method of claim 12, wherein the insulin autoantigen is insulin B chain peptide, and the insulin autoantigen-specific regulatory T cells are insulin B chain-specific regulatory T cells. 14. The method of claim 12, wherein the autoantigen-specific regulatory T cells are insulin B chain-specific regulatory T cells. 15. A method of evaluating the efficacy of a treatment for diabetes, the method comprising: administering to a subject a treatment for diabetes; obtaining a test sample comprising PBMCs from the subject; and detecting the presence or absence of insulin autoantigen-specific regulatory T cells in the test sample; wherein the presence or absence of insulin autoantigen-specific regulatory T cells in the test sample indicates whether the treatment is effective. 16. The method of claim 15, further comprising quantifying the insulin autoantigen-specific regulatory T cells in the test sample, wherein the quantity of insulin autoantigen specific regulatory T cells in the test sample indicates whether the treatment is effective. 17. The method of claim 15, further comprising, prior to administering the treatment intervention, obtaining a baseline sample comprising PBMCs from the subject; detecting the presence of insulin autoantigen-specific regulatory T cells in the baseline sample; and quantifying the insulin autoantigen-specific regulatory T cells in the baseline sample; wherein a significant increase in the quantity of insulin autoantigen-specific regulatory T cells from the baseline sample to the test sample indicates that the therapeutic intervention is effective. 18. A method of determining a subject's risk of developing type 1 diabetes mellitus (T1DM), the method comprising: obtaining a sample comprising PBMCs from the subject; and detecting the presence of insulin autoantigen-specific regulatory T cells in the sample; wherein the presence of insulin autoantigen-specific regulatory T cells in the sample indicates that the subject has a low risk of developing T1DM. 19. A method of treating an autoimmune disease in a mammalian subject, the method comprising: detecting the presence of autoantibodies to autoantigens associated with the autoimmune disease in the subject; administering to the subject an effective amount of a therapeutic composition comprising at least one autoantigen associated with the autoimmune disease in a water-in-oil emulsion; determining the level of autoantigen-specific regulatory T cells stimulated in the subject in response to the administered therapeutic composition, and administering a booster amount of the therapeutic composition to the subject if the level of the autoantigen-specific regulatory T cells declines. 20. The method of claim 19, wherein the autoimmune disease is diabetes, and the autoantigen is insulin B chain. 21. The method of claim 19, wherein the water-in-oil emulsion comprises IFA or MAS-1. 22. The method of claim 19, wherein the regulatory T cells are CD4+, CD25+ and FoxP3+. 23. The method of claim 19, wherein the regulatory T cells secrete IL-10, TGF-Beta, or both IL-10 and TGF-Beta.

Embodiments disclosed herein relate to methods and systems for performing an automated assay, and particularly to performing an assay on a plurality of samples on an automated instrument.

1. A method of performing an automated assay on a plurality of samples, said method comprising: providing an automated instrument comprising a first workstation and a second workstation, each of said first and second workstations configured to receive and processes a plurality of samples according to a plurality of different automated assay workflows, wherein each different automated assay workflow has an associated unique assay definition or user-defined protocol file; determining whether two discrete assay workflows are compatible or incompatible with each other for concurrent processing on the automated instrument; and performing said discrete assay workflows concurrently on said instrument when said assays are compatible. 2. The method of claim 1, wherein said assay definition or user defined protocol file comprises a first level compatibility index value, and wherein said determining step comprises: (a) selecting a first assay from among a first list of available assays; and (b) evaluating which of a plurality of other available assays have an assay definition file comprising the same first level compatibility index value as said first assay, wherein the same first level compatibility index value is indicative of first-level compatibility. 3. The method of claim 2, wherein said evaluating step comprises: (b1) identifying any assays which have first level compatibility index values different from the first compatibility index value of said first assay; and (b2) providing a second list of second assays, wherein said second list excludes any assay having a first level compatibility index value different from the first compatibility index value of said first assay. 4. The method of claim 2, wherein each assay definition file comprises a second level compatibility index value, and wherein said determining step further comprises: (c) evaluating which of a plurality of other available assays have an assay definition file comprising the same second level compatibility index value as said first assay, wherein the same second level compatibility index value is indicative of second-level compatibility. 5. The method of claim 4, wherein said evaluating step comprises: (c1) identifying any assays which have second level compatibility index values different from the second compatibility index value of said first assay; and (c2) providing a second list of second assays, wherein said second list excludes any assay having a second level compatibility index value different from the second compatibility index value of said first assay. 6. The method of claim 2, wherein said first level compatibility comprises compatibility of performing two assays concurrently at a single workstation, said parameters selected from the group consisting of: incubation time, lysis time, reagent volume, reagent type, incubation temperature, lysis temperature, workstation time demands, regulatory classification, business considerations, and a combination thereof. 7. The method of claim 4, wherein said second level compatibility comprises compatibility of performing two assays concurrently on said automated instrument, said parameters selected from the group consisting of: regulatory classification, workflow incompatibility, business considerations, and a combination thereof. 8. The method of claim 6, wherein said instrument prevents the concurrent performance of incompatible assays within the same workstation when the first compatibility indexes are different. 9. The method of claim 8, wherein said two discrete assay workflows are performed in the same workstation. 10. The method of claim 7, wherein said instrument is prevented from concurrently performing assays with different second compatibility index values. 11. (canceled) 12. (canceled) 13. (canceled) 14. The method of claim 1, wherein if said assays are compatible, said method further comprises one or more of the following: (d) initiating an assay-specific sample preparation script on the instrument; (e) comparing identifying indicia on a consumable package to a set of assay-specific identifying data stored on the instrument; (f) initiating an assay-specific load cartridge script on the instrument; (g) comparing fluorescence ratios in a loaded cartridge to a set of assay-specific fluorescence ratio data stored on the instrument to determine whether said cartridge was successfully loaded; (h) initiating an assay-specific reaction script on the instrument; (i) initiating an assay-specific data analysis algorithm on the instrument; (j) deriving a final call for the assay, based on one or more assay-specific result algorithms or scripts. 15. The method of claim 14, wherein said assay protocol comprises a reaction selected from the group selected from: Polymerase Chain Reaction (PCR), Transcription Mediated Amplification (TMA), Oligonucleotide Ligation Assay (OLA), Ligase Chain Reaction (LCR), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), and a hybridization reaction. 16. A system for performing an automated assay comprising: an automated instrument comprising a first workstation and a second workstation, each of said first and second workstations configured to receive and processes a plurality of samples according to a plurality of different automated assay workflows, wherein each different automated assay workflow has an associated unique assay definition file or user-defined protocol file; a processor; a storage capacity; and a program for performing an automated assay, said program comprising instructions for: determining whether two discrete assay workflows are compatible or incompatible with each other for concurrent processing on the automated instrument; and performing said discrete assay workflows concurrently on said instrument when said assays are compatible. 17. The system of claim 16, wherein said assay definition file or user defined protocol file comprises a first level compatibility index value, and wherein said determining step comprises: (a) selecting a first assay from among a first list of available assays; and (b) evaluating which of a plurality of other available assays have an assay definition file comprising the same first level compatibility index value as said first assay, wherein the same first level compatibility index value is indicative of first-level compatibility. 18. The system of claim 17, wherein said evaluating step comprises: (b1) identifying any assays which have first level compatibility index values different from the first compatibility index value of said first assay; and (b2) providing a second list of second assays, wherein said second list excludes any assay having a first level compatibility index value different from the first compatibility index value of said first assay. 19. The system of claim 17, wherein each assay definition file comprises a second level compatibility index value, and wherein said determining step further comprises: (c) evaluating which of a plurality of other available assays have an assay definition file comprising the same second level compatibility index value as said first assay, wherein the same second level compatibility index value is indicative of second-level compatibility. 20. The system of claim 19, wherein said evaluating step comprises: (c1) identifying any assays which have second level compatibility index values different from the second compatibility index value of said first assay; and (c2) providing a second list of second assays, wherein said second list excludes any assay having a second level compatibility index value different from the second compatibility index value of said first assay. 21. The system of claim 17, wherein said first level compatibility comprises compatibility of performing two assays concurrently at a single workstation, said parameters selected from the group consisting of: incubation time, lysis time, reagent volume, reagent type, incubation temperature, lysis temperature, workstation time demands, regulatory classification, business considerations, and a combination thereof. 22. (canceled) 23. (canceled) 24. (canceled) 25. (canceled) 26. (canceled) 27. (canceled) 28. (canceled) 29. The system of claim 16, wherein if said assays are compatible, said method further comprises one or more of the following: (d) initiating an assay-specific sample preparation script on the instrument; (e) comparing identifying indicia on a consumable package to a set of assay-specific identifying data stored on the instrument; (f) initiating an assay-specific load cartridge script on the instrument; (g) comparing fluorescence ratios in a loaded cartridge to a set of assay-specific fluorescence ratio data stored on the instrument to determine whether said cartridge was successfully loaded; (h) initiating an assay-specific reaction script on the instrument; (i) initiating an assay-specific data analysis algorithm on the instrument; (j) deriving a final call for the assay, based on one or more assay-specific result algorithms or scripts. 30. The system of claim 29, wherein said assay protocol comprises a reaction selected from the group selected from: Polymerase Chain Reaction (PCR), Transcription Mediated Amplification (TMA), Oligonucleotide Ligation Assay (OLA), Ligase Chain Reaction (LCR), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), and a hybridization reaction. 31. The system of claim 16, wherein said system further comprises a bar code reader. 32. (canceled) 33. (canceled)

Embodiments disclosed herein relate to methods and systems for performing an automated assay, and particularly to performing an assay on a plurality of samples on an automated instrument.1. A method of performing an automated assay on a plurality of samples, said method comprising: providing an automated instrument comprising a first workstation and a second workstation, each of said first and second workstations configured to receive and processes a plurality of samples according to a plurality of different automated assay workflows, wherein each different automated assay workflow has an associated unique assay definition or user-defined protocol file; determining whether two discrete assay workflows are compatible or incompatible with each other for concurrent processing on the automated instrument; and performing said discrete assay workflows concurrently on said instrument when said assays are compatible. 2. The method of claim 1, wherein said assay definition or user defined protocol file comprises a first level compatibility index value, and wherein said determining step comprises: (a) selecting a first assay from among a first list of available assays; and (b) evaluating which of a plurality of other available assays have an assay definition file comprising the same first level compatibility index value as said first assay, wherein the same first level compatibility index value is indicative of first-level compatibility. 3. The method of claim 2, wherein said evaluating step comprises: (b1) identifying any assays which have first level compatibility index values different from the first compatibility index value of said first assay; and (b2) providing a second list of second assays, wherein said second list excludes any assay having a first level compatibility index value different from the first compatibility index value of said first assay. 4. The method of claim 2, wherein each assay definition file comprises a second level compatibility index value, and wherein said determining step further comprises: (c) evaluating which of a plurality of other available assays have an assay definition file comprising the same second level compatibility index value as said first assay, wherein the same second level compatibility index value is indicative of second-level compatibility. 5. The method of claim 4, wherein said evaluating step comprises: (c1) identifying any assays which have second level compatibility index values different from the second compatibility index value of said first assay; and (c2) providing a second list of second assays, wherein said second list excludes any assay having a second level compatibility index value different from the second compatibility index value of said first assay. 6. The method of claim 2, wherein said first level compatibility comprises compatibility of performing two assays concurrently at a single workstation, said parameters selected from the group consisting of: incubation time, lysis time, reagent volume, reagent type, incubation temperature, lysis temperature, workstation time demands, regulatory classification, business considerations, and a combination thereof. 7. The method of claim 4, wherein said second level compatibility comprises compatibility of performing two assays concurrently on said automated instrument, said parameters selected from the group consisting of: regulatory classification, workflow incompatibility, business considerations, and a combination thereof. 8. The method of claim 6, wherein said instrument prevents the concurrent performance of incompatible assays within the same workstation when the first compatibility indexes are different. 9. The method of claim 8, wherein said two discrete assay workflows are performed in the same workstation. 10. The method of claim 7, wherein said instrument is prevented from concurrently performing assays with different second compatibility index values. 11. (canceled) 12. (canceled) 13. (canceled) 14. The method of claim 1, wherein if said assays are compatible, said method further comprises one or more of the following: (d) initiating an assay-specific sample preparation script on the instrument; (e) comparing identifying indicia on a consumable package to a set of assay-specific identifying data stored on the instrument; (f) initiating an assay-specific load cartridge script on the instrument; (g) comparing fluorescence ratios in a loaded cartridge to a set of assay-specific fluorescence ratio data stored on the instrument to determine whether said cartridge was successfully loaded; (h) initiating an assay-specific reaction script on the instrument; (i) initiating an assay-specific data analysis algorithm on the instrument; (j) deriving a final call for the assay, based on one or more assay-specific result algorithms or scripts. 15. The method of claim 14, wherein said assay protocol comprises a reaction selected from the group selected from: Polymerase Chain Reaction (PCR), Transcription Mediated Amplification (TMA), Oligonucleotide Ligation Assay (OLA), Ligase Chain Reaction (LCR), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), and a hybridization reaction. 16. A system for performing an automated assay comprising: an automated instrument comprising a first workstation and a second workstation, each of said first and second workstations configured to receive and processes a plurality of samples according to a plurality of different automated assay workflows, wherein each different automated assay workflow has an associated unique assay definition file or user-defined protocol file; a processor; a storage capacity; and a program for performing an automated assay, said program comprising instructions for: determining whether two discrete assay workflows are compatible or incompatible with each other for concurrent processing on the automated instrument; and performing said discrete assay workflows concurrently on said instrument when said assays are compatible. 17. The system of claim 16, wherein said assay definition file or user defined protocol file comprises a first level compatibility index value, and wherein said determining step comprises: (a) selecting a first assay from among a first list of available assays; and (b) evaluating which of a plurality of other available assays have an assay definition file comprising the same first level compatibility index value as said first assay, wherein the same first level compatibility index value is indicative of first-level compatibility. 18. The system of claim 17, wherein said evaluating step comprises: (b1) identifying any assays which have first level compatibility index values different from the first compatibility index value of said first assay; and (b2) providing a second list of second assays, wherein said second list excludes any assay having a first level compatibility index value different from the first compatibility index value of said first assay. 19. The system of claim 17, wherein each assay definition file comprises a second level compatibility index value, and wherein said determining step further comprises: (c) evaluating which of a plurality of other available assays have an assay definition file comprising the same second level compatibility index value as said first assay, wherein the same second level compatibility index value is indicative of second-level compatibility. 20. The system of claim 19, wherein said evaluating step comprises: (c1) identifying any assays which have second level compatibility index values different from the second compatibility index value of said first assay; and (c2) providing a second list of second assays, wherein said second list excludes any assay having a second level compatibility index value different from the second compatibility index value of said first assay. 21. The system of claim 17, wherein said first level compatibility comprises compatibility of performing two assays concurrently at a single workstation, said parameters selected from the group consisting of: incubation time, lysis time, reagent volume, reagent type, incubation temperature, lysis temperature, workstation time demands, regulatory classification, business considerations, and a combination thereof. 22. (canceled) 23. (canceled) 24. (canceled) 25. (canceled) 26. (canceled) 27. (canceled) 28. (canceled) 29. The system of claim 16, wherein if said assays are compatible, said method further comprises one or more of the following: (d) initiating an assay-specific sample preparation script on the instrument; (e) comparing identifying indicia on a consumable package to a set of assay-specific identifying data stored on the instrument; (f) initiating an assay-specific load cartridge script on the instrument; (g) comparing fluorescence ratios in a loaded cartridge to a set of assay-specific fluorescence ratio data stored on the instrument to determine whether said cartridge was successfully loaded; (h) initiating an assay-specific reaction script on the instrument; (i) initiating an assay-specific data analysis algorithm on the instrument; (j) deriving a final call for the assay, based on one or more assay-specific result algorithms or scripts. 30. The system of claim 29, wherein said assay protocol comprises a reaction selected from the group selected from: Polymerase Chain Reaction (PCR), Transcription Mediated Amplification (TMA), Oligonucleotide Ligation Assay (OLA), Ligase Chain Reaction (LCR), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), and a hybridization reaction. 31. The system of claim 16, wherein said system further comprises a bar code reader. 32. (canceled) 33. (canceled)

Described herein are compositions comprising combinations of metal salts, and methods of preparing and using the same.

1. An oral care composition comprising: a first metal salt, having a solubility of greater than 0.001 g/100 mL in water at 20° C.; a second metal salt, having a solubility of 0.001 g/100 mL, or less, in water at 20° C.; and a free water content of greater than about 10%, by weight; wherein the first metal salt and the second metal salt are independently selected from a zinc salt, a stannous salt and a copper salt; and wherein the second metal salt is present in an amount effective to provide a synergistic increase in delivery of the metal ion of the first or second metal salt. 2. The composition of claim 1, wherein the first metal salt and the second metal salt are salts of the same metal. 3. The composition of claim 1, wherein the second metal salt is present in an amount effective to provide a synergistic increase in delivery of the metal ion of the first metal salt. 4. The composition of claim 1, wherein the first metal salt is selected from: zinc citrate trihydrate, zinc chloride, zinc lactate, zinc nitrate, zinc acetate, zinc gluconate, zinc glycinate and zinc sulfate. 5. The composition of claim 1, wherein the second metal salt is selected from: zinc oxide, zinc phosphate, zinc pyrophosphate, zinc silicate, zinc oleate, zinc hydroxide, zinc peroxide, and zinc sulfide. 6. The composition of claim 1, comprising: from about 0.1 to about 5%, by weight, of said first metal salt; and from about 0.05 to about 2%, by weight, of said second metal salt. 7. The composition of claim 1, comprising: from about 0.5 to about 4%, by weight, of said first metal salt; and from about 0.1 to about 1.5%, by weight, of said second metal salt. 8. The composition of claim 1, comprising: from about 1 to about 3%, by weight, of said first metal salt; and from about 0.2 to about 0.75%, by weight, of said second metal salt. 9. The composition of claim 1, comprising: about 2%, by weight, of said first metal salt; and about 0.25%, by weight, of said second metal salt. 10. The composition of claim 1, comprising: about 2%, by weight, of said first metal salt; and about 0.5%, by weight, of said second metal salt. 11. The composition of claim 1, further comprising one or more components selected from a fluoride ion source; a tartar control agent; a buffering agent; an antibacterial agent; an abrasive; and a combination of two or more thereof. 12. The composition of claim 11, wherein at least one of the one or more components is a fluoride ion source selected from: stannous fluoride, sodium fluoride, potassium fluoride, sodium monofluorophosphate, sodium fluorosilicate, ammonium fluorosilicate, amine fluoride, ammonium fluoride, and a combination of two or more thereof. 13. The composition of claim 11, wherein at least one of the one or more components is a tartar control agent selected from: sodium tripolyphosphate, sodium tetrapolyphosphate, mono-, di-, tri- and tetrasodium pyrophosphates, sodium trimetaphosphate, sodium hexametaphosphate; potassium tripolyphosphate, potassium tetrapolyphosphate, mono-, di-, tri- and tetrapotassium pyrophosphates, potassium trimetaphosphate, potassium hexametaphosphate; and a combination of two or more thereof. 14. The composition of claim 1, wherein the composition comprises greater than about 15%, by weight, free water. 15. A method of treating or preventing a disease or condition of the oral cavity comprising contacting an oral cavity surface of a patient in need thereof with the composition of claim 1. 16. The method of claim 15, wherein the disease or condition of the oral cavity is halitosis. 17. The composition of claim 1, wherein the first metal salt is selected from: zinc citrate trihydrate, zinc chloride, zinc lactate, zinc nitrate, zinc acetate, zinc gluconate, zinc glycinate and zinc sulfate, the second metal salt is selected from: zinc oxide, zinc phosphate, zinc pyrophosphate, zinc silicate, zinc oleate, zinc hydroxide, zinc peroxide, and zinc sulfide; and, the composition comprises from about 0.1 to about 5%, by weight, of said first metal salt; and from about 0.05 to about 2%, by weight, of said second metal salt. 18. The composition of claim 17, wherein the composition comprises greater than about 15%, by weight, free water.

Described herein are compositions comprising combinations of metal salts, and methods of preparing and using the same.1. An oral care composition comprising: a first metal salt, having a solubility of greater than 0.001 g/100 mL in water at 20° C.; a second metal salt, having a solubility of 0.001 g/100 mL, or less, in water at 20° C.; and a free water content of greater than about 10%, by weight; wherein the first metal salt and the second metal salt are independently selected from a zinc salt, a stannous salt and a copper salt; and wherein the second metal salt is present in an amount effective to provide a synergistic increase in delivery of the metal ion of the first or second metal salt. 2. The composition of claim 1, wherein the first metal salt and the second metal salt are salts of the same metal. 3. The composition of claim 1, wherein the second metal salt is present in an amount effective to provide a synergistic increase in delivery of the metal ion of the first metal salt. 4. The composition of claim 1, wherein the first metal salt is selected from: zinc citrate trihydrate, zinc chloride, zinc lactate, zinc nitrate, zinc acetate, zinc gluconate, zinc glycinate and zinc sulfate. 5. The composition of claim 1, wherein the second metal salt is selected from: zinc oxide, zinc phosphate, zinc pyrophosphate, zinc silicate, zinc oleate, zinc hydroxide, zinc peroxide, and zinc sulfide. 6. The composition of claim 1, comprising: from about 0.1 to about 5%, by weight, of said first metal salt; and from about 0.05 to about 2%, by weight, of said second metal salt. 7. The composition of claim 1, comprising: from about 0.5 to about 4%, by weight, of said first metal salt; and from about 0.1 to about 1.5%, by weight, of said second metal salt. 8. The composition of claim 1, comprising: from about 1 to about 3%, by weight, of said first metal salt; and from about 0.2 to about 0.75%, by weight, of said second metal salt. 9. The composition of claim 1, comprising: about 2%, by weight, of said first metal salt; and about 0.25%, by weight, of said second metal salt. 10. The composition of claim 1, comprising: about 2%, by weight, of said first metal salt; and about 0.5%, by weight, of said second metal salt. 11. The composition of claim 1, further comprising one or more components selected from a fluoride ion source; a tartar control agent; a buffering agent; an antibacterial agent; an abrasive; and a combination of two or more thereof. 12. The composition of claim 11, wherein at least one of the one or more components is a fluoride ion source selected from: stannous fluoride, sodium fluoride, potassium fluoride, sodium monofluorophosphate, sodium fluorosilicate, ammonium fluorosilicate, amine fluoride, ammonium fluoride, and a combination of two or more thereof. 13. The composition of claim 11, wherein at least one of the one or more components is a tartar control agent selected from: sodium tripolyphosphate, sodium tetrapolyphosphate, mono-, di-, tri- and tetrasodium pyrophosphates, sodium trimetaphosphate, sodium hexametaphosphate; potassium tripolyphosphate, potassium tetrapolyphosphate, mono-, di-, tri- and tetrapotassium pyrophosphates, potassium trimetaphosphate, potassium hexametaphosphate; and a combination of two or more thereof. 14. The composition of claim 1, wherein the composition comprises greater than about 15%, by weight, free water. 15. A method of treating or preventing a disease or condition of the oral cavity comprising contacting an oral cavity surface of a patient in need thereof with the composition of claim 1. 16. The method of claim 15, wherein the disease or condition of the oral cavity is halitosis. 17. The composition of claim 1, wherein the first metal salt is selected from: zinc citrate trihydrate, zinc chloride, zinc lactate, zinc nitrate, zinc acetate, zinc gluconate, zinc glycinate and zinc sulfate, the second metal salt is selected from: zinc oxide, zinc phosphate, zinc pyrophosphate, zinc silicate, zinc oleate, zinc hydroxide, zinc peroxide, and zinc sulfide; and, the composition comprises from about 0.1 to about 5%, by weight, of said first metal salt; and from about 0.05 to about 2%, by weight, of said second metal salt. 18. The composition of claim 17, wherein the composition comprises greater than about 15%, by weight, free water.

The present invention relates to solid pharmaceutical compositions, in particular to oral contraceptives, comprising a progestogen, such as drospirenone; an estrogen, such as ethinylestradiol; a tetrahydrofolic acid or a pharmaceutically acceptable salt thereof, such as calcium 5-methyl-(6S)-tetrahydrofolate; and at least one pharmaceutical acceptable excipient or carrier. The compositions of the invention provide good stability of the tetrahydrofolic acid upon storage while still ensuring a fast and reliable release of the estrogen and the progestogen present in the composition.

1. A solid pharmaceutical composition comprising a progestogen, an estrogen, a tetrahydrofolic acid or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical acceptable excipient or carrier. 2.-42. (canceled) 43. A process for the manufacture of a composition according to claim 1 comprising the steps of: (i) subjecting a progestogen, an estrogen and at least one pharmaceutical acceptable excipient to a granulation process, (ii) mixing a tetrahydrofolic acid or a salt thereof with the granules formed in step (i), and (iii) optionally continuing the granulation process, and/or (iv) optionally collecting the granules. 44.-49. (canceled) 50. A method for female contraception comprising administering to a female a composition according to claim 1. 51.-54. (canceled) 55. A solid pharmaceutical composition comprising a progestogen, a tetrahydrofolic acid or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical acceptable excipient or carrier.

The present invention relates to solid pharmaceutical compositions, in particular to oral contraceptives, comprising a progestogen, such as drospirenone; an estrogen, such as ethinylestradiol; a tetrahydrofolic acid or a pharmaceutically acceptable salt thereof, such as calcium 5-methyl-(6S)-tetrahydrofolate; and at least one pharmaceutical acceptable excipient or carrier. The compositions of the invention provide good stability of the tetrahydrofolic acid upon storage while still ensuring a fast and reliable release of the estrogen and the progestogen present in the composition.1. A solid pharmaceutical composition comprising a progestogen, an estrogen, a tetrahydrofolic acid or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical acceptable excipient or carrier. 2.-42. (canceled) 43. A process for the manufacture of a composition according to claim 1 comprising the steps of: (i) subjecting a progestogen, an estrogen and at least one pharmaceutical acceptable excipient to a granulation process, (ii) mixing a tetrahydrofolic acid or a salt thereof with the granules formed in step (i), and (iii) optionally continuing the granulation process, and/or (iv) optionally collecting the granules. 44.-49. (canceled) 50. A method for female contraception comprising administering to a female a composition according to claim 1. 51.-54. (canceled) 55. A solid pharmaceutical composition comprising a progestogen, a tetrahydrofolic acid or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical acceptable excipient or carrier.

We have found a counter-intuitive way to improve the commercial-scale production of recombinant biological products in adherent-cell bioreactors, which reduces the risk of cell culture contamination, increases total yield and reduces the delay between seeding and harvest, thus minimizing expression product degradation, by inter alia inoculating an adherent culture bioreactor with suspension-adapted producer cells

1. A method for the production of a recombinant biological product, comprising: (b) obtaining suspension-adapted cells; and (c) inoculating the suspension-adapted cells obtained in (b) into a bioreactor having a carrier providing a surface area for adherent cell culture. 2. The method according to claim 1, further comprising: (a) before said obtaining step and before said inoculating step, expanding the suspension-adapted cells in non-adherent mode. 3. The method of claim 1, further comprising: (d) exposing said cells to a factor promoting adherence, in an amount effective to promote adherence, whereby after said inoculating step said cells adhere to said carrier in said bioreactor. 4. The method of claim 3, further comprising: (e) after said cells adhere to the carrier, introducing into said cells a transgene. 5. The method of claim 4, where said transgene is introduced into said cells by a viral vector. 6. A method according to claim 1, wherein said cells express a transgene in said bioreactor. 7. The method of claim 6, wherein said transgene is introduced into said cells after said inoculating step. 8. A method according to claim 6, wherein the transgene codes for a therapeutic protein. 9. The method according to claim 8, where the therapeutic protein comprises a polypeptide selected from the group consisting of: short-form VEGF-D3, endostatin, angiostatin, thymidine kinase, human interferon alpha-2b, ABCA4, ABC:D-1, myosin VITA, cyclooxygenase-2, PGF2-alpha receptor, dopamine, human hemoglobin subunit beta and an antibody subunit. 10. The method of claim 6, wherein the transgene codes for a polypeptide comprising a viral component selected from the group consisting of viral vector, viral-like particle, virus and viral vaccine. 11. The method of claim 10, where the polypeptide comprises a component of a viral vector. 12. The method of claim 11, where the viral vector is lentiviral. 13. The method of claim 11, wherein the polypeptide comprises at least about 1×1017 viral vector particles. 14. A gene therapy viral vector produced by the method of claim 1. 15. The gene therapy vector of' claim 14, wherein the gene therapy viral vector comprises a transgene coding for a polypeptide selected from the group consisting of: human interferon, human granulocyte-macrophage colony stimulating factor, Vascular Endothelial Growth Factor, endostatin, angiostatin, thymidine kinase, ABCA4. ABCD-1, myosin VIIA, cyclooxygenase-2, PGF2-alpha receptor, dopamine, human hemoglobin subunit beta and an antibody subunit. 16-17. (canceled) 18. The method according to claim 3, wherein said factor comprises a compound selected from the group consisting of: foetal bovine serum, fibronectin, collagen, laminin, calcium ions, proteoglycans of the extracellular matrix, non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. 19. A method according to claim 5, wherein the Multiplicity of' Infection (MOI) for said transfection is not more than about 10 viral particles per cell. 20-28. (canceled) 29. A method for treating cancer in a human comprising: b. Administering to said human the gene therapy viral vector of claim 14 in an amount effective to combat said cancer. 30. A method for treating cancer in a human comprising: a. Diagnosing cancer in a human; and then b. Administering to said human the gene therapy viral vector of claim 15 in an amount effective to combat said cancer. 31. The method of claim 30, wherein said cancer comprises bladder cancer. 32-34. (canceled)

We have found a counter-intuitive way to improve the commercial-scale production of recombinant biological products in adherent-cell bioreactors, which reduces the risk of cell culture contamination, increases total yield and reduces the delay between seeding and harvest, thus minimizing expression product degradation, by inter alia inoculating an adherent culture bioreactor with suspension-adapted producer cells1. A method for the production of a recombinant biological product, comprising: (b) obtaining suspension-adapted cells; and (c) inoculating the suspension-adapted cells obtained in (b) into a bioreactor having a carrier providing a surface area for adherent cell culture. 2. The method according to claim 1, further comprising: (a) before said obtaining step and before said inoculating step, expanding the suspension-adapted cells in non-adherent mode. 3. The method of claim 1, further comprising: (d) exposing said cells to a factor promoting adherence, in an amount effective to promote adherence, whereby after said inoculating step said cells adhere to said carrier in said bioreactor. 4. The method of claim 3, further comprising: (e) after said cells adhere to the carrier, introducing into said cells a transgene. 5. The method of claim 4, where said transgene is introduced into said cells by a viral vector. 6. A method according to claim 1, wherein said cells express a transgene in said bioreactor. 7. The method of claim 6, wherein said transgene is introduced into said cells after said inoculating step. 8. A method according to claim 6, wherein the transgene codes for a therapeutic protein. 9. The method according to claim 8, where the therapeutic protein comprises a polypeptide selected from the group consisting of: short-form VEGF-D3, endostatin, angiostatin, thymidine kinase, human interferon alpha-2b, ABCA4, ABC:D-1, myosin VITA, cyclooxygenase-2, PGF2-alpha receptor, dopamine, human hemoglobin subunit beta and an antibody subunit. 10. The method of claim 6, wherein the transgene codes for a polypeptide comprising a viral component selected from the group consisting of viral vector, viral-like particle, virus and viral vaccine. 11. The method of claim 10, where the polypeptide comprises a component of a viral vector. 12. The method of claim 11, where the viral vector is lentiviral. 13. The method of claim 11, wherein the polypeptide comprises at least about 1×1017 viral vector particles. 14. A gene therapy viral vector produced by the method of claim 1. 15. The gene therapy vector of' claim 14, wherein the gene therapy viral vector comprises a transgene coding for a polypeptide selected from the group consisting of: human interferon, human granulocyte-macrophage colony stimulating factor, Vascular Endothelial Growth Factor, endostatin, angiostatin, thymidine kinase, ABCA4. ABCD-1, myosin VIIA, cyclooxygenase-2, PGF2-alpha receptor, dopamine, human hemoglobin subunit beta and an antibody subunit. 16-17. (canceled) 18. The method according to claim 3, wherein said factor comprises a compound selected from the group consisting of: foetal bovine serum, fibronectin, collagen, laminin, calcium ions, proteoglycans of the extracellular matrix, non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. 19. A method according to claim 5, wherein the Multiplicity of' Infection (MOI) for said transfection is not more than about 10 viral particles per cell. 20-28. (canceled) 29. A method for treating cancer in a human comprising: b. Administering to said human the gene therapy viral vector of claim 14 in an amount effective to combat said cancer. 30. A method for treating cancer in a human comprising: a. Diagnosing cancer in a human; and then b. Administering to said human the gene therapy viral vector of claim 15 in an amount effective to combat said cancer. 31. The method of claim 30, wherein said cancer comprises bladder cancer. 32-34. (canceled)

The present invention relates to a method for the assembly and cloning of polynucleotides comprising highly similar polynucleotidic modules, that is highly versatile, does not require intermediate amplification step and can be easily automated for high throughput production of customized polynucleotidic modules.

1) A method of generating and assembling polynucleotides comprising arrays of at least two highly similar polynucleotidic modules comprising the steps of: a) generating at least one polynucleotidic building block comprising at least: one polynucleotidic module; a single cleavage site for a first restriction enzyme A, placed on one side of the polynucleotidic module; a single cleavage site for a second restriction enzyme B, placed on the other side of the polynucleotidic module; wherein A and B can produce compatible cohesive ends; wherein cleavage of said polynucleotidic building blocks with restriction enzyme A results in a polynucleotide comprising a polynucleotidic module flanked on one side by a cohesive end that can be re-ligated with a polynucleotide building block cleaved by restriction enzyme B without restoring a sequence cleavable by restriction enzyme A and/or B; wherein cleavage of said polynucleotidic building blocks with restriction enzyme B results in a polynucleotide comprising a polynucleotidic module flanked on one side by a cohesive end that can be re-ligated with a polynucleotide building block cleaved by restriction enzyme A without restoring a sequence cleavable by restriction enzyme A and/or B; b) generating “n” polynucleotides linked to a solid phase comprising at least: one polynucleotidic module; one end linked to a solid phase; a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase; a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; c) generating one C-terminal polynucleotidic building block comprising at least: one polynucleotidic module; a single cleavage site for a first restriction enzyme A, placed on one side of the polynucleotidic module; a single cleavage site for a second restriction enzyme B, placed on the other side of the polynucleotidic module; wherein cleavage of said polynucleotidic building block with restriction enzyme B results in a polynucleotide comprising a polynucleotide module flanked on one side by a cohesive end that cannot be re-ligated with a polynucleotide building block cleaved by restriction enzyme A and/or B; d) cutting said one C-terminal polynucleotidic building block of c) with restriction enzyme A; e) ligating the resulting C-terminal polynucleotidic module with the free end of one polynucleotide of b) immobilized on a solid phase, thereby producing a new immobilized polynucleotide comprising one additional polynucleotidic module; f) cutting the resulting new immobilized polynucleotide with restriction enzyme A, thus producing a new polynucleotide having a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme B; g) ligating the new polynucleotide with the free end of one polynucleotide of b) immobilized on a solid phase thus producing a new immobilized polynucleotide comprising one additional polynucleotidic module; 2) The method according to claim 1 wherein steps f) and g) are repeated N times to produce an immobilized polynucleotide having an array of n polynucleotidic modules wherein n=N+3. 3) A method according to claim 1 wherein step g) is replaced by the following steps: g′) cutting said at least one polynucleotidic building block of a) with restriction enzyme A; h) ligating the resulting polynucleotidic module with the free end of one polynucleotide of b) immobilized on a solid phase, thereby producing a new immobilized polynucleotide comprising one additional polynucleotidic module; i) cutting the resulting new immobilized polynucleotide with restriction enzyme B, thus producing a new immobilized polynucleotide having a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A; j) ligating on the new produced immobilized polynucleotide said new polynucleotide resulting from step f) thereby producing a new immobilized polynucleotide comprising “x” additional polynucleotidic modules wherein “x” is equal to the number of polynucleotidic modules present in said new polynucleotide resulting from step 0; 4) The method according to claim 3, further comprising the steps: a) cutting the resulting new immobilized polynucleotide of step j) with restriction enzyme A, thus producing a new polynucleotide having a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme B; b) ligating the new polynucleotide with the free end of one polynucleotide of b) immobilized on a solid phase thus producing a new immobilized polynucleotide comprising one additional polynucleotidic module; 5) The method according to claim 1, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) comprise a pre-assembly of more than one polynucleotidic module. 6) The method according to claim 1, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) comprises a fragment of building block. 7) The method according to claim 1, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) comprises a building block variant. 8) The method according to claim 1, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) comprises a polynucleotide sequence not highly similar to a polynucleotide module according to a). 9) The method according to claim 8, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) further comprises a fragment of building block according to a). 10) The method according to claim 1, wherein said one polynucleotide of b) has been generated by: b1) generating one polynucleotide linked to a solid phase comprising: one polynucleotidic module; one end linked to a solid phase; a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase; a single cleavage site for a second restriction enzyme B placed on the other side of the polynucleotide module; b2) cutting said polynucleotide linked to a solid phase with restriction enzyme B thereby obtaining a polynucleotide with a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; 11) The method according to claim 1, wherein said one polynucleotide of b) has been generated by: b1) generating one polynucleotide linked to a solid phase comprising: one end linked to a solid phase; a polynucleotide sequence not highly similar to a polynucleotide module according to a), wherein said polynucleotide sequence comprises a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase; a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; 12) The method according to claim 1 wherein said one polynucleotide of b) has been generated by: b1) generating one polynucleotide linked to a solid phase comprising: one end linked to a solid phase; a polynucleotide sequence not highly similar to a polynucleotide module according to a), wherein said polynucleotide sequence comprises a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase and a single cleavage site for a second restriction enzyme B placed on the other side of the polynucleotide module; b2) cutting said polynucleotide linked to a solid phase with restriction enzyme B thereby obtaining a polynucleotide with a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; 13) The method according to claim 1, wherein said one polynucleotide of b) has been generated by: b1) generating one polynucleotide linked to a solid phase comprising: one end linked to a solid phase; a polynucleotide sequence not highly similar to a polynucleotide module according to a), wherein said polynucleotide sequence comprises a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase; a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; b2) cutting a polynucleotidic building block described in a) with restriction enzyme A; b3) ligating the resulting polynucleotidic module with the free end of the polynucleotide immobilized on the solid phase; b4) cutting the resulting new immobilized polynucleotide with restriction enzyme B, thus producing a new immobilized polynucleotide comprising: one end linked to a solid phase; a polynucleotide sequence not highly similar to a polynucleotide module according to a); one polynucleotidic module; a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzymes A and/or B. 14) The method according to claim 1, wherein at least one polynucleotide linked to a solid phase of b) has been generated by a gene synthesis technology wherein said free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A has been obtained by using restriction enzymes or specific annealing and wherein said polynucleotide ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce at least a sequence cleavable by restriction enzyme A and/or B. 15) The method according to claim 8, wherein said polynucleotide sequence not highly similar to a polynucleotide module according to a) is linked to said solid phase. 16) The method according to claim 8, wherein at least one said polynucleotide linked to a solid phase of b) comprises a sequence not highly similar to a polynucleotide module according to a) encoding a N-terminal polypeptidic sequence of a TALE. 17) The method according to claim 8, wherein at least one said polynucleotidic building block of a) and/or said polynucleotidic building block of c) comprises a sequence not highly similar to a polynucleotide module according to a) encoding a C-terminal polypeptidic sequence of a TALE. 18) The method according to claim 1, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) further comprises at least one cleavage site for a restriction enzyme C located outward compared to restriction enzymes A and/or B cleavage sites. 19) The method according to claim 1, wherein the last polynucleotide of b) used comprises a single cleavage site for a restriction enzyme C placed on the side of the polynucleotide linked to a solid phase, located outward compared to restriction enzyme A cleavage site, wherein said cleavage with restriction enzyme C allows to unlink said polynucleotide from the solid phase. 20) The method according to claim 2, further comprising the step of unlinking said final polynucleotide comprising an array of polynucleotidic modules by cutting it with restriction enzyme C. 21) The method according to claim 2, further comprising the steps of: unlinking said final polynucleotide comprising an array of polynucleotidic modules of step g) by cutting it with restriction enzyme C; subcloning said final polynucleotide comprising an array of polynucleotidic modules into a plasmidic vector. 22) The method according to claim 21, further comprising the step of subcloning said final polynucleotide comprising an array of polynucleotidic modules from a plasmidic vector into another plasmidic vector by cutting it with restriction enzymes A and B; 23) The method according to claim 2, further comprising the steps of: unlinking said final polynucleotide comprising an array of polynucleotidic modules of step g) by cutting it with restriction enzymes A and/or B; subcloning said final polynucleotide comprising an array of polynucleotidic modules into a plasmidic vector. 24) The method according to claim 1, wherein said polynucleotidic modules to assemble share at least 85% similarity. 25) The method according to claim 1, wherein each polynucleotidic module encodes a Transcription Activator-like Effector (TALE) DNA binding repeat module. 26) The method according to claim 6, wherein said fragment of building block encodes at least a half Transcription Activator-like Effector (TALE) DNA binding repeat module. 27) The method according to claim 8, wherein said polynucleotide sequence not highly similar to a polynucleotide module according to a) encodes a C-terminal fragment of a TALE and wherein said fragment of building block encodes at least a half Transcription Activator-like Effector (TALE) DNA binding repeat module. 28) The method according to claim 1, wherein said restriction enzymes belong to typeIIS restriction enzymes. 29) The method according to claim 28, wherein said restriction enzymes are BbvI and SfaNI. 30) The method according to claim 18, wherein said restriction enzyme C is SfiI. 31) The method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) linked to a solid phase and/or said polynucleotidic building block of c) are part of a collection encoding polypeptidic repeated modules with Repeat Variable Dipeptide regions (RVDs) comprising a pair of amino acids responsible for recognizing one nucleotide selected from the group consisting of HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T. 32) A method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) linked to a solid phase and/or said polynucleotidic building block of c) are part of a collection encoding polypeptidic repeated modules with Repeat Variable Dipeptide regions (RVDs) comprising a pair of amino acids responsible for recognizing one nucleotide selected from the group consisting of HD for recognizing C, NG for recognizing T, NI for recognizing A, NN and NK for recognizing G. 33) A method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) linked to a solid phase and/or said polynucleotidic building block of c) are part of a collection encoding “y” polypeptidic repeated modules with Repeat Variable Dipeptide regions (RVDs) for recognizing “y” nucleotides via “y” pairs of amino acids wherein “y” is comprised between 1 and 8. 34) A method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) and/or said polynucleotidic building block of c) linked to a solid phase are part of a collection encoding two polypeptidic repeated modules with Repeat Variable Dipeptide regions (RVDs) for recognizing two nucleotides via two pairs of amino acids selected from the group listed in table 2. 35) A method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) and/or said polynucleotidic building block of c) linked to a solid phase are part of a collection encoding three polypeptidic repeated modules with Repeat Variable Dipeptide regions (RVDs) for recognizing three nucleotides via three pairs of amino acids selected from the group listed in table 3. 36) A method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) linked to a solid phase and/or said polynucleotidic building block of c) are part of a library of degenerated building blocks. 37) A method of conducting a high throughput custom-designed platform of TALE DNA binding domains comprising: a) receiving a DNA target sequence comprising “n” nucleotides, which TALE DNA binding domain has to bind; b) generating and assembling polynucleotidic repeated modules, each comprising a pair of amino acids for recognizing each one of the “n” nucleotides of said DNA target sequence according to the present invention, thus releasing a TALE DNA binding domain able to recognize said DNA target sequence; c) providing said custom-designed TALE DNA binding domains. 38) A method of conducting a high throughput custom-designed platform of chimeric protein derived from a TALE comprising: a) receiving a DNA target sequence comprising “n” nucleotides, which a chimeric protein derived from a TALE has to process; b) generating and assembling polynucleotidic repeated modules, each with RVDs comprising a pair of amino acids for recognizing each one of the “n” nucleotides of said DNA target sequence according to the method of claim 1, thus releasing a TALE binding domain able to recognize said DNA target sequence; c) fusing said DNA binding domain to a protein domain able to process said DNA target sequence; d) providing said custom-designed chimeric protein derived from a TALE.

The present invention relates to a method for the assembly and cloning of polynucleotides comprising highly similar polynucleotidic modules, that is highly versatile, does not require intermediate amplification step and can be easily automated for high throughput production of customized polynucleotidic modules.1) A method of generating and assembling polynucleotides comprising arrays of at least two highly similar polynucleotidic modules comprising the steps of: a) generating at least one polynucleotidic building block comprising at least: one polynucleotidic module; a single cleavage site for a first restriction enzyme A, placed on one side of the polynucleotidic module; a single cleavage site for a second restriction enzyme B, placed on the other side of the polynucleotidic module; wherein A and B can produce compatible cohesive ends; wherein cleavage of said polynucleotidic building blocks with restriction enzyme A results in a polynucleotide comprising a polynucleotidic module flanked on one side by a cohesive end that can be re-ligated with a polynucleotide building block cleaved by restriction enzyme B without restoring a sequence cleavable by restriction enzyme A and/or B; wherein cleavage of said polynucleotidic building blocks with restriction enzyme B results in a polynucleotide comprising a polynucleotidic module flanked on one side by a cohesive end that can be re-ligated with a polynucleotide building block cleaved by restriction enzyme A without restoring a sequence cleavable by restriction enzyme A and/or B; b) generating “n” polynucleotides linked to a solid phase comprising at least: one polynucleotidic module; one end linked to a solid phase; a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase; a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; c) generating one C-terminal polynucleotidic building block comprising at least: one polynucleotidic module; a single cleavage site for a first restriction enzyme A, placed on one side of the polynucleotidic module; a single cleavage site for a second restriction enzyme B, placed on the other side of the polynucleotidic module; wherein cleavage of said polynucleotidic building block with restriction enzyme B results in a polynucleotide comprising a polynucleotide module flanked on one side by a cohesive end that cannot be re-ligated with a polynucleotide building block cleaved by restriction enzyme A and/or B; d) cutting said one C-terminal polynucleotidic building block of c) with restriction enzyme A; e) ligating the resulting C-terminal polynucleotidic module with the free end of one polynucleotide of b) immobilized on a solid phase, thereby producing a new immobilized polynucleotide comprising one additional polynucleotidic module; f) cutting the resulting new immobilized polynucleotide with restriction enzyme A, thus producing a new polynucleotide having a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme B; g) ligating the new polynucleotide with the free end of one polynucleotide of b) immobilized on a solid phase thus producing a new immobilized polynucleotide comprising one additional polynucleotidic module; 2) The method according to claim 1 wherein steps f) and g) are repeated N times to produce an immobilized polynucleotide having an array of n polynucleotidic modules wherein n=N+3. 3) A method according to claim 1 wherein step g) is replaced by the following steps: g′) cutting said at least one polynucleotidic building block of a) with restriction enzyme A; h) ligating the resulting polynucleotidic module with the free end of one polynucleotide of b) immobilized on a solid phase, thereby producing a new immobilized polynucleotide comprising one additional polynucleotidic module; i) cutting the resulting new immobilized polynucleotide with restriction enzyme B, thus producing a new immobilized polynucleotide having a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A; j) ligating on the new produced immobilized polynucleotide said new polynucleotide resulting from step f) thereby producing a new immobilized polynucleotide comprising “x” additional polynucleotidic modules wherein “x” is equal to the number of polynucleotidic modules present in said new polynucleotide resulting from step 0; 4) The method according to claim 3, further comprising the steps: a) cutting the resulting new immobilized polynucleotide of step j) with restriction enzyme A, thus producing a new polynucleotide having a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme B; b) ligating the new polynucleotide with the free end of one polynucleotide of b) immobilized on a solid phase thus producing a new immobilized polynucleotide comprising one additional polynucleotidic module; 5) The method according to claim 1, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) comprise a pre-assembly of more than one polynucleotidic module. 6) The method according to claim 1, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) comprises a fragment of building block. 7) The method according to claim 1, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) comprises a building block variant. 8) The method according to claim 1, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) comprises a polynucleotide sequence not highly similar to a polynucleotide module according to a). 9) The method according to claim 8, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) further comprises a fragment of building block according to a). 10) The method according to claim 1, wherein said one polynucleotide of b) has been generated by: b1) generating one polynucleotide linked to a solid phase comprising: one polynucleotidic module; one end linked to a solid phase; a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase; a single cleavage site for a second restriction enzyme B placed on the other side of the polynucleotide module; b2) cutting said polynucleotide linked to a solid phase with restriction enzyme B thereby obtaining a polynucleotide with a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; 11) The method according to claim 1, wherein said one polynucleotide of b) has been generated by: b1) generating one polynucleotide linked to a solid phase comprising: one end linked to a solid phase; a polynucleotide sequence not highly similar to a polynucleotide module according to a), wherein said polynucleotide sequence comprises a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase; a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; 12) The method according to claim 1 wherein said one polynucleotide of b) has been generated by: b1) generating one polynucleotide linked to a solid phase comprising: one end linked to a solid phase; a polynucleotide sequence not highly similar to a polynucleotide module according to a), wherein said polynucleotide sequence comprises a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase and a single cleavage site for a second restriction enzyme B placed on the other side of the polynucleotide module; b2) cutting said polynucleotide linked to a solid phase with restriction enzyme B thereby obtaining a polynucleotide with a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; 13) The method according to claim 1, wherein said one polynucleotide of b) has been generated by: b1) generating one polynucleotide linked to a solid phase comprising: one end linked to a solid phase; a polynucleotide sequence not highly similar to a polynucleotide module according to a), wherein said polynucleotide sequence comprises a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase; a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; b2) cutting a polynucleotidic building block described in a) with restriction enzyme A; b3) ligating the resulting polynucleotidic module with the free end of the polynucleotide immobilized on the solid phase; b4) cutting the resulting new immobilized polynucleotide with restriction enzyme B, thus producing a new immobilized polynucleotide comprising: one end linked to a solid phase; a polynucleotide sequence not highly similar to a polynucleotide module according to a); one polynucleotidic module; a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzymes A and/or B. 14) The method according to claim 1, wherein at least one polynucleotide linked to a solid phase of b) has been generated by a gene synthesis technology wherein said free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A has been obtained by using restriction enzymes or specific annealing and wherein said polynucleotide ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce at least a sequence cleavable by restriction enzyme A and/or B. 15) The method according to claim 8, wherein said polynucleotide sequence not highly similar to a polynucleotide module according to a) is linked to said solid phase. 16) The method according to claim 8, wherein at least one said polynucleotide linked to a solid phase of b) comprises a sequence not highly similar to a polynucleotide module according to a) encoding a N-terminal polypeptidic sequence of a TALE. 17) The method according to claim 8, wherein at least one said polynucleotidic building block of a) and/or said polynucleotidic building block of c) comprises a sequence not highly similar to a polynucleotide module according to a) encoding a C-terminal polypeptidic sequence of a TALE. 18) The method according to claim 1, wherein at least one said polynucleotidic building block of a) and/or at least one polynucleotide linked to a solid phase of b) and/or said polynucleotidic building block of c) further comprises at least one cleavage site for a restriction enzyme C located outward compared to restriction enzymes A and/or B cleavage sites. 19) The method according to claim 1, wherein the last polynucleotide of b) used comprises a single cleavage site for a restriction enzyme C placed on the side of the polynucleotide linked to a solid phase, located outward compared to restriction enzyme A cleavage site, wherein said cleavage with restriction enzyme C allows to unlink said polynucleotide from the solid phase. 20) The method according to claim 2, further comprising the step of unlinking said final polynucleotide comprising an array of polynucleotidic modules by cutting it with restriction enzyme C. 21) The method according to claim 2, further comprising the steps of: unlinking said final polynucleotide comprising an array of polynucleotidic modules of step g) by cutting it with restriction enzyme C; subcloning said final polynucleotide comprising an array of polynucleotidic modules into a plasmidic vector. 22) The method according to claim 21, further comprising the step of subcloning said final polynucleotide comprising an array of polynucleotidic modules from a plasmidic vector into another plasmidic vector by cutting it with restriction enzymes A and B; 23) The method according to claim 2, further comprising the steps of: unlinking said final polynucleotide comprising an array of polynucleotidic modules of step g) by cutting it with restriction enzymes A and/or B; subcloning said final polynucleotide comprising an array of polynucleotidic modules into a plasmidic vector. 24) The method according to claim 1, wherein said polynucleotidic modules to assemble share at least 85% similarity. 25) The method according to claim 1, wherein each polynucleotidic module encodes a Transcription Activator-like Effector (TALE) DNA binding repeat module. 26) The method according to claim 6, wherein said fragment of building block encodes at least a half Transcription Activator-like Effector (TALE) DNA binding repeat module. 27) The method according to claim 8, wherein said polynucleotide sequence not highly similar to a polynucleotide module according to a) encodes a C-terminal fragment of a TALE and wherein said fragment of building block encodes at least a half Transcription Activator-like Effector (TALE) DNA binding repeat module. 28) The method according to claim 1, wherein said restriction enzymes belong to typeIIS restriction enzymes. 29) The method according to claim 28, wherein said restriction enzymes are BbvI and SfaNI. 30) The method according to claim 18, wherein said restriction enzyme C is SfiI. 31) The method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) linked to a solid phase and/or said polynucleotidic building block of c) are part of a collection encoding polypeptidic repeated modules with Repeat Variable Dipeptide regions (RVDs) comprising a pair of amino acids responsible for recognizing one nucleotide selected from the group consisting of HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T. 32) A method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) linked to a solid phase and/or said polynucleotidic building block of c) are part of a collection encoding polypeptidic repeated modules with Repeat Variable Dipeptide regions (RVDs) comprising a pair of amino acids responsible for recognizing one nucleotide selected from the group consisting of HD for recognizing C, NG for recognizing T, NI for recognizing A, NN and NK for recognizing G. 33) A method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) linked to a solid phase and/or said polynucleotidic building block of c) are part of a collection encoding “y” polypeptidic repeated modules with Repeat Variable Dipeptide regions (RVDs) for recognizing “y” nucleotides via “y” pairs of amino acids wherein “y” is comprised between 1 and 8. 34) A method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) and/or said polynucleotidic building block of c) linked to a solid phase are part of a collection encoding two polypeptidic repeated modules with Repeat Variable Dipeptide regions (RVDs) for recognizing two nucleotides via two pairs of amino acids selected from the group listed in table 2. 35) A method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) and/or said polynucleotidic building block of c) linked to a solid phase are part of a collection encoding three polypeptidic repeated modules with Repeat Variable Dipeptide regions (RVDs) for recognizing three nucleotides via three pairs of amino acids selected from the group listed in table 3. 36) A method according to claim 1 wherein said at least one polynucleotidic building blocks of a) and/or said polynucleotides of b) linked to a solid phase and/or said polynucleotidic building block of c) are part of a library of degenerated building blocks. 37) A method of conducting a high throughput custom-designed platform of TALE DNA binding domains comprising: a) receiving a DNA target sequence comprising “n” nucleotides, which TALE DNA binding domain has to bind; b) generating and assembling polynucleotidic repeated modules, each comprising a pair of amino acids for recognizing each one of the “n” nucleotides of said DNA target sequence according to the present invention, thus releasing a TALE DNA binding domain able to recognize said DNA target sequence; c) providing said custom-designed TALE DNA binding domains. 38) A method of conducting a high throughput custom-designed platform of chimeric protein derived from a TALE comprising: a) receiving a DNA target sequence comprising “n” nucleotides, which a chimeric protein derived from a TALE has to process; b) generating and assembling polynucleotidic repeated modules, each with RVDs comprising a pair of amino acids for recognizing each one of the “n” nucleotides of said DNA target sequence according to the method of claim 1, thus releasing a TALE binding domain able to recognize said DNA target sequence; c) fusing said DNA binding domain to a protein domain able to process said DNA target sequence; d) providing said custom-designed chimeric protein derived from a TALE.

The present invention relates to a method and apparatus for simulating blood flow through a cardiovascular structure, e.g. a blood cavity such as the left ventricle outflow tract, the aortic root including the AV, plus ascending aorta, a ventricle volume, the aorta or any other cavity where blood flows through, under patient-specific boundary conditions derived from the cardiac ejection output per heart stroke. The cardiac ejection output can be estimated from volumes of a heart chamber of the patient in different filling states at two or more different points in time. The results of the flow simulation can be used to derive at least one physiological parameter or can be visualized and virtual Doppler ultrasound images may be generated to allow a physician assessing the result.

1. An apparatus for simulating blood flow through a cardiovascular structure close to the heart of a patient, said apparatus comprising: an estimation circuit for estimating a cardiac ejection output per heart stroke based on a volume of at least one heart chamber of said patient in different filling states at two or more points in time, and for deriving at least one patient-specific boundary condition for the flow through said cardiovascular structure from the cardiac ejection output per heart stroke; and a simulation circuit for simulating a blood flow through a volumetric mesh of said cardiovascular structure under consideration of said patient-specific boundary conditions, to visualize said blood flow or to derive at least one physiological parameter of said patient. 2. The apparatus according to claim 1, further comprising a modeling circuit for generating said volumetric mesh of said cardiovascular structure based on partitioned segmented digital image of said cardiovascular structure. 3. The apparatus according to claim 1, wherein said digital image is a computed tomography image or a magnetic resonance image or an ultrasonic image. 4. The apparatus according to claim 1, wherein said apparatus is adapted to derive from said simulated blood flow at one or more of a pressure drop, an average blood residence time, a flow rate, a wall sheer stress and a blood swirl in said cardiovascular structure. 5. A method of simulating blood flow through a cardiovascular structure close to the heart of a patient, said method comprising: estimating a cardiac ejection output per heart stroke based on a volume of at least one heart chamber of said patient in different filling states at two or more points in time; deriving at least one patient-specific boundary condition for the flow through said cardiovascular structure from the cardiac ejection output per heart stroke; and simulating a blood flow through a volumetric mesh of said cardiovascular structure under consideration of said patient-specific boundary conditions, to visualize said blood flow or to derive at least one physiological parameter of said patient. 6. The method according to claim 5, further comprising partitioning said digital image by using a model-based segmentation to obtain a surface mesh of said cardiovascular structure. 7. The method according to claim 5, wherein said simulation is a computational fluid dynamics simulation or a fluid-solid interaction simulation. 8. The method according to claim 5, further comprising estimating said cardiac ejection output per heart stroke based on electrocardiography gated digital images. 9. The method according to claim 5, further comprising estimating said cardiac ejection output per heart stroke based on digital images of said at least one heart chamber in a maximum filling state and a minimum filling state. 10. The method according to claim 5, further comprising using said estimated cardiac ejection output per heart stroke to define a blood flow from a ventricular outflow tract to said cardiovascular structure. 11. The method according to claim 10, further comprising deriving said at least one patient-specific boundary condition by estimating a flow profile across said ventricular outflow tract and its temporal behavior. 12. The method according to claim 11, further comprising estimating said flow profile by defining a quadratic profile or a velocity profile of a pulsatile flow. 13. The method according to claim 5, further comprising estimating said cardiac ejection output per heart stroke based on volumes of said at least one heart chamber of said patient at an end of systole and at an end of a diastole. 14. The method according to claim 5, further comprising generating virtual Doppler ultrasound images based on said simulated blood flow. 15. A computer program product comprising code means for producing the steps of claim 5 when run on a computing device.

The present invention relates to a method and apparatus for simulating blood flow through a cardiovascular structure, e.g. a blood cavity such as the left ventricle outflow tract, the aortic root including the AV, plus ascending aorta, a ventricle volume, the aorta or any other cavity where blood flows through, under patient-specific boundary conditions derived from the cardiac ejection output per heart stroke. The cardiac ejection output can be estimated from volumes of a heart chamber of the patient in different filling states at two or more different points in time. The results of the flow simulation can be used to derive at least one physiological parameter or can be visualized and virtual Doppler ultrasound images may be generated to allow a physician assessing the result.1. An apparatus for simulating blood flow through a cardiovascular structure close to the heart of a patient, said apparatus comprising: an estimation circuit for estimating a cardiac ejection output per heart stroke based on a volume of at least one heart chamber of said patient in different filling states at two or more points in time, and for deriving at least one patient-specific boundary condition for the flow through said cardiovascular structure from the cardiac ejection output per heart stroke; and a simulation circuit for simulating a blood flow through a volumetric mesh of said cardiovascular structure under consideration of said patient-specific boundary conditions, to visualize said blood flow or to derive at least one physiological parameter of said patient. 2. The apparatus according to claim 1, further comprising a modeling circuit for generating said volumetric mesh of said cardiovascular structure based on partitioned segmented digital image of said cardiovascular structure. 3. The apparatus according to claim 1, wherein said digital image is a computed tomography image or a magnetic resonance image or an ultrasonic image. 4. The apparatus according to claim 1, wherein said apparatus is adapted to derive from said simulated blood flow at one or more of a pressure drop, an average blood residence time, a flow rate, a wall sheer stress and a blood swirl in said cardiovascular structure. 5. A method of simulating blood flow through a cardiovascular structure close to the heart of a patient, said method comprising: estimating a cardiac ejection output per heart stroke based on a volume of at least one heart chamber of said patient in different filling states at two or more points in time; deriving at least one patient-specific boundary condition for the flow through said cardiovascular structure from the cardiac ejection output per heart stroke; and simulating a blood flow through a volumetric mesh of said cardiovascular structure under consideration of said patient-specific boundary conditions, to visualize said blood flow or to derive at least one physiological parameter of said patient. 6. The method according to claim 5, further comprising partitioning said digital image by using a model-based segmentation to obtain a surface mesh of said cardiovascular structure. 7. The method according to claim 5, wherein said simulation is a computational fluid dynamics simulation or a fluid-solid interaction simulation. 8. The method according to claim 5, further comprising estimating said cardiac ejection output per heart stroke based on electrocardiography gated digital images. 9. The method according to claim 5, further comprising estimating said cardiac ejection output per heart stroke based on digital images of said at least one heart chamber in a maximum filling state and a minimum filling state. 10. The method according to claim 5, further comprising using said estimated cardiac ejection output per heart stroke to define a blood flow from a ventricular outflow tract to said cardiovascular structure. 11. The method according to claim 10, further comprising deriving said at least one patient-specific boundary condition by estimating a flow profile across said ventricular outflow tract and its temporal behavior. 12. The method according to claim 11, further comprising estimating said flow profile by defining a quadratic profile or a velocity profile of a pulsatile flow. 13. The method according to claim 5, further comprising estimating said cardiac ejection output per heart stroke based on volumes of said at least one heart chamber of said patient at an end of systole and at an end of a diastole. 14. The method according to claim 5, further comprising generating virtual Doppler ultrasound images based on said simulated blood flow. 15. A computer program product comprising code means for producing the steps of claim 5 when run on a computing device.

In certain embodiments, the invention relates to systems and methods for in vivo tomographic imaging of fluorescent probes and/or bioluminescent reporters, wherein a fluorescent probe and a bioluminescent reporter are spatially co-localized (e.g., located at distances equivalent to or smaller than the scattering mean free path of light) in a diffusive medium (e.g., biological tissue). Measurements obtained from bioluminescent and fluorescent modalities are combined per methods described herein.

1. A method for imaging a target region of a diffuse object, the method comprising: (a) administering a bioluminescent substrate and/or chemiluminescent substrate to the object; (b) detecting bioluminescent and/or chemiluminescent light emitted from the object by a bioluminescent reporter in the target region of the object; (c) administering a probe comprising a fluorescent species and/or a jointly fluorescent/bioluminescent species to the object; (d) directing excitation light into the object at multiple locations and/or at multiple angles, thereby exciting the fluorescent species; (e) detecting fluorescent light, the detected fluorescent light having been emitted by the fluorescent species in the target region of the diffuse object as a result of excitation by the excitation light; (f) detecting excitation light, the detected excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object; and (g) processing data corresponding to the detected bioluminescent light, the detected fluorescent light, and the detected excitation light to provide an image of the target region within the diffuse object. 2. The method of claim 1, wherein the bioluminescent reporter is endogenous. 3. The method of claim 2, wherein the bioluminescent reporter is expressed within the object by a bioluminescent cell line. 4. The method of claim 1, wherein the bioluminescent reporter is exogenously administered. 5. (canceled) 6. The method of claim 1, wherein, following step (c), at least some of the bioluminescent reporter is substantially co-located with at least some of the (fluorescent) probe in the target region of the object. 7. The method of claim 6, wherein, following step (c), the bioluminescent reporter is also present in the object at one or more locations that are not substantially co-located with the (fluorescent) probe. 8. The method of claim 1, wherein the probe in step (c) comprises both the fluorescent species and the bioluminescent reporter in tandem with the fluorescent species. 9. The method of claim 1, wherein the diffuse object comprises living biological tissue. 10. (canceled) 11. (canceled) 12. The method of claim 1, further comprising placing the object within a holder prior to steps (b), (d), (e), and (f). 13. The method of claim 12, wherein the image of the target region is a tomographic image and wherein a surface of the object is at least partially conformed by the holder such that the surface can be characterized by a continuous function, thereby facilitating tomographic reconstruction. 14.-39. (canceled) 40. The method of claim 1, wherein the target region of the object is a three-dimensional region and step (g) comprises providing a tomographic image that corresponds to the fluorescent species in the three-dimensional target region. 41. (canceled) 42. The method of claim 40, wherein the tomographic image indicates concentration of the probe as a function of position within the target region of the object. 43. (canceled) 44. The method of claim 1, wherein the target region of the object is a three-dimensional region and step (g) comprises providing a tomographic image that corresponds to the bioluminescent reporter in the three-dimensional target region. 45.-47. (canceled) 48. The method of claim 1, wherein step (g) comprises weighting data corresponding to the detected fluorescent light with data corresponding to the detected bioluminescent light and normalizing data corresponding to the detected fluorescent light with data corresponding to the detected excitation light. 49. The method of claim 1, wherein step (g) comprises weighting data corresponding to the detected fluorescent light with data corresponding to the detected bioluminescent light and then normalizing the resulting weighted data with data corresponding to the detected excitation light, then inverting an associated weight matrix to obtain a/the tomographic image of the fluorescent species in the target region of the object. 50. The method of claim 1, wherein step (g) comprises weighting data corresponding to the detected fluorescent light with data corresponding to the detected bioluminescent light, and weighting an associated weight matrix with data corresponding to the detected bioluminescent light, then inverting the weight matrix to obtain a/the tomographic image of the fluorescent species in the target region of the object. 51. The method of claim 1, wherein step (g) comprises weighting data corresponding to the detected bioluminescent light with data corresponding to the detected fluorescent light normalized with data corresponding to the detected excitation light, then inverting an associated weight matrix to obtain a/the tomographic image of the bioluminescent reporter in the target region of the object. 52. The method of claim 1, wherein the detecting in step (b) is performed at a different time than the detecting in steps (e) and (f). 53. (canceled) 54. (canceled) 55. A system for imaging a target region within a diffuse object, the system comprising: an excitation light source; an optical imaging apparatus configured to direct light from the excitation light source into the diffuse object at multiple locations and/or at multiple angles; one or more detectors, the one or more detectors individually or collectively configured to detect and/or measure, (i) bioluminescent (and/or chemiluminescent) light emitted from the object, (ii) fluorescent light emitted by a fluorescent species in the target region of the diffuse object as a result of excitation by the excitation light, and (iii) excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object; and a processor configured to process data corresponding to the detected bioluminescent light, the detected fluorescent light, and the detected excitation light to provide an image of the target region within the diffuse object. 56. An apparatus for reconstructing a tomographic representation of a probe within a target region of a diffuse object, the apparatus comprising: a memory that stores code defining a set of instructions; and a processor that executes the instructions thereby to process data corresponding to: (a) establish a forward model of excitation light propagation from an excitation light source to the probe within the target region of the object and of emission light propagation from the probe to a detector using (i) data corresponding to detected fluorescent light from the probe, (ii) data corresponding to detected excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object, and (iii) data corresponding to detected bioluminescent light emitted from a bioluminescent reporter, wherein at least some of the bioluminescent reporter is substantially co-located with at least some of the (fluorescent) probe in the target region of the object, and wherein the forward model is established as a weight matrix; and (b) invert the weight matrix to obtain the tomographic representation of the probe within the target region of the diffuse object. 57. (canceled) 58. (canceled)

In certain embodiments, the invention relates to systems and methods for in vivo tomographic imaging of fluorescent probes and/or bioluminescent reporters, wherein a fluorescent probe and a bioluminescent reporter are spatially co-localized (e.g., located at distances equivalent to or smaller than the scattering mean free path of light) in a diffusive medium (e.g., biological tissue). Measurements obtained from bioluminescent and fluorescent modalities are combined per methods described herein.1. A method for imaging a target region of a diffuse object, the method comprising: (a) administering a bioluminescent substrate and/or chemiluminescent substrate to the object; (b) detecting bioluminescent and/or chemiluminescent light emitted from the object by a bioluminescent reporter in the target region of the object; (c) administering a probe comprising a fluorescent species and/or a jointly fluorescent/bioluminescent species to the object; (d) directing excitation light into the object at multiple locations and/or at multiple angles, thereby exciting the fluorescent species; (e) detecting fluorescent light, the detected fluorescent light having been emitted by the fluorescent species in the target region of the diffuse object as a result of excitation by the excitation light; (f) detecting excitation light, the detected excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object; and (g) processing data corresponding to the detected bioluminescent light, the detected fluorescent light, and the detected excitation light to provide an image of the target region within the diffuse object. 2. The method of claim 1, wherein the bioluminescent reporter is endogenous. 3. The method of claim 2, wherein the bioluminescent reporter is expressed within the object by a bioluminescent cell line. 4. The method of claim 1, wherein the bioluminescent reporter is exogenously administered. 5. (canceled) 6. The method of claim 1, wherein, following step (c), at least some of the bioluminescent reporter is substantially co-located with at least some of the (fluorescent) probe in the target region of the object. 7. The method of claim 6, wherein, following step (c), the bioluminescent reporter is also present in the object at one or more locations that are not substantially co-located with the (fluorescent) probe. 8. The method of claim 1, wherein the probe in step (c) comprises both the fluorescent species and the bioluminescent reporter in tandem with the fluorescent species. 9. The method of claim 1, wherein the diffuse object comprises living biological tissue. 10. (canceled) 11. (canceled) 12. The method of claim 1, further comprising placing the object within a holder prior to steps (b), (d), (e), and (f). 13. The method of claim 12, wherein the image of the target region is a tomographic image and wherein a surface of the object is at least partially conformed by the holder such that the surface can be characterized by a continuous function, thereby facilitating tomographic reconstruction. 14.-39. (canceled) 40. The method of claim 1, wherein the target region of the object is a three-dimensional region and step (g) comprises providing a tomographic image that corresponds to the fluorescent species in the three-dimensional target region. 41. (canceled) 42. The method of claim 40, wherein the tomographic image indicates concentration of the probe as a function of position within the target region of the object. 43. (canceled) 44. The method of claim 1, wherein the target region of the object is a three-dimensional region and step (g) comprises providing a tomographic image that corresponds to the bioluminescent reporter in the three-dimensional target region. 45.-47. (canceled) 48. The method of claim 1, wherein step (g) comprises weighting data corresponding to the detected fluorescent light with data corresponding to the detected bioluminescent light and normalizing data corresponding to the detected fluorescent light with data corresponding to the detected excitation light. 49. The method of claim 1, wherein step (g) comprises weighting data corresponding to the detected fluorescent light with data corresponding to the detected bioluminescent light and then normalizing the resulting weighted data with data corresponding to the detected excitation light, then inverting an associated weight matrix to obtain a/the tomographic image of the fluorescent species in the target region of the object. 50. The method of claim 1, wherein step (g) comprises weighting data corresponding to the detected fluorescent light with data corresponding to the detected bioluminescent light, and weighting an associated weight matrix with data corresponding to the detected bioluminescent light, then inverting the weight matrix to obtain a/the tomographic image of the fluorescent species in the target region of the object. 51. The method of claim 1, wherein step (g) comprises weighting data corresponding to the detected bioluminescent light with data corresponding to the detected fluorescent light normalized with data corresponding to the detected excitation light, then inverting an associated weight matrix to obtain a/the tomographic image of the bioluminescent reporter in the target region of the object. 52. The method of claim 1, wherein the detecting in step (b) is performed at a different time than the detecting in steps (e) and (f). 53. (canceled) 54. (canceled) 55. A system for imaging a target region within a diffuse object, the system comprising: an excitation light source; an optical imaging apparatus configured to direct light from the excitation light source into the diffuse object at multiple locations and/or at multiple angles; one or more detectors, the one or more detectors individually or collectively configured to detect and/or measure, (i) bioluminescent (and/or chemiluminescent) light emitted from the object, (ii) fluorescent light emitted by a fluorescent species in the target region of the diffuse object as a result of excitation by the excitation light, and (iii) excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object; and a processor configured to process data corresponding to the detected bioluminescent light, the detected fluorescent light, and the detected excitation light to provide an image of the target region within the diffuse object. 56. An apparatus for reconstructing a tomographic representation of a probe within a target region of a diffuse object, the apparatus comprising: a memory that stores code defining a set of instructions; and a processor that executes the instructions thereby to process data corresponding to: (a) establish a forward model of excitation light propagation from an excitation light source to the probe within the target region of the object and of emission light propagation from the probe to a detector using (i) data corresponding to detected fluorescent light from the probe, (ii) data corresponding to detected excitation light having been transmitted through the region of the diffuse object or having been reflected from the region of the diffuse object, and (iii) data corresponding to detected bioluminescent light emitted from a bioluminescent reporter, wherein at least some of the bioluminescent reporter is substantially co-located with at least some of the (fluorescent) probe in the target region of the object, and wherein the forward model is established as a weight matrix; and (b) invert the weight matrix to obtain the tomographic representation of the probe within the target region of the diffuse object. 57. (canceled) 58. (canceled)

Disclosed are oral care compositions comprising an orally acceptable vehicle, a basic amino acid in free or salt form, particles of precipitated calcium carbonate, a source of zinc ions, and a surfactant system selected from at least one of a poloxamer nonionic surfactant and a betaine zwitterionic surfactant or a mixture thereof.

1. An oral care composition comprising an orally acceptable vehicle, a basic amino acid in free or salt form, calcium carbonate, a source of zinc ions, and at least one surfactant selected from a nonionic block copolymer surfactant and a betaine zwitterionic surfactant or a mixture thereof. 2. The oral care composition according to claim 1 wherein the nonionic block copolymer surfactant is present in an amount of from 0.25 to 2 wt % based on the weight of the oral care composition. 3. The oral care composition according to claim 2 wherein the nonionic block copolymer surfactant is present in an amount of from 0.5 to 1.5 wt % based on the weight of the oral care composition. 4. The oral care composition according to claim 3 wherein the nonionic block copolymer surfactant is present in an amount of about 1 wt % based on the weight of the oral care composition. 5. The oral care composition according to claim 1 wherein the nonionic block copolymer surfactant has a polyoxypropylene molecular mass of from 3000 to 5000 g/mol and a polyoxyethylene content of from 60 to 80 mol %. 6. The oral care composition according to claim 1, wherein the nonionic block copolymer surfactant comprises a poloxamer. 7. The oral care composition according to claim 6 wherein the poloxamer nonionic surfactant comprises poloxamer 407. 8. The oral care composition according to claim 1 wherein the betaine zwitterionic surfactant is present in an amount of from 0.25 to 2 wt % based on the weight of the oral care composition. 9. The oral care composition according to claim 8 wherein the betaine zwitterionic surfactant is present in an amount of from 0.5 to 1.5 wt % based on the weight of the oral care composition. 10. The oral care composition according to claim 9 wherein the betaine zwitterionic surfactant is present in an amount of about 1 wt % based on the weight of the oral care composition. 11. The oral care composition according to claim 1 wherein the betaine zwitterionic surfactant comprises a C8-C16 amidopropyl betaine. 12. The oral care composition according to claim 11 wherein the betaine zwitterionic surfactant comprises cocamidopropyl betaine. 13. The oral care composition according to claim 1 wherein the calcium carbonate is present in an amount of from 10 to 50 wt % based on the weight of the oral care composition. 14. The oral care composition according to claim 13 wherein the calcium carbonate is present in an amount of from 25 to 40 wt % based on the weight of the oral care composition. 15. The oral care composition according to claim 1 wherein the calcium carbonate has an average particle size of no greater than a dentin tubule of a mammalian tooth. 16. The oral care composition according to claim 1 wherein the calcium carbonate has an average particle size of from 1 to 5 microns. 17. The oral care composition according to claim 1 wherein the calcium carbonate comprises a mixture of first particles having a particle size range of from 1 to 7 microns and second particles having a particle size range of from 0.5 to 6 microns. 18. The oral care composition according to claim 17 wherein the first particles are present in an amount of from 5 to 20 wt % based on the weight of the oral care composition and the second particles are present in an amount of from 5 to 40 wt % based on the weight of the oral care composition. 19. The oral care composition according to claim 18 wherein the first particles are present in an amount of from 5 to 15 wt % based on the weight of the oral care composition and the second particles are present in an amount of from 20 to 30 wt % based on the weight of the oral care composition. 20. The oral care composition according to claim 1 wherein the basic amino acid in free or salt form comprises arginine bicarbonate. 21. The oral care composition according to claim 1 wherein the basic amino acid in free or salt form is present in an amount of from 5 to 15 wt % based on the weight of the oral care composition. 22. The oral care composition according to claim 21 wherein the basic amino acid in free or salt form is present in an amount of from 7 to 12 wt % based on the weight of the oral care composition. 23. The oral care composition according to claim 1 wherein the zinc ion source comprises at least one of zinc citrate, zinc lactate, zinc gluconate or zinc oxide, or any mixture of any two or more thereof. 24. The oral care composition according to claim 23 wherein the zinc ion source is present in an amount of from 0.5 to 3 wt % based on the weight of the oral care composition. 25. The oral care composition according to claim 24 wherein the zinc ion source is present in an amount of from 1 to 2 wt % based on the weight of the oral care composition. 26. The oral care composition according to claim 23 wherein the zinc ion source comprises a mixture of zinc citrate and zinc oxide. 27. The oral care composition according to claim 26 wherein zinc citrate is present in an amount of from 0.25 to 0.75 wt % based on the weight of the oral care composition and zinc oxide is present in an amount of from 0.75 to 1.25 wt % based on the weight of the oral care composition. 28. The oral care composition according to claim 1, wherein the basic amino acid in free or salt form comprises arginine bicarbonate, the zinc ion source comprises zinc oxide, and wherein the composition comprises a nonionic block copolymer surfactant comprising poloxamer 407. 29. The oral care composition according to claim 1, wherein the basic amino acid in free or salt form comprises arginine bicarbonate, the zinc ion source comprises zinc oxide, and wherein the composition comprises a betaine zwitterionic surfactant comprising cocamidopropyl betaine. 30. The oral care composition according to claim 1, wherein the basic amino acid in free or salt form comprises arginine bicarbonate, the zinc ion source comprises a mixture of zinc citrate and zinc oxide, and wherein the composition comprises a nonionic block copolymer surfactant comprising poloxamer 407 and a betaine zwitterionic surfactant comprising cocamidopropyl betaine. 31. The oral care composition according to claim 1 wherein the orally acceptable vehicle comprises sorbitol which is present in an amount of from 12 to 30 wt % based on the weight of the oral care composition. 32. The oral care composition according to claim 31 wherein the sorbitol is present in an amount of from 15 to 25 wt % based on the weight of the oral care composition. 33. The oral care composition according to claim 1 wherein the oral care composition does not comprise any sodium lauryl sulfate. 34. The oral care composition according to claim 33 wherein the oral care composition does not comprise any anionic surfactant. 35. The oral care composition according to claim 1 wherein the composition is formulated into a dentifrice in the form of a paste or gel. 36. The oral care composition according to claim 1 wherein the composition is formulated into a form adapted to be applied undiluted within the oral cavity directly to the surface of a mammalian tooth and to be retained within the cavity on the surface for a period of at least 1 hour for treating or preventing hypersensitivity of the tooth. 37. The oral care composition according to claim 1 for enhancing the uptake of zinc ions by the surface of a mammalian tooth in a method comprising applying the oral care composition to the tooth surface. 38. The oral care composition according to claim 1 for increasing the foaming of the oral care composition in an oral cavity in a method comprising applying the composition to the surface of a mammalian tooth in the oral cavity. 39. A method of reducing dental sensitivity comprising applying to the surface of a mammalian tooth an oral care composition of claim 1. 40. A method of occluding a dentin tubule of a mammalian tooth comprising applying to the tooth surface a composition according to claim 1. 41. A method of enhancing the uptake of zinc ions to a mammalian tooth comprising applying to the tooth surface a composition according to claim 1. 42-54. (canceled)

Disclosed are oral care compositions comprising an orally acceptable vehicle, a basic amino acid in free or salt form, particles of precipitated calcium carbonate, a source of zinc ions, and a surfactant system selected from at least one of a poloxamer nonionic surfactant and a betaine zwitterionic surfactant or a mixture thereof.1. An oral care composition comprising an orally acceptable vehicle, a basic amino acid in free or salt form, calcium carbonate, a source of zinc ions, and at least one surfactant selected from a nonionic block copolymer surfactant and a betaine zwitterionic surfactant or a mixture thereof. 2. The oral care composition according to claim 1 wherein the nonionic block copolymer surfactant is present in an amount of from 0.25 to 2 wt % based on the weight of the oral care composition. 3. The oral care composition according to claim 2 wherein the nonionic block copolymer surfactant is present in an amount of from 0.5 to 1.5 wt % based on the weight of the oral care composition. 4. The oral care composition according to claim 3 wherein the nonionic block copolymer surfactant is present in an amount of about 1 wt % based on the weight of the oral care composition. 5. The oral care composition according to claim 1 wherein the nonionic block copolymer surfactant has a polyoxypropylene molecular mass of from 3000 to 5000 g/mol and a polyoxyethylene content of from 60 to 80 mol %. 6. The oral care composition according to claim 1, wherein the nonionic block copolymer surfactant comprises a poloxamer. 7. The oral care composition according to claim 6 wherein the poloxamer nonionic surfactant comprises poloxamer 407. 8. The oral care composition according to claim 1 wherein the betaine zwitterionic surfactant is present in an amount of from 0.25 to 2 wt % based on the weight of the oral care composition. 9. The oral care composition according to claim 8 wherein the betaine zwitterionic surfactant is present in an amount of from 0.5 to 1.5 wt % based on the weight of the oral care composition. 10. The oral care composition according to claim 9 wherein the betaine zwitterionic surfactant is present in an amount of about 1 wt % based on the weight of the oral care composition. 11. The oral care composition according to claim 1 wherein the betaine zwitterionic surfactant comprises a C8-C16 amidopropyl betaine. 12. The oral care composition according to claim 11 wherein the betaine zwitterionic surfactant comprises cocamidopropyl betaine. 13. The oral care composition according to claim 1 wherein the calcium carbonate is present in an amount of from 10 to 50 wt % based on the weight of the oral care composition. 14. The oral care composition according to claim 13 wherein the calcium carbonate is present in an amount of from 25 to 40 wt % based on the weight of the oral care composition. 15. The oral care composition according to claim 1 wherein the calcium carbonate has an average particle size of no greater than a dentin tubule of a mammalian tooth. 16. The oral care composition according to claim 1 wherein the calcium carbonate has an average particle size of from 1 to 5 microns. 17. The oral care composition according to claim 1 wherein the calcium carbonate comprises a mixture of first particles having a particle size range of from 1 to 7 microns and second particles having a particle size range of from 0.5 to 6 microns. 18. The oral care composition according to claim 17 wherein the first particles are present in an amount of from 5 to 20 wt % based on the weight of the oral care composition and the second particles are present in an amount of from 5 to 40 wt % based on the weight of the oral care composition. 19. The oral care composition according to claim 18 wherein the first particles are present in an amount of from 5 to 15 wt % based on the weight of the oral care composition and the second particles are present in an amount of from 20 to 30 wt % based on the weight of the oral care composition. 20. The oral care composition according to claim 1 wherein the basic amino acid in free or salt form comprises arginine bicarbonate. 21. The oral care composition according to claim 1 wherein the basic amino acid in free or salt form is present in an amount of from 5 to 15 wt % based on the weight of the oral care composition. 22. The oral care composition according to claim 21 wherein the basic amino acid in free or salt form is present in an amount of from 7 to 12 wt % based on the weight of the oral care composition. 23. The oral care composition according to claim 1 wherein the zinc ion source comprises at least one of zinc citrate, zinc lactate, zinc gluconate or zinc oxide, or any mixture of any two or more thereof. 24. The oral care composition according to claim 23 wherein the zinc ion source is present in an amount of from 0.5 to 3 wt % based on the weight of the oral care composition. 25. The oral care composition according to claim 24 wherein the zinc ion source is present in an amount of from 1 to 2 wt % based on the weight of the oral care composition. 26. The oral care composition according to claim 23 wherein the zinc ion source comprises a mixture of zinc citrate and zinc oxide. 27. The oral care composition according to claim 26 wherein zinc citrate is present in an amount of from 0.25 to 0.75 wt % based on the weight of the oral care composition and zinc oxide is present in an amount of from 0.75 to 1.25 wt % based on the weight of the oral care composition. 28. The oral care composition according to claim 1, wherein the basic amino acid in free or salt form comprises arginine bicarbonate, the zinc ion source comprises zinc oxide, and wherein the composition comprises a nonionic block copolymer surfactant comprising poloxamer 407. 29. The oral care composition according to claim 1, wherein the basic amino acid in free or salt form comprises arginine bicarbonate, the zinc ion source comprises zinc oxide, and wherein the composition comprises a betaine zwitterionic surfactant comprising cocamidopropyl betaine. 30. The oral care composition according to claim 1, wherein the basic amino acid in free or salt form comprises arginine bicarbonate, the zinc ion source comprises a mixture of zinc citrate and zinc oxide, and wherein the composition comprises a nonionic block copolymer surfactant comprising poloxamer 407 and a betaine zwitterionic surfactant comprising cocamidopropyl betaine. 31. The oral care composition according to claim 1 wherein the orally acceptable vehicle comprises sorbitol which is present in an amount of from 12 to 30 wt % based on the weight of the oral care composition. 32. The oral care composition according to claim 31 wherein the sorbitol is present in an amount of from 15 to 25 wt % based on the weight of the oral care composition. 33. The oral care composition according to claim 1 wherein the oral care composition does not comprise any sodium lauryl sulfate. 34. The oral care composition according to claim 33 wherein the oral care composition does not comprise any anionic surfactant. 35. The oral care composition according to claim 1 wherein the composition is formulated into a dentifrice in the form of a paste or gel. 36. The oral care composition according to claim 1 wherein the composition is formulated into a form adapted to be applied undiluted within the oral cavity directly to the surface of a mammalian tooth and to be retained within the cavity on the surface for a period of at least 1 hour for treating or preventing hypersensitivity of the tooth. 37. The oral care composition according to claim 1 for enhancing the uptake of zinc ions by the surface of a mammalian tooth in a method comprising applying the oral care composition to the tooth surface. 38. The oral care composition according to claim 1 for increasing the foaming of the oral care composition in an oral cavity in a method comprising applying the composition to the surface of a mammalian tooth in the oral cavity. 39. A method of reducing dental sensitivity comprising applying to the surface of a mammalian tooth an oral care composition of claim 1. 40. A method of occluding a dentin tubule of a mammalian tooth comprising applying to the tooth surface a composition according to claim 1. 41. A method of enhancing the uptake of zinc ions to a mammalian tooth comprising applying to the tooth surface a composition according to claim 1. 42-54. (canceled)

Clostridium difficile disease involves a range of clinical presentations ranging from mild to self-limiting diarrhea to life-threatening pseudomembranous colitis and megacolon. Cases of C. difficile are treated differently depending on severity of disease. Mild and moderate cases may be treated with metronidazole while moderate-to-severe and relapsing cases are often treated with vancomycin or fidaxomicin. The presence of C. difficile disease is detected using a biomarker panel that includes C. difficile antigen (GDH), toxins A and B, and fecal lactoferrin. In patients suspected of C. difficile disease, if GDH is detected indicating the presence of C. difficile , and then toxins A and/or B are detected to indicate toxigenic C. difficile and support a diagnosis of C. difficile -associated disease, fecal lactoferrin concentrations are measured to determine severity of the disease by indicating the amount of intestinal inflammation.

1. A method of monitoring a patient with C. Difficile disease, the method comprising: obtaining a first fecal sample from a patient at a first time; obtaining a second fecal sample from the same patient at a second time later than the first time; comparing a first amount of one or more of lactoferrin or calprotectin in the first fecal sample with a second amount of one or more of lactoferrin or calprotectin in the second fecal sample to identify a change in level of one or more of lactoferrin or calprotectin between the first time and the second time; based on a change in level of one or more of lactoferrin or calprotectin, identifying a patient's change in C. Difficile disease severity; and administering a therapeutically effective amount of a treatment shown to be effective in treating C. Difficile to the patient based on identifying a patient's change in C. Difficile disease severity. 2. The method of claim 1, wherein a therapeutically effective amount of the treatment is stopping a treatment if the level of lactoferrin has dropped below 7.25 μg/mL for the second fecal sample of the patient. 3. The method of claim 1, wherein a therapeutically effective amount of the treatment is administering a therapeutically effective amount of a treatment shown to be effective in treating moderate C. Difficile if the level of lactoferrin is between 7.25 μg/mL and 99.99 μg/mL for the second fecal sample of the patient. 4. The method of claim 3, wherein the treatment shown to be effective in treating moderate C. Difficile comprises one or more of nitroimidazole antibiotics. 5. The method of claim 1, wherein a therapeutically effective amount of the treatment is administering a therapeutically effective amount of a treatment shown to be effective in treating moderate-to-severe C. Difficile if the level of lactoferrin is 100 μg/mL or greater for the second fecal sample of the patient, wherein a level of lactoferrin of 100 μg/mL or greater indicates severe intestinal inflammation. 6. The method of claim 5, wherein treatment shown to be effective in treating moderate-to-severe C. Difficile comprises treatment with one or more of glycopeptide antibiotics or macrocyclic antibiotics. 7. The method of claim 5, wherein treatment shown to be effective in treating moderate-to-severe C. Difficile comprises treatment with a native flora transplant. 8. A method of monitoring a patient with C. Difficile disease, the method comprising: obtaining a first fecal sample from a patient at a first time; obtaining a second fecal sample from the same patient at a second time later than the first time; comparing a first amount of one or more of lactoferrin or calprotectin in the first fecal sample with a second amount of one or more of lactoferrin or calprotectin in the second fecal sample to identify a change in level of one or more of lactoferrin or calprotectin between the first time and the second time; based on the change in level of one or more of lactoferrin or calprotectin, identifying a patient's change in C. Difficile disease severity; and administering a therapeutically effective amount of a treatment shown to be effective in treating C. Difficile to the patient after obtaining the second fecal sample, wherein the patient had a mild case of C. Difficile at the first time and an increased amount of one or more of lactoferrin or calprotectin at the second time, wherein the comparison of the first amount in the first sample and the second amount in the second sample indicates increased intestinal inflammation and worsening of the C. Difficile disease. 9. The method of claim 8, wherein a therapeutically effective amount of the treatment is administering a therapeutically effective amount of a treatment shown to be effective in treating moderate C. Difficile if the level of lactoferrin is between 7.25 μg/mL and 99.99 μg/mL for the second fecal sample of the patient. 10. The method of claim 9, wherein the treatment shown to be effective in treating moderate C. Difficile comprises treatment with one or more of nitroimidazole antibiotics. 11. The method of claim 8, wherein a therapeutically effective amount of the treatment is administering a therapeutically effective amount of the treatment shown to be effective in treating moderate-to-severe C. Difficile if the level of lactoferrin is 100 μg/mL or greater for the second fecal sample of the patient. 12. The method of claim 11, wherein the treatment shown to be effective in treating moderate-to-severe C. Difficile comprises treatment with one or more of glycopeptide antibiotics or macrocyclic antibiotics. 13. The method of claim 11, wherein the wherein the treatment shown to be effective in treating moderate-to-severe C. Difficile comprises treatment with a native flora transplant. 14. A diagnostic method of determining a presence of C. Difficile disease and a severity of C. Difficile disease, the method comprising: obtaining a fecal sample from a patient; determining a presence of a first biomarker from the same patient's fecal sample, wherein the presence of the first biomarker indicates the presence of C. Difficile disease; and determining a level of a second biomarker from the same patient's fecal sample, wherein the level of the second biomarker indicates the severity of the C. Difficile disease. 15. The method of claim 14, wherein the first biomarker comprises C. difficile glutamate dehydrogenase (GDH). 16. The method of claim 14, wherein the first biomarker comprises C. Difficile toxin A. 17. The method of claim 14, wherein the first biomarker comprises C. Difficile toxin B. 18. The method of claim 14, wherein the first biomarker comprises one or more of C. Difficile toxin A gene (tcdA) or C. Difficile toxin B gene (tcdB). 19. The method of claim 14, wherein the second biomarker comprises a level of lactoferrin in the patient's fecal sample. 20. The method of claim 14, wherein the second biomarker comprises a level of calprotectin in the patient's fecal sample.

Clostridium difficile disease involves a range of clinical presentations ranging from mild to self-limiting diarrhea to life-threatening pseudomembranous colitis and megacolon. Cases of C. difficile are treated differently depending on severity of disease. Mild and moderate cases may be treated with metronidazole while moderate-to-severe and relapsing cases are often treated with vancomycin or fidaxomicin. The presence of C. difficile disease is detected using a biomarker panel that includes C. difficile antigen (GDH), toxins A and B, and fecal lactoferrin. In patients suspected of C. difficile disease, if GDH is detected indicating the presence of C. difficile , and then toxins A and/or B are detected to indicate toxigenic C. difficile and support a diagnosis of C. difficile -associated disease, fecal lactoferrin concentrations are measured to determine severity of the disease by indicating the amount of intestinal inflammation.1. A method of monitoring a patient with C. Difficile disease, the method comprising: obtaining a first fecal sample from a patient at a first time; obtaining a second fecal sample from the same patient at a second time later than the first time; comparing a first amount of one or more of lactoferrin or calprotectin in the first fecal sample with a second amount of one or more of lactoferrin or calprotectin in the second fecal sample to identify a change in level of one or more of lactoferrin or calprotectin between the first time and the second time; based on a change in level of one or more of lactoferrin or calprotectin, identifying a patient's change in C. Difficile disease severity; and administering a therapeutically effective amount of a treatment shown to be effective in treating C. Difficile to the patient based on identifying a patient's change in C. Difficile disease severity. 2. The method of claim 1, wherein a therapeutically effective amount of the treatment is stopping a treatment if the level of lactoferrin has dropped below 7.25 μg/mL for the second fecal sample of the patient. 3. The method of claim 1, wherein a therapeutically effective amount of the treatment is administering a therapeutically effective amount of a treatment shown to be effective in treating moderate C. Difficile if the level of lactoferrin is between 7.25 μg/mL and 99.99 μg/mL for the second fecal sample of the patient. 4. The method of claim 3, wherein the treatment shown to be effective in treating moderate C. Difficile comprises one or more of nitroimidazole antibiotics. 5. The method of claim 1, wherein a therapeutically effective amount of the treatment is administering a therapeutically effective amount of a treatment shown to be effective in treating moderate-to-severe C. Difficile if the level of lactoferrin is 100 μg/mL or greater for the second fecal sample of the patient, wherein a level of lactoferrin of 100 μg/mL or greater indicates severe intestinal inflammation. 6. The method of claim 5, wherein treatment shown to be effective in treating moderate-to-severe C. Difficile comprises treatment with one or more of glycopeptide antibiotics or macrocyclic antibiotics. 7. The method of claim 5, wherein treatment shown to be effective in treating moderate-to-severe C. Difficile comprises treatment with a native flora transplant. 8. A method of monitoring a patient with C. Difficile disease, the method comprising: obtaining a first fecal sample from a patient at a first time; obtaining a second fecal sample from the same patient at a second time later than the first time; comparing a first amount of one or more of lactoferrin or calprotectin in the first fecal sample with a second amount of one or more of lactoferrin or calprotectin in the second fecal sample to identify a change in level of one or more of lactoferrin or calprotectin between the first time and the second time; based on the change in level of one or more of lactoferrin or calprotectin, identifying a patient's change in C. Difficile disease severity; and administering a therapeutically effective amount of a treatment shown to be effective in treating C. Difficile to the patient after obtaining the second fecal sample, wherein the patient had a mild case of C. Difficile at the first time and an increased amount of one or more of lactoferrin or calprotectin at the second time, wherein the comparison of the first amount in the first sample and the second amount in the second sample indicates increased intestinal inflammation and worsening of the C. Difficile disease. 9. The method of claim 8, wherein a therapeutically effective amount of the treatment is administering a therapeutically effective amount of a treatment shown to be effective in treating moderate C. Difficile if the level of lactoferrin is between 7.25 μg/mL and 99.99 μg/mL for the second fecal sample of the patient. 10. The method of claim 9, wherein the treatment shown to be effective in treating moderate C. Difficile comprises treatment with one or more of nitroimidazole antibiotics. 11. The method of claim 8, wherein a therapeutically effective amount of the treatment is administering a therapeutically effective amount of the treatment shown to be effective in treating moderate-to-severe C. Difficile if the level of lactoferrin is 100 μg/mL or greater for the second fecal sample of the patient. 12. The method of claim 11, wherein the treatment shown to be effective in treating moderate-to-severe C. Difficile comprises treatment with one or more of glycopeptide antibiotics or macrocyclic antibiotics. 13. The method of claim 11, wherein the wherein the treatment shown to be effective in treating moderate-to-severe C. Difficile comprises treatment with a native flora transplant. 14. A diagnostic method of determining a presence of C. Difficile disease and a severity of C. Difficile disease, the method comprising: obtaining a fecal sample from a patient; determining a presence of a first biomarker from the same patient's fecal sample, wherein the presence of the first biomarker indicates the presence of C. Difficile disease; and determining a level of a second biomarker from the same patient's fecal sample, wherein the level of the second biomarker indicates the severity of the C. Difficile disease. 15. The method of claim 14, wherein the first biomarker comprises C. difficile glutamate dehydrogenase (GDH). 16. The method of claim 14, wherein the first biomarker comprises C. Difficile toxin A. 17. The method of claim 14, wherein the first biomarker comprises C. Difficile toxin B. 18. The method of claim 14, wherein the first biomarker comprises one or more of C. Difficile toxin A gene (tcdA) or C. Difficile toxin B gene (tcdB). 19. The method of claim 14, wherein the second biomarker comprises a level of lactoferrin in the patient's fecal sample. 20. The method of claim 14, wherein the second biomarker comprises a level of calprotectin in the patient's fecal sample.

The present invention provides an oral care composition for encouraging proper tooth cleaning, containing particulate materials which can be breakable under a brushing action with a brushing force from 0.1N to 5N. The particulate materials can have a particle size distribution characterized by (1) a change ratio of mean particle size before and after the brushing action is at least 20%, (2) a change ratio of D90 before and after the brushing action is at least 20%, (3) at least 5% of the particulate materials have a particle size greater than 200 μm before the brushing action, and (4) no more than 30% of the particulate materials have a particle size greater than 200 μm after the brushing action. The oral care composition can have a viscosity ranging from 10 to 90 BKU.

1. An oral care composition, comprising particulate materials which are breakable under a brushing action with a brushing force from 0.1N to 5N, wherein the particulate materials have a particle size distribution characterized by (1) a change ratio of mean particle size before and after the brushing action is at least 20%, (2) a change ratio of D90 before and after the brushing action is at least 20%, (3) at least 5% of the particulate materials have a particle size greater than 200 μm before the brushing action, and (4) no more than 30% of the particulate materials have a particle size greater than 200 μm after the brushing action, and wherein the oral care composition has a viscosity ranging from 10 to 90 BKU. 2. The oral care composition according to claim 1, wherein said particulate materials are silica agglomerates. 3. The oral care composition according to claim 1, wherein said oral care composition has a viscosity ranging from 15 to 70 BKU. 4. The oral care composition according to claim 1, wherein said oral care composition comprises from 1% to 30% by weight of said particulate materials. 5. The oral care composition according to claim 1, wherein said particulate materials have a particle size distribution characterized by (1) a change ratio of mean particle size before and after the brushing action is at least 30% or 40%, (2) a change ratio of D90 before and after the brushing action is at least 30% or 40%, (3) at least 10% or 20% of the particulate materials have a particle size greater than 200 μm before the brushing action, and (4) no more than 20% or 10% of the particulate materials have a particle size greater than 200 μm after the brushing action. 6. The oral care composition according to claim 1, wherein said particulate materials are breakable under a brushing action with a brushing force from 1N to 4N. 7. The oral care composition according to claim 1, wherein said oral care composition further comprises a thickening agent selected from the group consisting of polysaccharides and derivatives thereof, carbomers, natural and synthetic gums, acrylamide polymers, acrylic acid polymers, vinyl polymers, polyamines, ethylene oxide polymers, mineral oils, petrolatums, clays and organomodified clays, and mixtures thereof. 8. The oral care composition according to claim 7, wherein the thickening agent is selected from the group consisting of methylcellulose, ethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, carrageenan, xanthan gum, guar gum, tragacanth gum, alginate, acacia gum, gelatin, and mixtures thereof. 9. The oral care composition according to claim 8, wherein said thickening agent is present in an amount from 0.2% to 5% by weight of said oral care composition. 10. The oral care composition according to claim 9, wherein the thickening agent is present in said oral care composition in an amount from 0.4% to 1.5% by weight, and wherein the thickening agent is selected from the group consisting of hydroxyethylcellulose, carrageenan, sodium carboxymethylcellulose, and mixtures thereof. 11. The oral care composition according to claim 1, wherein said oral care composition is in a form selected from the group consisting of toothpaste, tooth powder, tooth gel, and mixtures thereof. 12. A method of encouraging proper tooth cleaning, comprising the steps of applying an oral care composition according to claim 1 onto a subject's tooth surface and brushing said tooth surface with said oral care composition. 13. Use of particulate materials in manufacturing an oral care composition, wherein said particulate materials are breakable under a brushing action with a brushing force from 0.1N to 5N, and wherein the particulate materials have a particle size distribution characterized by (1) a change ratio of mean particle size before and after the brushing action is at least 20%, (2) a change ratio of D90 before and after the brushing action is at least 20%, (3) at least 5% of the particulate materials have a particle size greater than 200 μm before the brushing action, and (4) no more than 30% of the particulate materials have a particle size greater than 200 μm after the brushing action. 14. The use according to claim 13, wherein said particulate materials are silica agglomerates. 15. The use according to claim 14, wherein said oral care composition has a viscosity ranging from 10 to 90 BKU. 16. The use according to claim 15, wherein said oral care composition is in a form selected from the group consisting of toothpaste, tooth powder, tooth gel, and mixtures thereof.

The present invention provides an oral care composition for encouraging proper tooth cleaning, containing particulate materials which can be breakable under a brushing action with a brushing force from 0.1N to 5N. The particulate materials can have a particle size distribution characterized by (1) a change ratio of mean particle size before and after the brushing action is at least 20%, (2) a change ratio of D90 before and after the brushing action is at least 20%, (3) at least 5% of the particulate materials have a particle size greater than 200 μm before the brushing action, and (4) no more than 30% of the particulate materials have a particle size greater than 200 μm after the brushing action. The oral care composition can have a viscosity ranging from 10 to 90 BKU.1. An oral care composition, comprising particulate materials which are breakable under a brushing action with a brushing force from 0.1N to 5N, wherein the particulate materials have a particle size distribution characterized by (1) a change ratio of mean particle size before and after the brushing action is at least 20%, (2) a change ratio of D90 before and after the brushing action is at least 20%, (3) at least 5% of the particulate materials have a particle size greater than 200 μm before the brushing action, and (4) no more than 30% of the particulate materials have a particle size greater than 200 μm after the brushing action, and wherein the oral care composition has a viscosity ranging from 10 to 90 BKU. 2. The oral care composition according to claim 1, wherein said particulate materials are silica agglomerates. 3. The oral care composition according to claim 1, wherein said oral care composition has a viscosity ranging from 15 to 70 BKU. 4. The oral care composition according to claim 1, wherein said oral care composition comprises from 1% to 30% by weight of said particulate materials. 5. The oral care composition according to claim 1, wherein said particulate materials have a particle size distribution characterized by (1) a change ratio of mean particle size before and after the brushing action is at least 30% or 40%, (2) a change ratio of D90 before and after the brushing action is at least 30% or 40%, (3) at least 10% or 20% of the particulate materials have a particle size greater than 200 μm before the brushing action, and (4) no more than 20% or 10% of the particulate materials have a particle size greater than 200 μm after the brushing action. 6. The oral care composition according to claim 1, wherein said particulate materials are breakable under a brushing action with a brushing force from 1N to 4N. 7. The oral care composition according to claim 1, wherein said oral care composition further comprises a thickening agent selected from the group consisting of polysaccharides and derivatives thereof, carbomers, natural and synthetic gums, acrylamide polymers, acrylic acid polymers, vinyl polymers, polyamines, ethylene oxide polymers, mineral oils, petrolatums, clays and organomodified clays, and mixtures thereof. 8. The oral care composition according to claim 7, wherein the thickening agent is selected from the group consisting of methylcellulose, ethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, carrageenan, xanthan gum, guar gum, tragacanth gum, alginate, acacia gum, gelatin, and mixtures thereof. 9. The oral care composition according to claim 8, wherein said thickening agent is present in an amount from 0.2% to 5% by weight of said oral care composition. 10. The oral care composition according to claim 9, wherein the thickening agent is present in said oral care composition in an amount from 0.4% to 1.5% by weight, and wherein the thickening agent is selected from the group consisting of hydroxyethylcellulose, carrageenan, sodium carboxymethylcellulose, and mixtures thereof. 11. The oral care composition according to claim 1, wherein said oral care composition is in a form selected from the group consisting of toothpaste, tooth powder, tooth gel, and mixtures thereof. 12. A method of encouraging proper tooth cleaning, comprising the steps of applying an oral care composition according to claim 1 onto a subject's tooth surface and brushing said tooth surface with said oral care composition. 13. Use of particulate materials in manufacturing an oral care composition, wherein said particulate materials are breakable under a brushing action with a brushing force from 0.1N to 5N, and wherein the particulate materials have a particle size distribution characterized by (1) a change ratio of mean particle size before and after the brushing action is at least 20%, (2) a change ratio of D90 before and after the brushing action is at least 20%, (3) at least 5% of the particulate materials have a particle size greater than 200 μm before the brushing action, and (4) no more than 30% of the particulate materials have a particle size greater than 200 μm after the brushing action. 14. The use according to claim 13, wherein said particulate materials are silica agglomerates. 15. The use according to claim 14, wherein said oral care composition has a viscosity ranging from 10 to 90 BKU. 16. The use according to claim 15, wherein said oral care composition is in a form selected from the group consisting of toothpaste, tooth powder, tooth gel, and mixtures thereof.

Compositions containing luteolin, quercetin, and kaempferol are provided. The compositions are useful killing cancer cells and treating cancer. Exemplary cancers that can be treated include, but are not limited to prostate cancer and head and neck cancer.

1. A composition comprising luteolin, quercetin and kaempferol at a molar ratio of 1:1:2. 2. The composition of claim 1, further comprising a pharmaceutically acceptable excipient. 3. The composition of claim 1 formulated for oral administration. 4. The composition of claim 1 formulated for parenteral administration. 5. The composition of claim 1 formulated as a capsule. 6. The composition of claim 1, wherein at least one hydroxyl group of the luteolin, quercetin, or kaempferol is modified to increase water solubility of the composition. 7. The composition of claim 2 wherein the excipient is oil. 8. The composition of claim 7, wherein the oil is vegetable oil. 9. A method of inhibiting or killing cancer cells comprising: exposing the cancer cells to an inhibitory dose of the composition of claim 1. 10. The method of claim 9, wherein the cancer cells are metastatic castration-resistant prostate cancer cells or head and neck cancer cells. 11. A method for treating a solid tumor in a mammalian subject comprising: administering to the subject a therapeutically effective dose of the composition according to claim 1. 12. The method of claim 11, further comprising the step of repeating the administration at intervals of at least three times per week. 13. The method of claim 12, wherein administration is for a period of at least five weeks. 14. The method of any one of claim 11, wherein the subject is a human subject. 15. The method of claim 9, wherein the composition is administered either as a single regimen or combined with a second cancer treatment regimen. 16. The method of claim 15, wherein the second cancer treatment regimen is selected from the group consisting of hormonal therapy, chemotherapy, and radiotherapy. 17. The method of any one of claim 16, wherein the cancer is prostate cancer or head and neck cancer. 18. The method of claim 9, wherein the composition is administered on a daily basis at a daily dose between 1 to 200 mg/kg body weight.

Compositions containing luteolin, quercetin, and kaempferol are provided. The compositions are useful killing cancer cells and treating cancer. Exemplary cancers that can be treated include, but are not limited to prostate cancer and head and neck cancer.1. A composition comprising luteolin, quercetin and kaempferol at a molar ratio of 1:1:2. 2. The composition of claim 1, further comprising a pharmaceutically acceptable excipient. 3. The composition of claim 1 formulated for oral administration. 4. The composition of claim 1 formulated for parenteral administration. 5. The composition of claim 1 formulated as a capsule. 6. The composition of claim 1, wherein at least one hydroxyl group of the luteolin, quercetin, or kaempferol is modified to increase water solubility of the composition. 7. The composition of claim 2 wherein the excipient is oil. 8. The composition of claim 7, wherein the oil is vegetable oil. 9. A method of inhibiting or killing cancer cells comprising: exposing the cancer cells to an inhibitory dose of the composition of claim 1. 10. The method of claim 9, wherein the cancer cells are metastatic castration-resistant prostate cancer cells or head and neck cancer cells. 11. A method for treating a solid tumor in a mammalian subject comprising: administering to the subject a therapeutically effective dose of the composition according to claim 1. 12. The method of claim 11, further comprising the step of repeating the administration at intervals of at least three times per week. 13. The method of claim 12, wherein administration is for a period of at least five weeks. 14. The method of any one of claim 11, wherein the subject is a human subject. 15. The method of claim 9, wherein the composition is administered either as a single regimen or combined with a second cancer treatment regimen. 16. The method of claim 15, wherein the second cancer treatment regimen is selected from the group consisting of hormonal therapy, chemotherapy, and radiotherapy. 17. The method of any one of claim 16, wherein the cancer is prostate cancer or head and neck cancer. 18. The method of claim 9, wherein the composition is administered on a daily basis at a daily dose between 1 to 200 mg/kg body weight.

The invention relates to particular substituted heterocycle fused gamma-carbolines, their prodrugs, in free, solid, pharmaceutically acceptable salt and/or substantially pure form as described herein, pharmaceutical compositions thereof, and methods of use in the treatment of diseases involving the 5-HT2A receptor, the serotonin transporter (SERT), pathways involving the dopamine D1 and D2 receptor signaling system, and/or the μ-opioid receptor.

1. A method for the treatment or prophylaxis of a central nervous system disorder, comprising administering to a patient in need thereof a compound of a Formula wherein: X is —NH— or —N(CH3)—; L is selected from O, NH, NRa, and S; Z is —CH(O—R1)—, —O— or —C(O)—; R1 is H, —C(O)—C1-21 alkyl (e.g., —C(O)—C1-5alkyl, —C(O)—C6-15 alkyl or —C(O)—C16-21 alkyl), preferably said alkyl is a straight chain, optionally saturated or unsaturated and optionally substituted with one or more hydroxy or C1-22 alkoxy (e.g., ethoxy) groups, for example R1 is C(O)—C3 alkyl, —C(O)C6 alkyl, —C(O)—C7 alkyl, —C(O)—C9 alkyl, —C(O)—C11 alkyl, —C(O)—C13 alkyl or —C(O)—C15 alkyl; Ra is: halogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, or C3-6 cycloalkyl, each of which can be independently substituted with up to three independently selected Rb groups, for example C1-3 haloalkyl or C1-3 hydroxyalkyl; or aryl optionally substituted with up to five independently selected Rb; and each Rb is independently selected from H, halogen, NH2, NO2, OH, C(O)OH, CN, SO3, and C1-4 alkyl; in free or salt form; optionally in an isolated or purified free or salt form; wherein the disease or disorder is selected from obsessive-compulsive disorder (OCD), obsessive-compulsive personality disorder (OCPD), general anxiety disorder, social anxiety disorder, panic disorder, agoraphobia, compulsive gambling disorder, compulsive eating disorder, body dysmorphic disorder, hypochondriasis, pathological grooming disorder, kleptomania, pyromania, attention deficit-hyperactivity disorder (ADHD), attention deficit disorder (ADD), impulse control disorder, and related disorders, and pain disorder or conditions associated with pain, such as cephalic pain, neuropathic pain, idiopathic pain, chronic pain, fibromyalgia, and chronic fatigue, and related disorders, opiate dependency, cocaine dependency, amphetamine dependency, alcohol dependency, and combinations thereof. 2. The method according to claim 1, wherein L is O. 3. The method according to claim 1, wherein Z is —CH(O—R1)—. 4. The method according to claim 1, wherein Z is —C(═O)—. 5. The method according to claim 1, wherein Z is —O—. 6. The method according to claim 1, wherein X is —NH—. 7. The method according to claim 1, wherein X is —N(CH3)—. 8. The method according to claim 1, wherein the compound is selected from the group consisting of: 9. The method according to claim 1, wherein the compound is selected from the group consisting of: 10. The method according to claim 1, wherein the compound is in the form of a salt, optionally in the form of a pharmaceutically acceptable salt. 11. The method according to claim 1, wherein in the compound is administered to the patient in the form of a pharmaceutically acceptable composition comprising the compound in free or pharmaceutically acceptable salt form, in admixture with a pharmaceutically acceptable diluent or carrier. 12. The method of claim 11, wherein the pharmaceutically acceptable diluent or carrier comprises a polymeric matrix. 13. The method according to claim 12, wherein the polymeric matrix is a biodegradable poly(d,l-lactide-co-glycolide) microsphere. 14-18. (canceled) 19. A compound according to Formula I: wherein: X is —NH; L is O; Z is —O— or —C(O)—; in free form, salt form, or pharmaceutically acceptable salt form. 20. The compound according to claim 19, wherein Z is —C(═O)—. 21. The compound according to claim 19, wherein Z is —O—. 22. A pharmaceutical composition comprising the compound according to claim 19 in admixture with a pharmaceutically acceptable diluent or carrier. 23. A pharmaceutical composition comprising the compound according to claim 21 in admixture with a pharmaceutically acceptable diluent or carrier. 24. The composition according to claim 22, wherein the pharmaceutically acceptable diluent or carrier comprises a polymeric matrix. 25. The composition according to claim 24, wherein the polymeric matrix comprises a biodegradable poly(d,l-lactide-co-glycolide) microsphere.

The invention relates to particular substituted heterocycle fused gamma-carbolines, their prodrugs, in free, solid, pharmaceutically acceptable salt and/or substantially pure form as described herein, pharmaceutical compositions thereof, and methods of use in the treatment of diseases involving the 5-HT2A receptor, the serotonin transporter (SERT), pathways involving the dopamine D1 and D2 receptor signaling system, and/or the μ-opioid receptor.1. A method for the treatment or prophylaxis of a central nervous system disorder, comprising administering to a patient in need thereof a compound of a Formula wherein: X is —NH— or —N(CH3)—; L is selected from O, NH, NRa, and S; Z is —CH(O—R1)—, —O— or —C(O)—; R1 is H, —C(O)—C1-21 alkyl (e.g., —C(O)—C1-5alkyl, —C(O)—C6-15 alkyl or —C(O)—C16-21 alkyl), preferably said alkyl is a straight chain, optionally saturated or unsaturated and optionally substituted with one or more hydroxy or C1-22 alkoxy (e.g., ethoxy) groups, for example R1 is C(O)—C3 alkyl, —C(O)C6 alkyl, —C(O)—C7 alkyl, —C(O)—C9 alkyl, —C(O)—C11 alkyl, —C(O)—C13 alkyl or —C(O)—C15 alkyl; Ra is: halogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, or C3-6 cycloalkyl, each of which can be independently substituted with up to three independently selected Rb groups, for example C1-3 haloalkyl or C1-3 hydroxyalkyl; or aryl optionally substituted with up to five independently selected Rb; and each Rb is independently selected from H, halogen, NH2, NO2, OH, C(O)OH, CN, SO3, and C1-4 alkyl; in free or salt form; optionally in an isolated or purified free or salt form; wherein the disease or disorder is selected from obsessive-compulsive disorder (OCD), obsessive-compulsive personality disorder (OCPD), general anxiety disorder, social anxiety disorder, panic disorder, agoraphobia, compulsive gambling disorder, compulsive eating disorder, body dysmorphic disorder, hypochondriasis, pathological grooming disorder, kleptomania, pyromania, attention deficit-hyperactivity disorder (ADHD), attention deficit disorder (ADD), impulse control disorder, and related disorders, and pain disorder or conditions associated with pain, such as cephalic pain, neuropathic pain, idiopathic pain, chronic pain, fibromyalgia, and chronic fatigue, and related disorders, opiate dependency, cocaine dependency, amphetamine dependency, alcohol dependency, and combinations thereof. 2. The method according to claim 1, wherein L is O. 3. The method according to claim 1, wherein Z is —CH(O—R1)—. 4. The method according to claim 1, wherein Z is —C(═O)—. 5. The method according to claim 1, wherein Z is —O—. 6. The method according to claim 1, wherein X is —NH—. 7. The method according to claim 1, wherein X is —N(CH3)—. 8. The method according to claim 1, wherein the compound is selected from the group consisting of: 9. The method according to claim 1, wherein the compound is selected from the group consisting of: 10. The method according to claim 1, wherein the compound is in the form of a salt, optionally in the form of a pharmaceutically acceptable salt. 11. The method according to claim 1, wherein in the compound is administered to the patient in the form of a pharmaceutically acceptable composition comprising the compound in free or pharmaceutically acceptable salt form, in admixture with a pharmaceutically acceptable diluent or carrier. 12. The method of claim 11, wherein the pharmaceutically acceptable diluent or carrier comprises a polymeric matrix. 13. The method according to claim 12, wherein the polymeric matrix is a biodegradable poly(d,l-lactide-co-glycolide) microsphere. 14-18. (canceled) 19. A compound according to Formula I: wherein: X is —NH; L is O; Z is —O— or —C(O)—; in free form, salt form, or pharmaceutically acceptable salt form. 20. The compound according to claim 19, wherein Z is —C(═O)—. 21. The compound according to claim 19, wherein Z is —O—. 22. A pharmaceutical composition comprising the compound according to claim 19 in admixture with a pharmaceutically acceptable diluent or carrier. 23. A pharmaceutical composition comprising the compound according to claim 21 in admixture with a pharmaceutically acceptable diluent or carrier. 24. The composition according to claim 22, wherein the pharmaceutically acceptable diluent or carrier comprises a polymeric matrix. 25. The composition according to claim 24, wherein the polymeric matrix comprises a biodegradable poly(d,l-lactide-co-glycolide) microsphere.

The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more oligonucleotide probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligated oligonucleotide product. In accordance with the invention, the presence of the target(s) of interest can be determined by measuring the presence or amount of ligated oligonucleotide product.

1. A method comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first target domain and a second target domain; ii) a first ligation probe comprising: 1) a first probe domain substantially complementary to said first target domain; and 2) a 5′-ligation moiety; and iii) a second ligation probe comprising: 1) a second probe domain substantially complementary to said second target domain; 2) a 3′ ligation moiety; wherein said first target domain and said second target domain are separated by at least one nucleotide, and wherein at least one of said first and said second ligation probes comprises an anchor sequence; b) ligating said first and said second ligation probes in the absence of a ligase enzyme to form a ligation product; c) capturing said ligated product on a substrate comprising a capture probe substantially complementary to said anchor sequence; and d) detecting the presence of said ligated product. 2. A method comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first target domain and a second target domain; ii) a first ligation probe comprising: 1) a first probe domain substantially complementary to said first target domain; and 2) a 5′-ligation moiety comprising a thioester; and iii) a second ligation probe comprising: 1) a second probe domain substantially complementary to said second target domain; 2) a 3′ ligation moiety comprising a nucleophile; wherein at least one of said target domains do not comprise PNA, and b) ligating said first and said second ligation probes in the absence of a ligase enzyme to form a ligation product. 3. A method according to claim 2 wherein at least one of said first and second ligation probes further comprises an anchor probe, and said method further comprises a) capturing said ligated product on a substrate comprising a capture probe substantially complementary to said anchor sequence; and b detecting the presence of said ligated product. 4. A method of chemical ligation, said method comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first target domain and a second target domain; ii) a first ligation probe comprising: 1) a first probe domain substantially complementary to said first target domain; and 2) a 5′-ligation moiety; and iii) a second ligation probe comprising: 1) a second probe domain substantially complementary to said second target domain; 2) a 3′ ligation moiety; wherein at least one of said first and said second ligation probes comprises at least a first linker between a probe domain and said ligation moiety, b) ligating said first and said second ligation probes in the absence of a ligase enzyme.

The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more oligonucleotide probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligated oligonucleotide product. In accordance with the invention, the presence of the target(s) of interest can be determined by measuring the presence or amount of ligated oligonucleotide product.1. A method comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first target domain and a second target domain; ii) a first ligation probe comprising: 1) a first probe domain substantially complementary to said first target domain; and 2) a 5′-ligation moiety; and iii) a second ligation probe comprising: 1) a second probe domain substantially complementary to said second target domain; 2) a 3′ ligation moiety; wherein said first target domain and said second target domain are separated by at least one nucleotide, and wherein at least one of said first and said second ligation probes comprises an anchor sequence; b) ligating said first and said second ligation probes in the absence of a ligase enzyme to form a ligation product; c) capturing said ligated product on a substrate comprising a capture probe substantially complementary to said anchor sequence; and d) detecting the presence of said ligated product. 2. A method comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first target domain and a second target domain; ii) a first ligation probe comprising: 1) a first probe domain substantially complementary to said first target domain; and 2) a 5′-ligation moiety comprising a thioester; and iii) a second ligation probe comprising: 1) a second probe domain substantially complementary to said second target domain; 2) a 3′ ligation moiety comprising a nucleophile; wherein at least one of said target domains do not comprise PNA, and b) ligating said first and said second ligation probes in the absence of a ligase enzyme to form a ligation product. 3. A method according to claim 2 wherein at least one of said first and second ligation probes further comprises an anchor probe, and said method further comprises a) capturing said ligated product on a substrate comprising a capture probe substantially complementary to said anchor sequence; and b detecting the presence of said ligated product. 4. A method of chemical ligation, said method comprising: a) providing a ligation substrate comprising: i) a target sequence comprising a first target domain and a second target domain; ii) a first ligation probe comprising: 1) a first probe domain substantially complementary to said first target domain; and 2) a 5′-ligation moiety; and iii) a second ligation probe comprising: 1) a second probe domain substantially complementary to said second target domain; 2) a 3′ ligation moiety; wherein at least one of said first and said second ligation probes comprises at least a first linker between a probe domain and said ligation moiety, b) ligating said first and said second ligation probes in the absence of a ligase enzyme.

In one aspect, the present invention provides heterodimeric antibodies comprising a first monomer comprising a first heavy chain constant domain comprising a first variant Fc domain and a first antigen binding domain and a second monomer comprising a second heavy chain constant domain comprising a second variant Fc domain and a second antigen binding domain. In an additional aspect the heterodimeric antibody comprises a first monomer comprising a heavy chain comprising a first Fc domain and a single chain Fv region (scFv) that binds a first antigen, wherein the scFv comprises a charged scFv linker. The heterodimeric antibody further comprises a second monomer comprising a first heavy chain comprising a second Fc domain and a first variable heavy chain and a first light chain.

1. A heterodimeric antibody comprising: a) a first monomer comprising: i) a first heavy chain constant domain comprising a first variant Fc domain; and ii) a first antigen binding domain; and b) a second monomer comprising: i) a second heavy chain constant domain comprising a second variant Fc domain; and ii) a second antigen binding domain; wherein one of said first and second variant Fc domains comprise amino acid substitution(s) selected from the group consisting of those depicted in FIG. 6. 2. A heterodimeric antibody according to claim 1 wherein said first antigen binding domain is a scFv covalent attached to said first heavy chain constant domain. 3. A heterodimeric antibody according to claim 1 wherein said heterodimeric antibody has a structure selected from the structures of FIGS. 1B to 1L and 2A to 2M. 4. A heterodimeric antibody according to claim 1 wherein said first and/or second Fc domain further comprises amino acid substitution(s) selected from the group consisting of 434A, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y, 252Y/254T/256E, 259I/308F/428L, 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 236R, 328R, 236R/328R, 236N/267E, 243L, 298A and 299T. 5. A heterodimeric antibody according to claim 1 wherein one of said first and said second variant Fc domains comprises the amino acid substitutions 364K/E357Q and the other of said first and said second variant Fc domains comprises the amino acid substitutions 368D/370S. 6. A heterodimeric antibody according to claim 1 wherein said first and/or second Fc domain further comprises amino acid substitution(s) selected from the group consisting of those listed in FIG. 7. 7. A heterodimeric antibody according to claim 1 wherein said first monomer comprises a heavy chain constant domain covalently attached to an scFv and said second monomer comprises a heavy chain and a light chain. 8. A heterodimeric antibody according to a claim 1 wherein one of said monomers comprises N208D/Q295E/N384D/Q418E/N421D. 9. A nucleic acid composition comprising nucleic acids encoding said first and second monomer according to claim 1. 10. A host cell comprising the nucleic acid composition of claim 9. 11. A method of making a heterodimeric antibody according to claim 1 comprising culturing the host cell of claim K10 under conditions wherein said heterodimeric antibody is produced and recovering said heterodimeric antibody. 12. A method of treating an individual in need thereof by administering a heterodimeric antibody according to claim 1. 13. A heterodimeric antibody comprising: a) a first monomer comprising a heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds a first antigen; wherein said scFv comprises a charged scFv linker; and b) a second monomer comprising: I) a first heavy chain comprising: 1) a second Fc domain; 2) a first variable heavy chain; and ii) a first light chain. 14-18. (canceled) 19. A heterodimeric antibody composition comprising: a) a first monomer comprising: i) a first heavy chain sequence comprising: A) a first variant Fc domain as compared to a human Fc domain; and B) a first antigen-binding domain that binds to a first antigen; and ii) a second heavy chain sequence comprising: A) a second variant Fc domain as compared to a human Fc domain; and B) a second antigen binding domain that binds to a second antigen; Wherein said first and second variant Fc domains comprise a set of amino acid substitutions selected from the group consisting of the amino acid sets depicted in FIG. 3. 20. A heterodimeric antibody composition comprising: a) a first monomer comprising: i) a first heavy chain sequence comprising: A) a first variant Fc domain as compared to a human Fc domain; and B) a first antigen-binding domain that binds to a first antigen; and ii) a second heavy chain sequence comprising: A) a second variant Fc domain as compared to a human Fc domain; and B) a second antigen binding domain that binds to CD19 and comprises a variable heavy chain domain comprising the amino acid sequence of H1.227 (SEQ ID NO:X) and a variable light chain selected from the group consisting of the amino acid sequence of L1.198 (SEQ ID NO:X) and the amino acid sequence of 1.199 (SEQ ID NO:X) as depicted in FIG. 21. 21. A heterodimeric antibody composition comprising: a) a first monomer comprising: i) a first heavy chain sequence comprising: A) a first variant Fc domain as compared to a human Fc domain; and B) a first antigen binding domain comprising an anti-CD3 variable region having a sequence comprising a vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 having the sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436), a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 having the sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is L or H (SEQ ID NO:440); and b) a second monomer comprising: ii) a second heavy chain sequence comprising: A) a second variant Fc domain as compared to a human Fc domain; and B) an anti-CD19 antigen binding domain comprising a variable heavy chain domain comprising the amino acid sequence of H1.227 (SEQ ID NO:X) and a variable light chain selected from the group consisting of the amino acid sequence of L1.198 (SEQ ID NO:X) and the amino acid sequence of 1.199 (SEQ ID NO:X) as depicted in FIG. 21. 22-24. (canceled) 25. A heterodimeric antibody comprising: a) a first monomer comprising a heavy chain comprising: i) a first variant Fc domain; and ii) a single chain Fv region (scFv) that binds a first antigen; wherein said scFv comprises a charged scFv linker; and b) a second monomer comprising: I) a first heavy chain comprising: 1) a second variant Fc domain; 2) a first variable heavy chain; and ii) a first light chain; wherein said first and second variant Fc domains comprise amino acid substitution(s) selected from the group consisting of those depicted in FIG. 7. 26. A heterodimeric antibody composition comprising: a) a first monomer comprising: i) a first antigen binding domain comprising an anti-CD3 variable region having a sequence comprising a vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 having the sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436), a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 having the sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is L or H (SEQ ID NO:440); ii) a first heavy chain sequence comprising a first variant Fc domain as compared to a human Fc domain; and b) a second monomer comprising: i) a second antigen-binding domain; and ii) a second heavy chain sequence comprising a second variant Fc domain as compared to a human Fc domain; wherein said first and second variant Fc domains have different amino acid sequences. 27-54. (canceled) 55. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds CD3 comprising aa vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 having the sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436), a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 having the sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is L or H (SEQ ID NO:440); and b) a second heavy chain comprising: i) a second Fc domain; ii) a first variable heavy chain; and ii) a first variable light chain; wherein said first and second Fc domains are different. 56. A method of making a heterodimeric antibody comprising: a) providing a first nucleic acid encoding a first heavy chain comprising: i) a first heavy chain comprising: 1a first Fc domain; and 2 a single chain Fv region (scFv) that binds a first antigen; wherein said scFv comprises a charged linker; and b) providing a second nucleic acid encoding a second heavy chain comprising: i) a second Fc domain; ii) a first variable heavy chain; and c) providing a third nucleic acid comprising a light chain; d) expressing said first, second and third nucleic acids in a host cell to produce a first, second and third amino acid sequence, respectively; e) loading said first, second and third amino acid sequences onto an ion exchange column; and f) collecting the heterodimeric fraction. 57. A composition comprising a heterodimer protein comprising: a) a first monomer comprising: i) a first variant heavy chain constant region; ii) a first fusion partner; and b) a second monomer comprising: i) a second variant heavy chain constant region; ii) a second fusion partner; wherein the pIs of said first and second variant heavy chain constant regions are at least 0.5 logs apart. 58. A composition comprising a heterodimer protein comprising: a) a first monomer comprising: i) a first variant heavy chain constant region; ii) a first fusion partner; and b) a second monomer comprising: i) a second variant heavy chain constant region; ii) a second fusion partner; wherein the Fc region of said first and second constant regions comprise a set of amino acid substitutions from FIGS. 3 and 12. 59-74. (canceled)

In one aspect, the present invention provides heterodimeric antibodies comprising a first monomer comprising a first heavy chain constant domain comprising a first variant Fc domain and a first antigen binding domain and a second monomer comprising a second heavy chain constant domain comprising a second variant Fc domain and a second antigen binding domain. In an additional aspect the heterodimeric antibody comprises a first monomer comprising a heavy chain comprising a first Fc domain and a single chain Fv region (scFv) that binds a first antigen, wherein the scFv comprises a charged scFv linker. The heterodimeric antibody further comprises a second monomer comprising a first heavy chain comprising a second Fc domain and a first variable heavy chain and a first light chain.1. A heterodimeric antibody comprising: a) a first monomer comprising: i) a first heavy chain constant domain comprising a first variant Fc domain; and ii) a first antigen binding domain; and b) a second monomer comprising: i) a second heavy chain constant domain comprising a second variant Fc domain; and ii) a second antigen binding domain; wherein one of said first and second variant Fc domains comprise amino acid substitution(s) selected from the group consisting of those depicted in FIG. 6. 2. A heterodimeric antibody according to claim 1 wherein said first antigen binding domain is a scFv covalent attached to said first heavy chain constant domain. 3. A heterodimeric antibody according to claim 1 wherein said heterodimeric antibody has a structure selected from the structures of FIGS. 1B to 1L and 2A to 2M. 4. A heterodimeric antibody according to claim 1 wherein said first and/or second Fc domain further comprises amino acid substitution(s) selected from the group consisting of 434A, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y, 252Y/254T/256E, 259I/308F/428L, 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 236R, 328R, 236R/328R, 236N/267E, 243L, 298A and 299T. 5. A heterodimeric antibody according to claim 1 wherein one of said first and said second variant Fc domains comprises the amino acid substitutions 364K/E357Q and the other of said first and said second variant Fc domains comprises the amino acid substitutions 368D/370S. 6. A heterodimeric antibody according to claim 1 wherein said first and/or second Fc domain further comprises amino acid substitution(s) selected from the group consisting of those listed in FIG. 7. 7. A heterodimeric antibody according to claim 1 wherein said first monomer comprises a heavy chain constant domain covalently attached to an scFv and said second monomer comprises a heavy chain and a light chain. 8. A heterodimeric antibody according to a claim 1 wherein one of said monomers comprises N208D/Q295E/N384D/Q418E/N421D. 9. A nucleic acid composition comprising nucleic acids encoding said first and second monomer according to claim 1. 10. A host cell comprising the nucleic acid composition of claim 9. 11. A method of making a heterodimeric antibody according to claim 1 comprising culturing the host cell of claim K10 under conditions wherein said heterodimeric antibody is produced and recovering said heterodimeric antibody. 12. A method of treating an individual in need thereof by administering a heterodimeric antibody according to claim 1. 13. A heterodimeric antibody comprising: a) a first monomer comprising a heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds a first antigen; wherein said scFv comprises a charged scFv linker; and b) a second monomer comprising: I) a first heavy chain comprising: 1) a second Fc domain; 2) a first variable heavy chain; and ii) a first light chain. 14-18. (canceled) 19. A heterodimeric antibody composition comprising: a) a first monomer comprising: i) a first heavy chain sequence comprising: A) a first variant Fc domain as compared to a human Fc domain; and B) a first antigen-binding domain that binds to a first antigen; and ii) a second heavy chain sequence comprising: A) a second variant Fc domain as compared to a human Fc domain; and B) a second antigen binding domain that binds to a second antigen; Wherein said first and second variant Fc domains comprise a set of amino acid substitutions selected from the group consisting of the amino acid sets depicted in FIG. 3. 20. A heterodimeric antibody composition comprising: a) a first monomer comprising: i) a first heavy chain sequence comprising: A) a first variant Fc domain as compared to a human Fc domain; and B) a first antigen-binding domain that binds to a first antigen; and ii) a second heavy chain sequence comprising: A) a second variant Fc domain as compared to a human Fc domain; and B) a second antigen binding domain that binds to CD19 and comprises a variable heavy chain domain comprising the amino acid sequence of H1.227 (SEQ ID NO:X) and a variable light chain selected from the group consisting of the amino acid sequence of L1.198 (SEQ ID NO:X) and the amino acid sequence of 1.199 (SEQ ID NO:X) as depicted in FIG. 21. 21. A heterodimeric antibody composition comprising: a) a first monomer comprising: i) a first heavy chain sequence comprising: A) a first variant Fc domain as compared to a human Fc domain; and B) a first antigen binding domain comprising an anti-CD3 variable region having a sequence comprising a vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 having the sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436), a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 having the sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is L or H (SEQ ID NO:440); and b) a second monomer comprising: ii) a second heavy chain sequence comprising: A) a second variant Fc domain as compared to a human Fc domain; and B) an anti-CD19 antigen binding domain comprising a variable heavy chain domain comprising the amino acid sequence of H1.227 (SEQ ID NO:X) and a variable light chain selected from the group consisting of the amino acid sequence of L1.198 (SEQ ID NO:X) and the amino acid sequence of 1.199 (SEQ ID NO:X) as depicted in FIG. 21. 22-24. (canceled) 25. A heterodimeric antibody comprising: a) a first monomer comprising a heavy chain comprising: i) a first variant Fc domain; and ii) a single chain Fv region (scFv) that binds a first antigen; wherein said scFv comprises a charged scFv linker; and b) a second monomer comprising: I) a first heavy chain comprising: 1) a second variant Fc domain; 2) a first variable heavy chain; and ii) a first light chain; wherein said first and second variant Fc domains comprise amino acid substitution(s) selected from the group consisting of those depicted in FIG. 7. 26. A heterodimeric antibody composition comprising: a) a first monomer comprising: i) a first antigen binding domain comprising an anti-CD3 variable region having a sequence comprising a vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 having the sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436), a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 having the sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is L or H (SEQ ID NO:440); ii) a first heavy chain sequence comprising a first variant Fc domain as compared to a human Fc domain; and b) a second monomer comprising: i) a second antigen-binding domain; and ii) a second heavy chain sequence comprising a second variant Fc domain as compared to a human Fc domain; wherein said first and second variant Fc domains have different amino acid sequences. 27-54. (canceled) 55. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds CD3 comprising aa vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 having the sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436), a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 having the sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is L or H (SEQ ID NO:440); and b) a second heavy chain comprising: i) a second Fc domain; ii) a first variable heavy chain; and ii) a first variable light chain; wherein said first and second Fc domains are different. 56. A method of making a heterodimeric antibody comprising: a) providing a first nucleic acid encoding a first heavy chain comprising: i) a first heavy chain comprising: 1a first Fc domain; and 2 a single chain Fv region (scFv) that binds a first antigen; wherein said scFv comprises a charged linker; and b) providing a second nucleic acid encoding a second heavy chain comprising: i) a second Fc domain; ii) a first variable heavy chain; and c) providing a third nucleic acid comprising a light chain; d) expressing said first, second and third nucleic acids in a host cell to produce a first, second and third amino acid sequence, respectively; e) loading said first, second and third amino acid sequences onto an ion exchange column; and f) collecting the heterodimeric fraction. 57. A composition comprising a heterodimer protein comprising: a) a first monomer comprising: i) a first variant heavy chain constant region; ii) a first fusion partner; and b) a second monomer comprising: i) a second variant heavy chain constant region; ii) a second fusion partner; wherein the pIs of said first and second variant heavy chain constant regions are at least 0.5 logs apart. 58. A composition comprising a heterodimer protein comprising: a) a first monomer comprising: i) a first variant heavy chain constant region; ii) a first fusion partner; and b) a second monomer comprising: i) a second variant heavy chain constant region; ii) a second fusion partner; wherein the Fc region of said first and second constant regions comprise a set of amino acid substitutions from FIGS. 3 and 12. 59-74. (canceled)

The invention provides novel heterodimeric proteins including heterodimeric antibodies.

1. (canceled) 2. A composition comprising an anti-CD3 variable region having a sequence comprising a vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 having the sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436), a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 having the sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is L or H (SEQ ID NO:440). 3-4. (canceled) 5. A composition according to claim 2 wherein said composition comprises a first amino acid sequence comprising the variable heavy CDRs and a second amino acid sequence comprising the variable light CDRs. 6. A composition according to claim 2 wherein said composition comprises a scFv. 7-13. (canceled) 14. A nucleic acid composition encoding the composition of claim 2. 15-17. (canceled) 18. A host cell comprising an nucleic acid composition according to claim 14. 19. A method of making a composition comprising an anti-CD3 variable region comprising culturing a host cell according to claim 18 under conditions wherein said composition is expressed. 20. A method of treating a patient in need thereof by administering a composition according to claim 2. 21. (canceled) 22. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds CD3 comprising aa vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 having the sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436), a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 having the sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is L or H (SEQ ID NO:440); and b) a second heavy chain comprising: i) a second Fc domain; and ii) a first variable heavy chain; and c) a light chain; wherein said first and second Fc domains are different. 23-26. (canceled) 27. A composition according to claim 22 wherein said composition comprises a scFv. 28. (canceled) 29. A composition according to claim 27 wherein said scFv comprises a charged scFv linker. 30-33. (canceled) 34. A nucleic acid composition encoding a heterodimeric antibody according to claim 27 comprising: a) a first nucleic acid encoding said scFv; b) a second nucleic acid encoding said second heavy chain; and c) a third nucleic acid encoding said light chain. 35-38. (canceled) 39. A host cell comprising an nucleic acid composition according to claim 34. 40. A method of making a heterodimeric antibody comprising culturing a host cell according to claim 39 under conditions wherein said composition is expressed. 41. A method of treating a patient in need thereof by administering a heterodimeric antibody according to claim 22. 42. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds a first antigen; wherein said scFv comprises a charged scFv linker; and b) a second heavy chain comprising: i) a second Fc domain; ii) a first variable heavy chain; and c) a light chain. 43-44. (canceled) 45. A heterodimeric antibody according to claim 42 wherein said first and second Fc domains each further comprising at least one heterodimerization variant. 46. A nucleic acid composition encoding a heterodimeric antibody according to claim 42. 47. An expression vector composition comprising the nucleic acid composition of claim 46. 48. A method of making a heterodimeric antibody comprising culturing a host cell according to claim 47 under conditions wherein said composition is expressed. 49. A method of treating a patient in need thereof by administering a heterodimeric antibody according to claim 42. 50. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds a first antigen; and b) a second heavy chain comprising: i) a second Fc domain; and ii) a first variable heavy chain; and c) a light chain; wherein said first and second Fc domains comprise a variant set selected from the variants listed in FIG. 9. 51-52. (canceled) 53. A heterodimeric antibody according to claim 50 further comprising at least one ablation variant. 54. A heterodimeric antibody according to claim 50 further comprising at least one pI variant. 55-56. (canceled) 57. A nucleic acid composition encoding a heterodimeric antibody according to claim 50. 58. A host cell comprising the nucleic acid composition of 57. 59. A method of making a heterodimeric antibody comprising culturing a host cell according to claim 58 under conditions wherein said composition is expressed. 60. A method of treating a patient in need thereof by administering a heterodimeric antibody according to claim 50. 61. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds a first antigen; and b) a second heavy chain comprising: i) a second Fe domain; and ii) a first variable heavy chain; and c) a light chain; wherein one of said first and second Fc domains comprises an Fcγ receptor ablation variant set. 62-66. (canceled) 67. A nucleic acid composition encoding a heterodimeric antibody according to claim 61. 68. A host cell comprising the nucleic acid(s) of claim 67. 69. A method of making a heterodimeric antibody comprising culturing a host cell according to claim 68 under conditions wherein said composition is expressed. 70. A method of treating a patient in need thereof by administering a heterodimeric antibody according to claim 61. 71. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region that binds a first antigen; and b) a second heavy chain comprising: i) a second Fc domain; and ii) a first variable heavy chain; and c) a light chain; wherein said first and second Fc domains comprise a set of variants as shown in FIG. 9, 32 or 33. 72. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a first single chain Fv region (scFv) that binds a first antigen; wherein said first scFv comprises a first charged scFv linker; and b) a second heavy chain comprising: i) a second Fc domain; ii) a second scFv that binds a second antigen. 73-80. (canceled) 81. A method of making a heterodimeric antibody comprising: a) providing a first nucleic acid encoding a first heavy chain comprising: i) a first heavy chain comprising: 1 a first Fc domain; and 2 a single chain Fv region (scFv) that binds a first antigen; wherein said scFv comprises a charged linker; and b) providing a second nucleic acid encoding a second heavy chain comprising: i) a second Fc domain; ii) a first variable heavy chain; and c) providing a third nucleic acid comprising a light chain; d) expressing said first, second and third nucleic acids in a host cell to produce a first, second and third amino acid sequence, respectively; e) loading said first, second and third amino acid sequences onto an ion exchange column; and f) collecting the heterodimeric fraction.

The invention provides novel heterodimeric proteins including heterodimeric antibodies.1. (canceled) 2. A composition comprising an anti-CD3 variable region having a sequence comprising a vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 having the sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436), a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 having the sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is L or H (SEQ ID NO:440). 3-4. (canceled) 5. A composition according to claim 2 wherein said composition comprises a first amino acid sequence comprising the variable heavy CDRs and a second amino acid sequence comprising the variable light CDRs. 6. A composition according to claim 2 wherein said composition comprises a scFv. 7-13. (canceled) 14. A nucleic acid composition encoding the composition of claim 2. 15-17. (canceled) 18. A host cell comprising an nucleic acid composition according to claim 14. 19. A method of making a composition comprising an anti-CD3 variable region comprising culturing a host cell according to claim 18 under conditions wherein said composition is expressed. 20. A method of treating a patient in need thereof by administering a composition according to claim 2. 21. (canceled) 22. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds CD3 comprising aa vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 having the sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436), a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 having the sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is L or H (SEQ ID NO:440); and b) a second heavy chain comprising: i) a second Fc domain; and ii) a first variable heavy chain; and c) a light chain; wherein said first and second Fc domains are different. 23-26. (canceled) 27. A composition according to claim 22 wherein said composition comprises a scFv. 28. (canceled) 29. A composition according to claim 27 wherein said scFv comprises a charged scFv linker. 30-33. (canceled) 34. A nucleic acid composition encoding a heterodimeric antibody according to claim 27 comprising: a) a first nucleic acid encoding said scFv; b) a second nucleic acid encoding said second heavy chain; and c) a third nucleic acid encoding said light chain. 35-38. (canceled) 39. A host cell comprising an nucleic acid composition according to claim 34. 40. A method of making a heterodimeric antibody comprising culturing a host cell according to claim 39 under conditions wherein said composition is expressed. 41. A method of treating a patient in need thereof by administering a heterodimeric antibody according to claim 22. 42. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds a first antigen; wherein said scFv comprises a charged scFv linker; and b) a second heavy chain comprising: i) a second Fc domain; ii) a first variable heavy chain; and c) a light chain. 43-44. (canceled) 45. A heterodimeric antibody according to claim 42 wherein said first and second Fc domains each further comprising at least one heterodimerization variant. 46. A nucleic acid composition encoding a heterodimeric antibody according to claim 42. 47. An expression vector composition comprising the nucleic acid composition of claim 46. 48. A method of making a heterodimeric antibody comprising culturing a host cell according to claim 47 under conditions wherein said composition is expressed. 49. A method of treating a patient in need thereof by administering a heterodimeric antibody according to claim 42. 50. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds a first antigen; and b) a second heavy chain comprising: i) a second Fc domain; and ii) a first variable heavy chain; and c) a light chain; wherein said first and second Fc domains comprise a variant set selected from the variants listed in FIG. 9. 51-52. (canceled) 53. A heterodimeric antibody according to claim 50 further comprising at least one ablation variant. 54. A heterodimeric antibody according to claim 50 further comprising at least one pI variant. 55-56. (canceled) 57. A nucleic acid composition encoding a heterodimeric antibody according to claim 50. 58. A host cell comprising the nucleic acid composition of 57. 59. A method of making a heterodimeric antibody comprising culturing a host cell according to claim 58 under conditions wherein said composition is expressed. 60. A method of treating a patient in need thereof by administering a heterodimeric antibody according to claim 50. 61. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region (scFv) that binds a first antigen; and b) a second heavy chain comprising: i) a second Fe domain; and ii) a first variable heavy chain; and c) a light chain; wherein one of said first and second Fc domains comprises an Fcγ receptor ablation variant set. 62-66. (canceled) 67. A nucleic acid composition encoding a heterodimeric antibody according to claim 61. 68. A host cell comprising the nucleic acid(s) of claim 67. 69. A method of making a heterodimeric antibody comprising culturing a host cell according to claim 68 under conditions wherein said composition is expressed. 70. A method of treating a patient in need thereof by administering a heterodimeric antibody according to claim 61. 71. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a single chain Fv region that binds a first antigen; and b) a second heavy chain comprising: i) a second Fc domain; and ii) a first variable heavy chain; and c) a light chain; wherein said first and second Fc domains comprise a set of variants as shown in FIG. 9, 32 or 33. 72. A heterodimeric antibody comprising: a) a first heavy chain comprising: i) a first Fc domain; and ii) a first single chain Fv region (scFv) that binds a first antigen; wherein said first scFv comprises a first charged scFv linker; and b) a second heavy chain comprising: i) a second Fc domain; ii) a second scFv that binds a second antigen. 73-80. (canceled) 81. A method of making a heterodimeric antibody comprising: a) providing a first nucleic acid encoding a first heavy chain comprising: i) a first heavy chain comprising: 1 a first Fc domain; and 2 a single chain Fv region (scFv) that binds a first antigen; wherein said scFv comprises a charged linker; and b) providing a second nucleic acid encoding a second heavy chain comprising: i) a second Fc domain; ii) a first variable heavy chain; and c) providing a third nucleic acid comprising a light chain; d) expressing said first, second and third nucleic acids in a host cell to produce a first, second and third amino acid sequence, respectively; e) loading said first, second and third amino acid sequences onto an ion exchange column; and f) collecting the heterodimeric fraction.

The present invention is directed to optimized anti-CD3 variable sequences for use in a variety of bispecific formats, including those that utilize scFv components. The invention further relates to nucleic acids encoding for the polypeptide, to vectors comprising the same and to host cells comprising the vector. In another aspect, the invention provides for a pharmaceutical composition comprising the mentioned polypeptide and medical uses of the polypeptide.

1-33. (canceled) 34. An anti-CD3 binding domain comprising a variable heavy sequence and a variable light sequence pair selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:17 and SEQ ID NO:18; SEQ ID NO:21 and SEQ ID NO:22; SEQ ID NO:25 and SEQ ID NO:26; SEQ ID NO:29 and SEQ ID NO:30; SEQ ID NO:33 and SEQ ID NO:34; SEQ ID NO:37 and SEQ ID NO:38; SEQ ID NO:41 and SEQ ID NO:42; SEQ ID NO:45 and SEQ ID NO:46; SEQ ID NO:49 and SEQ ID NO:50; SEQ ID NO:53 and SEQ ID NO:54; SEQ ID NO:57 and SEQ ID NO:58; SEQ ID NO:61 and SEQ ID NO:62; SEQ ID NO:65 and SEQ ID NO:66; SEQ ID NO:69 and SEQ ID NO:70; SEQ ID NO:73 and SEQ ID NO:74; SEQ ID NO:77 and SEQ ID NO:78; SEQ ID NO:81 and SEQ ID NO:82; SEQ ID NO:85 and SEQ ID NO:86; SEQ ID NO:89 and SEQ ID NO:90; SEQ ID NO:93 and SEQ ID NO:94; SEQ ID NO:97 and SEQ ID NO:98; SEQ ID NO:101 and SEQ ID NO:102; SEQ ID NO:105 and SEQ ID NO:106; SEQ ID NO:109 and SEQ ID NO:110; SEQ ID NO:113 and SEQ ID NO:114; SEQ ID NO:117 and SEQ ID NO:118; SEQ ID NO:121 and SEQ ID NO:122; SEQ ID NO:125 and SEQ ID NO:126; SEQ ID NO:129 and SEQ ID NO:130; SEQ ID NO:133 and SEQ ID NO:134; SEQ ID NO:137 and SEQ ID NO:138; SEQ ID NO:141 and SEQ ID NO:142; SEQ ID NO:145 and SEQ ID NO:146; SEQ ID NO:149 and SEQ ID NO:150; SEQ ID NO:153 and SEQ ID NO:154; SEQ ID NO:157 and SEQ ID NO:158; SEQ ID NO:161 and SEQ ID NO:162; SEQ ID NO:165 and SEQ ID NO:166; SEQ ID NO:169 and SEQ ID NO:170; SEQ ID NO:173 and SEQ ID NO:174; SEQ ID NO:177 and SEQ ID NO:178; SEQ ID NO:181 and SEQ ID NO:182; SEQ ID NO:185 and SEQ ID NO:186; SEQ ID NO:189 and SEQ ID NO:190; SEQ ID NO:193 and SEQ ID NO:194; SEQ ID NO:197 and SEQ ID NO:198; SEQ ID NO:201 and SEQ ID NO:202; SEQ ID NO:205 and SEQ ID NO:206; SEQ ID NO:209 and SEQ ID NO:210; SEQ ID NO:213 and SEQ ID NO:214; SEQ ID NO:217 and SEQ ID NO:218; SEQ ID NO:221 and SEQ ID NO:222; SEQ ID NO:225 and SEQ ID NO:226; SEQ ID NO:229 and SEQ ID NO:230; SEQ ID NO:233 and SEQ ID NO:234; SEQ ID NO:237 and SEQ ID NO:238; SEQ ID NO:241 and SEQ ID NO:242; SEQ ID NO:245 and SEQ ID NO:246; SEQ ID NO:249 and SEQ ID NO:250; SEQ ID NO:253 and SEQ ID NO:254; SEQ ID NO:257 and SEQ ID NO:258; SEQ ID NO:261 and SEQ ID NO:262; SEQ ID NO:265 and SEQ ID NO:266; SEQ ID NO:269 and SEQ ID NO:270; SEQ ID NO:273 and SEQ ID NO:274; SEQ ID NO:277 and SEQ ID NO:278; SEQ ID NO:281 and SEQ ID NO:282; SEQ ID NO:285 and SEQ ID NO:286; SEQ ID NO:289 and SEQ ID NO:290; SEQ ID NO:293 and SEQ ID NO:294; SEQ ID NO:297 and SEQ ID NO:298; SEQ ID NO:301 and SEQ ID NO:302; SEQ ID NO:305 and SEQ ID NO:306; SEQ ID NO:309 and SEQ ID NO:310; SEQ ID NO:313 and SEQ ID NO:314; SEQ ID NO:317 and SEQ ID NO:318; SEQ ID NO:321 and SEQ ID NO:322; SEQ ID NO:325 and SEQ ID NO:326; SEQ ID NO:329 and SEQ ID NO:330; SEQ ID NO:333 and SEQ ID NO:334; SEQ ID NO:337 and SEQ ID NO:338; SEQ ID NO:341 and SEQ ID NO:342; SEQ ID NO:345 and SEQ ID NO:346; SEQ ID NO:349 and SEQ ID NO:350; SEQ ID NO:353 and SEQ ID NO:354; SEQ ID NO:357 and SEQ ID NO:358; SEQ ID NO:361 and SEQ ID NO:362; SEQ ID NO:365 and SEQ ID NO:366; SEQ ID NO:369 and SEQ ID NO:370; SEQ ID NO:373 and SEQ ID NO:374; SEQ ID NO:377 and SEQ ID NO:378; SEQ ID NO:381 and SEQ ID NO:382; SEQ ID NO:385 and SEQ ID NO:386; SEQ ID NO:389 and SEQ ID NO:390; SEQ ID NO:393 and SEQ ID NO:394; SEQ ID NO:397 and SEQ ID NO:398; SEQ ID NO:401 and SEQ ID NO:402; SEQ ID NO:405 and SEQ ID NO:406; and SEQ ID NO:409 and SEQ ID NO:410; 35. A composition according to claim 34 wherein said variable heavy sequence has SEQ ID NO:397 and said variable light sequence has SEQ ID NO:398. 36. A composition according to claim 34 wherein said variable heavy sequence and said variable light sequence are linked together to form an scFv domain. 37. A composition according to claim 36 wherein said composition further comprises an Fc region. 38. A composition according to claim 37 wherein said Fc region is a variable Fc region as compared to a wild-type human Fc region, wherein said variable Fc region has a an amino acid substitution selected from the group consisting of 239D, 239E, 236R, 330L, 332D, 332E, 239D/332E, 239D/332E/330L, 267D, 267E, 328F, 328R, 267E/328F, 243L, 298A, 236R/328R, 434A, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y, 252Y/254T/256E and 259I/308F/428L. 39. A composition according to claim 34 wherein said composition has a structure selected from the group consisting of FIG. 7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10E and FIGS. 11A-E, wherein the Fc domain of said structure comprises a set of corresponding variants selected from FIG. 12. 40. A composition according to claim 34, wherein said composition has a structure selected from the group consisting of FIG. 7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10E and FIGS. 11A-E, wherein the Fc domain of said structure comprises a set of corresponding variants selected from FIG. 13. 41. A composition according to claim 34, wherein said composition has a structure selected from the group consisting of FIG. 7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10E and FIGS. 11A-E, wherein the Fc domain of said structure comprises a set of corresponding variants selected from FIG. 14. 42. A nucleic acid encoding a composition according to claim 34. 43. An expression vector comprising a nucleic acid according to claim 42. 44. A host cell comprising a nucleic acid encoding a composition according to claim 34. 45. A method of making a composition by providing a host cell according to claim 44 and culturing it under conditions wherein said composition is expressed. 46. A method according to claim 45 further comprising running said composition on an ion exchange column. 47. A method of treating a patient in need thereof by administering a composition according to claim 34.

The present invention is directed to optimized anti-CD3 variable sequences for use in a variety of bispecific formats, including those that utilize scFv components. The invention further relates to nucleic acids encoding for the polypeptide, to vectors comprising the same and to host cells comprising the vector. In another aspect, the invention provides for a pharmaceutical composition comprising the mentioned polypeptide and medical uses of the polypeptide.1-33. (canceled) 34. An anti-CD3 binding domain comprising a variable heavy sequence and a variable light sequence pair selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:17 and SEQ ID NO:18; SEQ ID NO:21 and SEQ ID NO:22; SEQ ID NO:25 and SEQ ID NO:26; SEQ ID NO:29 and SEQ ID NO:30; SEQ ID NO:33 and SEQ ID NO:34; SEQ ID NO:37 and SEQ ID NO:38; SEQ ID NO:41 and SEQ ID NO:42; SEQ ID NO:45 and SEQ ID NO:46; SEQ ID NO:49 and SEQ ID NO:50; SEQ ID NO:53 and SEQ ID NO:54; SEQ ID NO:57 and SEQ ID NO:58; SEQ ID NO:61 and SEQ ID NO:62; SEQ ID NO:65 and SEQ ID NO:66; SEQ ID NO:69 and SEQ ID NO:70; SEQ ID NO:73 and SEQ ID NO:74; SEQ ID NO:77 and SEQ ID NO:78; SEQ ID NO:81 and SEQ ID NO:82; SEQ ID NO:85 and SEQ ID NO:86; SEQ ID NO:89 and SEQ ID NO:90; SEQ ID NO:93 and SEQ ID NO:94; SEQ ID NO:97 and SEQ ID NO:98; SEQ ID NO:101 and SEQ ID NO:102; SEQ ID NO:105 and SEQ ID NO:106; SEQ ID NO:109 and SEQ ID NO:110; SEQ ID NO:113 and SEQ ID NO:114; SEQ ID NO:117 and SEQ ID NO:118; SEQ ID NO:121 and SEQ ID NO:122; SEQ ID NO:125 and SEQ ID NO:126; SEQ ID NO:129 and SEQ ID NO:130; SEQ ID NO:133 and SEQ ID NO:134; SEQ ID NO:137 and SEQ ID NO:138; SEQ ID NO:141 and SEQ ID NO:142; SEQ ID NO:145 and SEQ ID NO:146; SEQ ID NO:149 and SEQ ID NO:150; SEQ ID NO:153 and SEQ ID NO:154; SEQ ID NO:157 and SEQ ID NO:158; SEQ ID NO:161 and SEQ ID NO:162; SEQ ID NO:165 and SEQ ID NO:166; SEQ ID NO:169 and SEQ ID NO:170; SEQ ID NO:173 and SEQ ID NO:174; SEQ ID NO:177 and SEQ ID NO:178; SEQ ID NO:181 and SEQ ID NO:182; SEQ ID NO:185 and SEQ ID NO:186; SEQ ID NO:189 and SEQ ID NO:190; SEQ ID NO:193 and SEQ ID NO:194; SEQ ID NO:197 and SEQ ID NO:198; SEQ ID NO:201 and SEQ ID NO:202; SEQ ID NO:205 and SEQ ID NO:206; SEQ ID NO:209 and SEQ ID NO:210; SEQ ID NO:213 and SEQ ID NO:214; SEQ ID NO:217 and SEQ ID NO:218; SEQ ID NO:221 and SEQ ID NO:222; SEQ ID NO:225 and SEQ ID NO:226; SEQ ID NO:229 and SEQ ID NO:230; SEQ ID NO:233 and SEQ ID NO:234; SEQ ID NO:237 and SEQ ID NO:238; SEQ ID NO:241 and SEQ ID NO:242; SEQ ID NO:245 and SEQ ID NO:246; SEQ ID NO:249 and SEQ ID NO:250; SEQ ID NO:253 and SEQ ID NO:254; SEQ ID NO:257 and SEQ ID NO:258; SEQ ID NO:261 and SEQ ID NO:262; SEQ ID NO:265 and SEQ ID NO:266; SEQ ID NO:269 and SEQ ID NO:270; SEQ ID NO:273 and SEQ ID NO:274; SEQ ID NO:277 and SEQ ID NO:278; SEQ ID NO:281 and SEQ ID NO:282; SEQ ID NO:285 and SEQ ID NO:286; SEQ ID NO:289 and SEQ ID NO:290; SEQ ID NO:293 and SEQ ID NO:294; SEQ ID NO:297 and SEQ ID NO:298; SEQ ID NO:301 and SEQ ID NO:302; SEQ ID NO:305 and SEQ ID NO:306; SEQ ID NO:309 and SEQ ID NO:310; SEQ ID NO:313 and SEQ ID NO:314; SEQ ID NO:317 and SEQ ID NO:318; SEQ ID NO:321 and SEQ ID NO:322; SEQ ID NO:325 and SEQ ID NO:326; SEQ ID NO:329 and SEQ ID NO:330; SEQ ID NO:333 and SEQ ID NO:334; SEQ ID NO:337 and SEQ ID NO:338; SEQ ID NO:341 and SEQ ID NO:342; SEQ ID NO:345 and SEQ ID NO:346; SEQ ID NO:349 and SEQ ID NO:350; SEQ ID NO:353 and SEQ ID NO:354; SEQ ID NO:357 and SEQ ID NO:358; SEQ ID NO:361 and SEQ ID NO:362; SEQ ID NO:365 and SEQ ID NO:366; SEQ ID NO:369 and SEQ ID NO:370; SEQ ID NO:373 and SEQ ID NO:374; SEQ ID NO:377 and SEQ ID NO:378; SEQ ID NO:381 and SEQ ID NO:382; SEQ ID NO:385 and SEQ ID NO:386; SEQ ID NO:389 and SEQ ID NO:390; SEQ ID NO:393 and SEQ ID NO:394; SEQ ID NO:397 and SEQ ID NO:398; SEQ ID NO:401 and SEQ ID NO:402; SEQ ID NO:405 and SEQ ID NO:406; and SEQ ID NO:409 and SEQ ID NO:410; 35. A composition according to claim 34 wherein said variable heavy sequence has SEQ ID NO:397 and said variable light sequence has SEQ ID NO:398. 36. A composition according to claim 34 wherein said variable heavy sequence and said variable light sequence are linked together to form an scFv domain. 37. A composition according to claim 36 wherein said composition further comprises an Fc region. 38. A composition according to claim 37 wherein said Fc region is a variable Fc region as compared to a wild-type human Fc region, wherein said variable Fc region has a an amino acid substitution selected from the group consisting of 239D, 239E, 236R, 330L, 332D, 332E, 239D/332E, 239D/332E/330L, 267D, 267E, 328F, 328R, 267E/328F, 243L, 298A, 236R/328R, 434A, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y, 252Y/254T/256E and 259I/308F/428L. 39. A composition according to claim 34 wherein said composition has a structure selected from the group consisting of FIG. 7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10E and FIGS. 11A-E, wherein the Fc domain of said structure comprises a set of corresponding variants selected from FIG. 12. 40. A composition according to claim 34, wherein said composition has a structure selected from the group consisting of FIG. 7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10E and FIGS. 11A-E, wherein the Fc domain of said structure comprises a set of corresponding variants selected from FIG. 13. 41. A composition according to claim 34, wherein said composition has a structure selected from the group consisting of FIG. 7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10E and FIGS. 11A-E, wherein the Fc domain of said structure comprises a set of corresponding variants selected from FIG. 14. 42. A nucleic acid encoding a composition according to claim 34. 43. An expression vector comprising a nucleic acid according to claim 42. 44. A host cell comprising a nucleic acid encoding a composition according to claim 34. 45. A method of making a composition by providing a host cell according to claim 44 and culturing it under conditions wherein said composition is expressed. 46. A method according to claim 45 further comprising running said composition on an ion exchange column. 47. A method of treating a patient in need thereof by administering a composition according to claim 34.

The present disclosure relates to liquid and dried compositions comprising a live, attenuated or genetically modified herpesvirus and methods of preparing such compositions, in one aspect, the composition comprises at least two or more pharmaceutically acceptable excepients, at least one of which is histidine and at least one of which is a sugar or sugar alcohol. The compositions retain a sufficiently high infectious titre following storage or large-scale manufacturing steps, such as lyophilization.

1. A composition comprising a live, attenuated or genetically modified herpesvirus, and at least two or more pharmaceutically acceptable excipients, at least one of which is histidine and at least one of which is a sugar or sugar alcohol. 2. The composition of claim 1, wherein the composition further comprises at least one or more additional pharmaceutically acceptable excipients selected from buffers, salts, surfactants, bulking agents, chelating agents, gelling agents, urea, albumin or combinations thereof. 3. The composition of claim 1, further comprising one or more buffers selected from the group consisting of TRIS (tris(hydroxymethyl)-aminomethane) and TRIS-acetate, and wherein the composition has a pH of about 6.5 to 7.5. 4. The composition of claim 2, further comprising one or more salts selected from the group consisting of sodium glutamate, potassium glutamate, sodium chloride, potassium chloride, and calcium chloride. 5. The composition of claim 2, further comprising urea and/or albumin. 6. The composition of claim 1, wherein the sugar or sugar alcohol is selected from the group consisting of sucrose, trehalose and sorbitol. 7. The composition of claim 6, comprising one or more sugar or sugar alcohols. 8. The composition of claim 6, wherein the sugar comprises sucrose. 9. The composition of claim 8, comprising 1 mM to 50 mM histidine and 5% to 10% w/v sucrose and having a pH of about 6.5 to 7.5. 10. The composition of claim 9, further comprising 1 mM to 100 mM potassium glutamate or sodium glutamate. 11. The composition of claim 9, further comprising 80 mM to 160 mM sodium chloride and has a pH of about 6.5 to 7.5. 12. The composition of claim 1, wherein the composition comprises 10 mM histidine, 10% w/v sucrose, 160 mM NaCl, 50 mM potassium glutamate and a pH of about 6.5 to 7.5. 13. The composition of claim 1, wherein the composition is a liquid. 14. The composition of claim 13, wherein the liquid composition has a titre loss of less than 0.3 Log10 pfu/mL following storage at 2-8° C. for 1-2 weeks. 15. The composition of claim 13, wherein the liquid composition has a titre loss of less than 0.5 Log10 pfu/mL following storage at 25° C.±2° C. for 1 week. 16. The composition of claim 1, wherein the composition is in a dried form. 17. The composition of claim 1, wherein the live, attenuated or genetically modified herpesvirus is a herpes simplex virus 2 (HSV-2). 18. The composition of claim 17, wherein the HSV-2 is a replication defective HSV-529 strain. 19. The composition of claim 17, wherein the HSV-2 contains less than 10 ng of host cell DNA per 1×107 pfu/mL. 20. The dried composition of claim 16, wherein following storage for 6 months at 5° C.±2° C., the composition retains an infectious titre within 0.25 Log10 pfu/mL of the titre measured after the composition is dried, when the viral titres in the composition are between 107 to 105 pfu/mL before drying. 21. The dried composition of claim 16, wherein following storage for 6 months at 5° C.±2° C., the composition retains an infectious titre within 0.2 Log10 pfu/mL of the titre measured after the composition is dried, when the viral titres in the composition are between 107 to 105 pfu/mL before drying. 22. The dried composition of claim 16, wherein following storage for 6 months at 5° C.±2° C., the dried composition retains an infectious titre within 0.1 Log10 pfu/mL of the titre measured after the composition is dried, when the viral titres in the composition are between 107 to 105 pfu/mL before drying. 23. The dried composition of claim 16, wherein following storage for 6 months at 25° C.±2° C., the dried composition retains an infectious titre within 0.5 Log10 pfu/mL of the titre measured after the composition is dried, when the viral titres in the composition range from 107 to 105 pfu/mL before drying. 24. The composition of claim 16, wherein the composition is freeze-dried, foam-dried or spray-dried. 25. The composition of claim 24, wherein the residual moisture content of the composition is 6% or less. 26. The composition of claim 1, wherein the herpesvirus is grown using serum-free media. 27. The composition of claim 1, wherein the composition is a pharmaceutical composition. 28. The composition of claim 27, wherein the composition is a vaccine. 29. A method of manufacturing a dried composition comprising a live, attenuated or genetically modified herpesvirus and stabilizer that comprises at least two or more pharmaceutically acceptable excipients, at least one of which is histidine and at least one of which is a sugar or sugar alcohol, the method comprising: (i) admixing the live, attenuated or genetically modified herpesvirus with the stabilizer; and (ii) drying the admixture. 30. The method of claim 29, wherein the admixture is dried by means of a method selected from the group of consisting of freeze-drying, foam-drying, and spray-drying. 31. The method of claim 30 comprising a replication-defective herpes simplex virus. 32. A method of preparing a vaccine, comprising reconstituting the dried composition of claim 16 with an aqueous solution. 33. A kit comprising a first container containing the dried composition of claim 16 and a second container containing an aqueous solution for reconstituting the dried composition.

The present disclosure relates to liquid and dried compositions comprising a live, attenuated or genetically modified herpesvirus and methods of preparing such compositions, in one aspect, the composition comprises at least two or more pharmaceutically acceptable excepients, at least one of which is histidine and at least one of which is a sugar or sugar alcohol. The compositions retain a sufficiently high infectious titre following storage or large-scale manufacturing steps, such as lyophilization.1. A composition comprising a live, attenuated or genetically modified herpesvirus, and at least two or more pharmaceutically acceptable excipients, at least one of which is histidine and at least one of which is a sugar or sugar alcohol. 2. The composition of claim 1, wherein the composition further comprises at least one or more additional pharmaceutically acceptable excipients selected from buffers, salts, surfactants, bulking agents, chelating agents, gelling agents, urea, albumin or combinations thereof. 3. The composition of claim 1, further comprising one or more buffers selected from the group consisting of TRIS (tris(hydroxymethyl)-aminomethane) and TRIS-acetate, and wherein the composition has a pH of about 6.5 to 7.5. 4. The composition of claim 2, further comprising one or more salts selected from the group consisting of sodium glutamate, potassium glutamate, sodium chloride, potassium chloride, and calcium chloride. 5. The composition of claim 2, further comprising urea and/or albumin. 6. The composition of claim 1, wherein the sugar or sugar alcohol is selected from the group consisting of sucrose, trehalose and sorbitol. 7. The composition of claim 6, comprising one or more sugar or sugar alcohols. 8. The composition of claim 6, wherein the sugar comprises sucrose. 9. The composition of claim 8, comprising 1 mM to 50 mM histidine and 5% to 10% w/v sucrose and having a pH of about 6.5 to 7.5. 10. The composition of claim 9, further comprising 1 mM to 100 mM potassium glutamate or sodium glutamate. 11. The composition of claim 9, further comprising 80 mM to 160 mM sodium chloride and has a pH of about 6.5 to 7.5. 12. The composition of claim 1, wherein the composition comprises 10 mM histidine, 10% w/v sucrose, 160 mM NaCl, 50 mM potassium glutamate and a pH of about 6.5 to 7.5. 13. The composition of claim 1, wherein the composition is a liquid. 14. The composition of claim 13, wherein the liquid composition has a titre loss of less than 0.3 Log10 pfu/mL following storage at 2-8° C. for 1-2 weeks. 15. The composition of claim 13, wherein the liquid composition has a titre loss of less than 0.5 Log10 pfu/mL following storage at 25° C.±2° C. for 1 week. 16. The composition of claim 1, wherein the composition is in a dried form. 17. The composition of claim 1, wherein the live, attenuated or genetically modified herpesvirus is a herpes simplex virus 2 (HSV-2). 18. The composition of claim 17, wherein the HSV-2 is a replication defective HSV-529 strain. 19. The composition of claim 17, wherein the HSV-2 contains less than 10 ng of host cell DNA per 1×107 pfu/mL. 20. The dried composition of claim 16, wherein following storage for 6 months at 5° C.±2° C., the composition retains an infectious titre within 0.25 Log10 pfu/mL of the titre measured after the composition is dried, when the viral titres in the composition are between 107 to 105 pfu/mL before drying. 21. The dried composition of claim 16, wherein following storage for 6 months at 5° C.±2° C., the composition retains an infectious titre within 0.2 Log10 pfu/mL of the titre measured after the composition is dried, when the viral titres in the composition are between 107 to 105 pfu/mL before drying. 22. The dried composition of claim 16, wherein following storage for 6 months at 5° C.±2° C., the dried composition retains an infectious titre within 0.1 Log10 pfu/mL of the titre measured after the composition is dried, when the viral titres in the composition are between 107 to 105 pfu/mL before drying. 23. The dried composition of claim 16, wherein following storage for 6 months at 25° C.±2° C., the dried composition retains an infectious titre within 0.5 Log10 pfu/mL of the titre measured after the composition is dried, when the viral titres in the composition range from 107 to 105 pfu/mL before drying. 24. The composition of claim 16, wherein the composition is freeze-dried, foam-dried or spray-dried. 25. The composition of claim 24, wherein the residual moisture content of the composition is 6% or less. 26. The composition of claim 1, wherein the herpesvirus is grown using serum-free media. 27. The composition of claim 1, wherein the composition is a pharmaceutical composition. 28. The composition of claim 27, wherein the composition is a vaccine. 29. A method of manufacturing a dried composition comprising a live, attenuated or genetically modified herpesvirus and stabilizer that comprises at least two or more pharmaceutically acceptable excipients, at least one of which is histidine and at least one of which is a sugar or sugar alcohol, the method comprising: (i) admixing the live, attenuated or genetically modified herpesvirus with the stabilizer; and (ii) drying the admixture. 30. The method of claim 29, wherein the admixture is dried by means of a method selected from the group of consisting of freeze-drying, foam-drying, and spray-drying. 31. The method of claim 30 comprising a replication-defective herpes simplex virus. 32. A method of preparing a vaccine, comprising reconstituting the dried composition of claim 16 with an aqueous solution. 33. A kit comprising a first container containing the dried composition of claim 16 and a second container containing an aqueous solution for reconstituting the dried composition.

Disclosed are active ingredient combinations of alkylamidothiazoles and one or more cosmetically or dermatologically relevant fragrances.

1.-15. (canceled) 16. An active ingredient combination of one or more alkylamidothiazoles and one or more cosmetically or dermatologically relevant fragrances. 17. The active ingredient combination of claim 16, wherein the one or more fragrances comprise one or more of coumarin, Iraldein alpha iff, farnesol, lilial, bitter orange oil, Florosa, hexylsalicylate, phenylethyl alcohol, benzyl benzoate M, hydroxycitronellal, Macrolide Supra, phenoxanol, Geraniol Supra, dihydromyrcenol, cinnamaldehyde, lyral, isoeugenol, anis alcohol, terpineol pure, bergamot oil, hedione, vanillin, thymol, linalyl acetate, linalool aroma, hexenol cis-3, tetrahydromuguol, Limonene D pure, benzyl salicylate, benzyl cinnamate, Iso E Super, citronellol 950, benzyl alcohol DD, ethylvanillin, eugenol, methyl heptine carbonate, Citral 95, hexylcinnamaldehyde alpha, benzyl acetate, ethyllinalool, Iraldein gamma Coeur 262654, amyl cinnamaldehyde, alpha-isomethylionone, methyl benzoate, alpha-methylionone, 2-tert-pentylcyclohexyl acetate, 7-acetyl-1,1,3,4,4,6-hexamethyltetralin, adipic diester, amyl salicylate, amyl cinnamyl alcohol, amyl C butylphenylmethylpropionalcinnamal, benzoin, bitter orange oil, sweet orange oil, cardamom oil, cedrol, cinnamyl alcohol, citronellyl methyl crotonate, lemon oil, diethyl succinate, Evernia Furfuracea Extract, Evernia Prunastri Extract, guaiac wood oil, hexylcinnamal, lavender oil, lime oil, mandarin oil, menthyl PCA, methylheptenone, nutmeg oil, rosemary oil, Tonka bean oil, triethyl citrate. 18. The active ingredient combination of claim 16, wherein the one or more fragrances comprise one or more of coumarin, Iraldein alpha iff, farnesol, lilial, bitter orange oil, Florosa, hexyl salicylate, phenylethyl alcohol, Benzylbenzoate M, hydroxycitronellal, Macrolide Supra, phenoxanol, Geraniol Supra, dihydromyrcenol, cinnamaldehyde, lyral, isoeugenol, terpineol pure, vanillin, thymol, linalyl acetate, linalool aroma, Limonene D pure, benzyl salicylate, Iso E Super, citronellol 950, benzyl alcohol DD, eugenol, Citral 95, ethyllinalool, Iraldein gamma Coeur 262654, alpha-isomethylionone, methyl benzoate, alpha-methylionone, bitter orange oil, sweet orange oil, lemon oil, Evernia Prunastri Extract, hexylcinnamal, menthyl PCA, rosemary oil, triethyl citrate. 19. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which R1, R2, X and Y are different, partly identical or completely identical and, independently of one another, represent: R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24-alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylaryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24 alkyl-morpholino, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alkyl-piperazino-N-alkyl; R2=H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-hydroxyalkyl (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched); X=—H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-aryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), —C1-C24-heteroaryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), -aryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), -phenyl, -2,4-dihydroxyphenyl, -2,3-dihydroxyphenyl, -2,4-dimethoxyphenyl, -2,3-dimethoxyphenyl; Y=H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-aryl, —C1-C24-heteroaryl, —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), -aryl, -phenyl, -2,4-dihydroxyphenyl, -2,3-dihydroxyphenyl, -2,4-dimethoxyphenyl, -2,3-dimethoxyphenyl, —COO-alkyl, —COO-alkenyl, —COO-cycloalkyl, —COO-aryl, —COO-heteroaryl; and X, Y can also form a fused aromatic ring system and can form with one another aromatic or aliphatic homo- or heterocyclic ring systems with up to n ring-forming atoms, where n can assume values from 5 to 8, and the respective ring systems can in turn be substituted with up to n-1 alkyl groups, hydroxyl groups, carboxyl groups, amino groups, nitrile functions, sulfur-containing substituents, ester groups and/or ether groups, and wherein the one or more alkylamidothiazoles are be present as a free base and/or as a cosmetically and dermatologically acceptable salt thereof. 20. The active ingredient combination of claim 19, wherein X represents a substituted phenyl group. 21. The active ingredient combination of claim 19, wherein the one or more alkylamidothiazoles comprise one or more alkylamidothiazoles of formula where the substituents Z independently represent —H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN, acetyl. 22. The active ingredient combination of claim 21, wherein the one or more alkylamidothiazoles comprise one or more alkylamidothiazoles of formula where the substituent Z represents —H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN. 23. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which Y=H; R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24 alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylaryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24-alkyl-morpholine, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alkyl-piperazino-N-alkyl; R2=H, —C1-C24-alkyl (linear and branched); Z=—H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN, acetyl. 24. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which Y=H; R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24-alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkyl-aryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24 alkyl-morpholino, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alkyl-piperazino-N-alkyl; R2=H. 25. The active ingredient combination of claim 16, wherein the combination comprises one or more of the following alkylamidothiazoles: 26. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles in the form of one or more of a halide, a carbonate, an ascorbate, a sulfate, an acetate, a phosphate. 27. A cosmetic or dermatological preparation, wherein the preparation comprises the active ingredient combination of claim 16. 28. The preparation of claim 27, wherein the preparation comprises from 0.000001% to 10% by weight of the active ingredient combination, based on a total weight of the preparation. 29. The preparation of claim 28, wherein the preparation comprises from 0.0001% to 3% by weight of the active ingredient combination. 30. The preparation of claim 28, wherein the preparation comprises from 0.001% to 1% by weight of the active ingredient combination. 31. The preparation of claim 27, wherein the preparation comprises a total of from 0.00001% by weight to 10% by weight of the one or more fragrances, based on a total weight of the preparation. 32. The preparation of claim 31, wherein the preparation comprises a total of from 0.001% by weight to 5% by weight of the one or more preservatives. 33. The preparation of claim 31, wherein the preparation comprises a total of from 0.005% by weight to 3% by weight of the one or more preservatives. 34. A method of lightening human skin, wherein the method comprises applying to human skin to be lightened the preparation of claim 28. 35. A method of lightening human skin, wherein the method comprises applying to human skin to be lightened the active ingredient combination of claim 16.

Disclosed are active ingredient combinations of alkylamidothiazoles and one or more cosmetically or dermatologically relevant fragrances.1.-15. (canceled) 16. An active ingredient combination of one or more alkylamidothiazoles and one or more cosmetically or dermatologically relevant fragrances. 17. The active ingredient combination of claim 16, wherein the one or more fragrances comprise one or more of coumarin, Iraldein alpha iff, farnesol, lilial, bitter orange oil, Florosa, hexylsalicylate, phenylethyl alcohol, benzyl benzoate M, hydroxycitronellal, Macrolide Supra, phenoxanol, Geraniol Supra, dihydromyrcenol, cinnamaldehyde, lyral, isoeugenol, anis alcohol, terpineol pure, bergamot oil, hedione, vanillin, thymol, linalyl acetate, linalool aroma, hexenol cis-3, tetrahydromuguol, Limonene D pure, benzyl salicylate, benzyl cinnamate, Iso E Super, citronellol 950, benzyl alcohol DD, ethylvanillin, eugenol, methyl heptine carbonate, Citral 95, hexylcinnamaldehyde alpha, benzyl acetate, ethyllinalool, Iraldein gamma Coeur 262654, amyl cinnamaldehyde, alpha-isomethylionone, methyl benzoate, alpha-methylionone, 2-tert-pentylcyclohexyl acetate, 7-acetyl-1,1,3,4,4,6-hexamethyltetralin, adipic diester, amyl salicylate, amyl cinnamyl alcohol, amyl C butylphenylmethylpropionalcinnamal, benzoin, bitter orange oil, sweet orange oil, cardamom oil, cedrol, cinnamyl alcohol, citronellyl methyl crotonate, lemon oil, diethyl succinate, Evernia Furfuracea Extract, Evernia Prunastri Extract, guaiac wood oil, hexylcinnamal, lavender oil, lime oil, mandarin oil, menthyl PCA, methylheptenone, nutmeg oil, rosemary oil, Tonka bean oil, triethyl citrate. 18. The active ingredient combination of claim 16, wherein the one or more fragrances comprise one or more of coumarin, Iraldein alpha iff, farnesol, lilial, bitter orange oil, Florosa, hexyl salicylate, phenylethyl alcohol, Benzylbenzoate M, hydroxycitronellal, Macrolide Supra, phenoxanol, Geraniol Supra, dihydromyrcenol, cinnamaldehyde, lyral, isoeugenol, terpineol pure, vanillin, thymol, linalyl acetate, linalool aroma, Limonene D pure, benzyl salicylate, Iso E Super, citronellol 950, benzyl alcohol DD, eugenol, Citral 95, ethyllinalool, Iraldein gamma Coeur 262654, alpha-isomethylionone, methyl benzoate, alpha-methylionone, bitter orange oil, sweet orange oil, lemon oil, Evernia Prunastri Extract, hexylcinnamal, menthyl PCA, rosemary oil, triethyl citrate. 19. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which R1, R2, X and Y are different, partly identical or completely identical and, independently of one another, represent: R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24-alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylaryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24 alkyl-morpholino, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alkyl-piperazino-N-alkyl; R2=H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-hydroxyalkyl (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched); X=—H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-aryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), —C1-C24-heteroaryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), -aryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), -phenyl, -2,4-dihydroxyphenyl, -2,3-dihydroxyphenyl, -2,4-dimethoxyphenyl, -2,3-dimethoxyphenyl; Y=H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-aryl, —C1-C24-heteroaryl, —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), -aryl, -phenyl, -2,4-dihydroxyphenyl, -2,3-dihydroxyphenyl, -2,4-dimethoxyphenyl, -2,3-dimethoxyphenyl, —COO-alkyl, —COO-alkenyl, —COO-cycloalkyl, —COO-aryl, —COO-heteroaryl; and X, Y can also form a fused aromatic ring system and can form with one another aromatic or aliphatic homo- or heterocyclic ring systems with up to n ring-forming atoms, where n can assume values from 5 to 8, and the respective ring systems can in turn be substituted with up to n-1 alkyl groups, hydroxyl groups, carboxyl groups, amino groups, nitrile functions, sulfur-containing substituents, ester groups and/or ether groups, and wherein the one or more alkylamidothiazoles are be present as a free base and/or as a cosmetically and dermatologically acceptable salt thereof. 20. The active ingredient combination of claim 19, wherein X represents a substituted phenyl group. 21. The active ingredient combination of claim 19, wherein the one or more alkylamidothiazoles comprise one or more alkylamidothiazoles of formula where the substituents Z independently represent —H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN, acetyl. 22. The active ingredient combination of claim 21, wherein the one or more alkylamidothiazoles comprise one or more alkylamidothiazoles of formula where the substituent Z represents —H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN. 23. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which Y=H; R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24 alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylaryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24-alkyl-morpholine, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alkyl-piperazino-N-alkyl; R2=H, —C1-C24-alkyl (linear and branched); Z=—H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN, acetyl. 24. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which Y=H; R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24-alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkyl-aryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24 alkyl-morpholino, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alkyl-piperazino-N-alkyl; R2=H. 25. The active ingredient combination of claim 16, wherein the combination comprises one or more of the following alkylamidothiazoles: 26. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles in the form of one or more of a halide, a carbonate, an ascorbate, a sulfate, an acetate, a phosphate. 27. A cosmetic or dermatological preparation, wherein the preparation comprises the active ingredient combination of claim 16. 28. The preparation of claim 27, wherein the preparation comprises from 0.000001% to 10% by weight of the active ingredient combination, based on a total weight of the preparation. 29. The preparation of claim 28, wherein the preparation comprises from 0.0001% to 3% by weight of the active ingredient combination. 30. The preparation of claim 28, wherein the preparation comprises from 0.001% to 1% by weight of the active ingredient combination. 31. The preparation of claim 27, wherein the preparation comprises a total of from 0.00001% by weight to 10% by weight of the one or more fragrances, based on a total weight of the preparation. 32. The preparation of claim 31, wherein the preparation comprises a total of from 0.001% by weight to 5% by weight of the one or more preservatives. 33. The preparation of claim 31, wherein the preparation comprises a total of from 0.005% by weight to 3% by weight of the one or more preservatives. 34. A method of lightening human skin, wherein the method comprises applying to human skin to be lightened the preparation of claim 28. 35. A method of lightening human skin, wherein the method comprises applying to human skin to be lightened the active ingredient combination of claim 16.

The present invention relates to systems and methods for administering high concentrations of nitric oxide (NO) gas to a patient without the need to provide supplemental oxygen to the patient. The systems and methods can be used to administer high therapeutic amounts of NO gas, for example a gas comprising 160 ppm NO, while forming little or no residual nitrogen dioxide (NO 2 ). The method is based on using a NO gas source with a relatively high NO concentration, for example 5,000 ppm, while rapidly mixing the gas from the NO gas source with air immediately prior to administering the gas to a patient.

1. A method for administering nitric oxide (NO) gas to a patient, comprising: receiving, at a gas mixing and administration device, a controlled quantity of high concentration NO gas flow from a high concentration NO gas source; receiving, at the gas mixing and administration device, air flow from an air source into the gas mixer; mixing, at the gas mixing and administration device, the received high concentration NO and air in close proximity to the subject's point of administration to form a delivery gas having an NO concentration of between 100-200 ppm and an oxygen concentration of at least about 20%; and administering, via an exit in the gas mixing and administration device, the delivery gas to a patient. 2. The method of claim 1, wherein the received NO gas has a concentration in the range of about 3000 to 10,000 ppm. 3. The method of claim 2, wherein the received NO gas concentration is about 5000 ppm. 4. The method of claim 1, wherein the NO concentration of the delivery gas is about 160 ppm. 5. The method of claim 1, wherein the high concentration NO gas flow received from the high concentration NO gas source is from a gas metering device. 6. The method of claim 1, wherein the delivery gas is administered to the patient via a breathing tube connected to the gas mixing and administration device. 7. The method of claim 1, further comprising: restricting, via a first one way valve, the delivery gas flow in the gas mixing and administration device to only forward flow in a direction towards the patient. 8. The method of claim 1, further comprising: restricting, via a first one way valve, patient expiratory flow from entering the gas mixing and administration device. 9. The method of claim 1, wherein the air source is comprises atmospheric air. 10. The method of claim 7, wherein the atmospheric air has an oxygen concentration of about 21%. 11. The method of claim 1, wherein the delivery gas is formed without adding supplemental oxygen. 12. The method of claim 1, wherein the nitrogen dioxide (NO2) concentration of the delivery gas is less than 5 ppm. 13. The method of claim 1, wherein the NO2 concentration of the delivery gas is less than 3 ppm. 14. A method for administering nitric oxide (NO) gas to a patient, comprising: receiving, at a gas mixing and administration device, inspiratory air flow from an air source; receiving, at the gas mixing and administration device, high concentration NO gas flow from a high concentration NO gas source, the high concentration NO gas flow being proportional to the inspiratory air flow; mixing, at the gas mixing and administration device, the received high concentration NO and air in close proximity to the subject's point of administration to form a delivery gas having an NO concentration of between 100-200 ppm and an oxygen concentration of at least about 20%; and administering the delivery gas to a patient. 15. A method for administering inhaled therapeutic nitric oxide (NO) gas to a patient in need thereof, comprising: receiving, at an air inlet in a gas mixing and administration device, air flow from an air source; receiving, at an NO inlet in the gas mixing and administration device, high concentration NO gas flow from a high concentration NO gas source, the flow of high concentration NO gas being proportional to the air flow enabling reduction of the high concentration NO gas to a desired therapeutic NO concentration, wherein the NO inlet is a short distance from an exit of the gas mixing and administration device and, in turn, entrance to the patients airway; mixing, in close proximity to the exit of the gas mixing and administration device and, in turn, entrance to the patients airway, the received NO and air flow to form a delivery gas having the desired therapeutic NO concentration of between 100-200 ppm and an oxygen concentration of at least about 20%; and administering, via the exit in the gas mixing and administration device, the delivery gas to the patient. 16. A system for high-dose administration of nitric oxide gas to a patient, comprising: a NO gas source having a NO concentration in the range of 3000 to 10,000 ppm; a gas metering device, wherein the NO gas source is connected to the gas metering device via a first conduit; a gas mixing device, wherein the gas metering device is connected to the gas mixing device via a second conduit, and wherein NO source gas from the NO gas source is transferred to the gas mixing device via the first conduit, gas metering device, and the second conduit; an air source connected to the gas mixing device via a third conduit; and a delivery gas dispensing mechanism for administering a mixed delivery gas to a patient. 17. The system of claim 12, wherein when the NO source gas is mixed with air in the mixing device, a delivery gas is formed having a NO concentration in the range of about 100 to 200 ppm. 18. The system of claim 13, wherein the delivery gas has an oxygen concentration of at least about 20%. 19. The system of claim 13, wherein the delivery gas has a NO2 concentration of less than 3 ppm. 20. The system of claim 13, wherein the delivery gas has a NO2 concentration of less than 5 ppm. 21. The system of claim 12, wherein the delivery gas dispensing mechanism is a conduit connected to the gas mixing device. 22. The system of claim 12, wherein the delivery gas dispensing mechanism is a breathing mask connected to the gas mixing device. 23. The system of claim 12, wherein NO concentration of the NO gas source is 5,000 ppm.

The present invention relates to systems and methods for administering high concentrations of nitric oxide (NO) gas to a patient without the need to provide supplemental oxygen to the patient. The systems and methods can be used to administer high therapeutic amounts of NO gas, for example a gas comprising 160 ppm NO, while forming little or no residual nitrogen dioxide (NO 2 ). The method is based on using a NO gas source with a relatively high NO concentration, for example 5,000 ppm, while rapidly mixing the gas from the NO gas source with air immediately prior to administering the gas to a patient.1. A method for administering nitric oxide (NO) gas to a patient, comprising: receiving, at a gas mixing and administration device, a controlled quantity of high concentration NO gas flow from a high concentration NO gas source; receiving, at the gas mixing and administration device, air flow from an air source into the gas mixer; mixing, at the gas mixing and administration device, the received high concentration NO and air in close proximity to the subject's point of administration to form a delivery gas having an NO concentration of between 100-200 ppm and an oxygen concentration of at least about 20%; and administering, via an exit in the gas mixing and administration device, the delivery gas to a patient. 2. The method of claim 1, wherein the received NO gas has a concentration in the range of about 3000 to 10,000 ppm. 3. The method of claim 2, wherein the received NO gas concentration is about 5000 ppm. 4. The method of claim 1, wherein the NO concentration of the delivery gas is about 160 ppm. 5. The method of claim 1, wherein the high concentration NO gas flow received from the high concentration NO gas source is from a gas metering device. 6. The method of claim 1, wherein the delivery gas is administered to the patient via a breathing tube connected to the gas mixing and administration device. 7. The method of claim 1, further comprising: restricting, via a first one way valve, the delivery gas flow in the gas mixing and administration device to only forward flow in a direction towards the patient. 8. The method of claim 1, further comprising: restricting, via a first one way valve, patient expiratory flow from entering the gas mixing and administration device. 9. The method of claim 1, wherein the air source is comprises atmospheric air. 10. The method of claim 7, wherein the atmospheric air has an oxygen concentration of about 21%. 11. The method of claim 1, wherein the delivery gas is formed without adding supplemental oxygen. 12. The method of claim 1, wherein the nitrogen dioxide (NO2) concentration of the delivery gas is less than 5 ppm. 13. The method of claim 1, wherein the NO2 concentration of the delivery gas is less than 3 ppm. 14. A method for administering nitric oxide (NO) gas to a patient, comprising: receiving, at a gas mixing and administration device, inspiratory air flow from an air source; receiving, at the gas mixing and administration device, high concentration NO gas flow from a high concentration NO gas source, the high concentration NO gas flow being proportional to the inspiratory air flow; mixing, at the gas mixing and administration device, the received high concentration NO and air in close proximity to the subject's point of administration to form a delivery gas having an NO concentration of between 100-200 ppm and an oxygen concentration of at least about 20%; and administering the delivery gas to a patient. 15. A method for administering inhaled therapeutic nitric oxide (NO) gas to a patient in need thereof, comprising: receiving, at an air inlet in a gas mixing and administration device, air flow from an air source; receiving, at an NO inlet in the gas mixing and administration device, high concentration NO gas flow from a high concentration NO gas source, the flow of high concentration NO gas being proportional to the air flow enabling reduction of the high concentration NO gas to a desired therapeutic NO concentration, wherein the NO inlet is a short distance from an exit of the gas mixing and administration device and, in turn, entrance to the patients airway; mixing, in close proximity to the exit of the gas mixing and administration device and, in turn, entrance to the patients airway, the received NO and air flow to form a delivery gas having the desired therapeutic NO concentration of between 100-200 ppm and an oxygen concentration of at least about 20%; and administering, via the exit in the gas mixing and administration device, the delivery gas to the patient. 16. A system for high-dose administration of nitric oxide gas to a patient, comprising: a NO gas source having a NO concentration in the range of 3000 to 10,000 ppm; a gas metering device, wherein the NO gas source is connected to the gas metering device via a first conduit; a gas mixing device, wherein the gas metering device is connected to the gas mixing device via a second conduit, and wherein NO source gas from the NO gas source is transferred to the gas mixing device via the first conduit, gas metering device, and the second conduit; an air source connected to the gas mixing device via a third conduit; and a delivery gas dispensing mechanism for administering a mixed delivery gas to a patient. 17. The system of claim 12, wherein when the NO source gas is mixed with air in the mixing device, a delivery gas is formed having a NO concentration in the range of about 100 to 200 ppm. 18. The system of claim 13, wherein the delivery gas has an oxygen concentration of at least about 20%. 19. The system of claim 13, wherein the delivery gas has a NO2 concentration of less than 3 ppm. 20. The system of claim 13, wherein the delivery gas has a NO2 concentration of less than 5 ppm. 21. The system of claim 12, wherein the delivery gas dispensing mechanism is a conduit connected to the gas mixing device. 22. The system of claim 12, wherein the delivery gas dispensing mechanism is a breathing mask connected to the gas mixing device. 23. The system of claim 12, wherein NO concentration of the NO gas source is 5,000 ppm.

According to the invention a transdermal therapeutic system for administering fentanyl or an analogue thereof through the skin is provided that has a pressure-sensitive adhesive matrix layer containing a mixture of two polyisobutylenes with specific storage moduli.

1. A transdermal therapeutic system for the administration of an active ingredient through the skin comprising or consisting of a) a back layer, b) a pressure-sensitive adhesive matrix layer containing the active ingredient; and c) a stripping layer (release liner), wherein the active ingredient is fentanyl or an analogue of the fentanyl selected from alfentanil, carfentanil, lofentanil, remifentanil, and trefentanil or a salt of one of these active ingredients and wherein the matrix layer as the pressure-sensitive adhesive polymer contains a mixture of a polyisobutylene A and a polyisobutylene B, wherein the polyisobutylene A has a storage modulus G′ the value of which is substantially constant in the temperature range of from 10° C. to 40° C. and wherein the polyisobutylene B has a storage modulus G′ the value of which continuously decreases with increasing temperature in the temperature range of from 10° C. to 40° C., wherein the storage modulus G′ is measured in the linear viscoelastic range at a frequency of 10 rad/sec using a rheometer with parallel plate geometry and parallel plates; and wherein the pressure-sensitive adhesive matrix layer contains undissolved active ingredient in the form of active ingredient particles. 2. The transdermal therapeutic system according to claim 1, wherein the active ingredient is fentanyl. 3. The transdermal therapeutic system according to claim 1, wherein for the polyisobutylene A in the temperature range of from 10° C. to 40° C. all values of the storage modulus G′ deviate from the value for the storage modulus G′ at 40° C. by no more than 50%. 4. The transdermal therapeutic system according to claim 1, wherein for the polyisobutylene B the storage modulus G′ at 10° C. is at least two times the storage modulus G′ at 80° C. 5. The transdermal therapeutic system according claim 1, wherein the content of the polyisobutylene A to the polyisobutylene B in the matrix layer is in the range of from 20% (A) : 80% (B) to 40% (A) : 60% (B), each based on the total weight of the polyisobutylenes A and B. 6. The transdermal therapeutic system according to claim 1, wherein the polyisobutylene A and the polyisobutylene B each are individual polyisobutylenes of different average molecular weights. 7. The transdermal therapeutic system according to claim 1, wherein the matrix layer contains a permeation enhancer that optionally is isopropyl myristate or oleyl oleate. 8. The transdermal therapeutic system according to claim 1, wherein the matrix layer contains a tackifier that optionally is a polybutene or hydrogenated or non-hydrogenated rosin ester. 9. The transdermal therapeutic system according to claim 1, characterized in that the amount of active ingredient is sufficient for an application time of 3 days and the active ingredient is present in the matrix layer at a concentration in the range of from 3-15% by weight, based on the weight of the matrix layer. 10. The transdermal therapeutic system according to claim 1, characterized in that in the pressure-sensitive adhesive matrix layer in addition to the active ingredient, the polyisobutylene A, and the polyisobutylene B, only a tackifier, optionally a polybutene or a hydrogenated rosin ester, and a permeation enhancer, optionally isopropyl myristate or oleyl oleate, are present. 11. The transdermal therapeutic system according to claim 10, wherein the tackifier is present in an amount in the range of from 23 to 28% and the permeation enhancer is present in an amount in the range of from 8 to 15%, each based on the total weight of the matrix layer. 12. A method for the preparation of a transdermal therapeutic system according to claim 1, wherein the active ingredient is dispersed in the permeation enhancer, the polyisobutylene A and the polyisobutylene B each are distributed in a suitable solvent, subsequently both polymer-containing solutions are homogeneously mixed, the polymer-containing solutions are mixed with the dispersed active ingredient and optionally further components until a uniform mass is formed, the thus obtained mass is applied to the stripping layer or the back layer, the solvent is removed, the back layer or the stripping layer, respectively, is laminated thereon; and transdermal therapeutic systems of the desired size are cut out or punched out. 13. The transdermal therapeutic system obtained according to the method according to claim 12. 14. The transdermal therapeutic system according to 13 to alleviate pain during an intended wearing time of optionally 3 to 7 days, wherein the matrix layer after application to the skin for the duration of the intended wearing time has a residual content of active ingredient below 35% of the initial content of active ingredient. 15. The transdermal therapeutic system according to claim 14, wherein the transdermal therapeutic system has a delivery rate of the active ingredient that corresponds to that of a transdermal therapeutic system approved by at least one medicine agency. 16. A used transdermal therapeutic system obtained by removing the transdermal therapeutic system according to claim 1 that was applied to the skin for the duration of an intended wearing time of optionally 3 to 7 days. 17. A method of utilizing of the pressure-sensitive adhesive matrix layer in the transdermal therapeutic system as defined in claim 1 for the preparation of the transdermal therapeutic system alleviating pain during an intended wearing time of optionally 3 to 7 days, wherein the matrix layer of the transdermal therapeutic system to be prepared after application to the skin for the intended wearing time has a residual content of active ingredient below 35 of the initial content of active ingredient. 18. A method of utilizing of the pressure-sensitive adhesive matrix layer in the transdermal therapeutic system as defined in claim 1 for the preparation of the transdermal therapeutic system protected from abuse or misuse. 19. A method of utilizing of the pressure-sensitive adhesive matrix layer in the transdermal therapeutic system as defined in claim 1 to reduce the size of the transdermal therapeutic system at a substantially constant release profile. 20. The method according to claim 19, wherein the transdermal therapeutic system is or was a commercially available system, optionally Matrifen® and Durogesic DTrans®. 21. A method of utilizing of the pressure-sensitive adhesive matrix layer in the transdermal therapeutic system as defined in claim 1 for providing the transdermal therapeutic system having a delivery rate of more than 100 μg/h. 22. The method according to claim 21, wherein the transdermal therapeutic system has a size of less than 50 cm2.

According to the invention a transdermal therapeutic system for administering fentanyl or an analogue thereof through the skin is provided that has a pressure-sensitive adhesive matrix layer containing a mixture of two polyisobutylenes with specific storage moduli.1. A transdermal therapeutic system for the administration of an active ingredient through the skin comprising or consisting of a) a back layer, b) a pressure-sensitive adhesive matrix layer containing the active ingredient; and c) a stripping layer (release liner), wherein the active ingredient is fentanyl or an analogue of the fentanyl selected from alfentanil, carfentanil, lofentanil, remifentanil, and trefentanil or a salt of one of these active ingredients and wherein the matrix layer as the pressure-sensitive adhesive polymer contains a mixture of a polyisobutylene A and a polyisobutylene B, wherein the polyisobutylene A has a storage modulus G′ the value of which is substantially constant in the temperature range of from 10° C. to 40° C. and wherein the polyisobutylene B has a storage modulus G′ the value of which continuously decreases with increasing temperature in the temperature range of from 10° C. to 40° C., wherein the storage modulus G′ is measured in the linear viscoelastic range at a frequency of 10 rad/sec using a rheometer with parallel plate geometry and parallel plates; and wherein the pressure-sensitive adhesive matrix layer contains undissolved active ingredient in the form of active ingredient particles. 2. The transdermal therapeutic system according to claim 1, wherein the active ingredient is fentanyl. 3. The transdermal therapeutic system according to claim 1, wherein for the polyisobutylene A in the temperature range of from 10° C. to 40° C. all values of the storage modulus G′ deviate from the value for the storage modulus G′ at 40° C. by no more than 50%. 4. The transdermal therapeutic system according to claim 1, wherein for the polyisobutylene B the storage modulus G′ at 10° C. is at least two times the storage modulus G′ at 80° C. 5. The transdermal therapeutic system according claim 1, wherein the content of the polyisobutylene A to the polyisobutylene B in the matrix layer is in the range of from 20% (A) : 80% (B) to 40% (A) : 60% (B), each based on the total weight of the polyisobutylenes A and B. 6. The transdermal therapeutic system according to claim 1, wherein the polyisobutylene A and the polyisobutylene B each are individual polyisobutylenes of different average molecular weights. 7. The transdermal therapeutic system according to claim 1, wherein the matrix layer contains a permeation enhancer that optionally is isopropyl myristate or oleyl oleate. 8. The transdermal therapeutic system according to claim 1, wherein the matrix layer contains a tackifier that optionally is a polybutene or hydrogenated or non-hydrogenated rosin ester. 9. The transdermal therapeutic system according to claim 1, characterized in that the amount of active ingredient is sufficient for an application time of 3 days and the active ingredient is present in the matrix layer at a concentration in the range of from 3-15% by weight, based on the weight of the matrix layer. 10. The transdermal therapeutic system according to claim 1, characterized in that in the pressure-sensitive adhesive matrix layer in addition to the active ingredient, the polyisobutylene A, and the polyisobutylene B, only a tackifier, optionally a polybutene or a hydrogenated rosin ester, and a permeation enhancer, optionally isopropyl myristate or oleyl oleate, are present. 11. The transdermal therapeutic system according to claim 10, wherein the tackifier is present in an amount in the range of from 23 to 28% and the permeation enhancer is present in an amount in the range of from 8 to 15%, each based on the total weight of the matrix layer. 12. A method for the preparation of a transdermal therapeutic system according to claim 1, wherein the active ingredient is dispersed in the permeation enhancer, the polyisobutylene A and the polyisobutylene B each are distributed in a suitable solvent, subsequently both polymer-containing solutions are homogeneously mixed, the polymer-containing solutions are mixed with the dispersed active ingredient and optionally further components until a uniform mass is formed, the thus obtained mass is applied to the stripping layer or the back layer, the solvent is removed, the back layer or the stripping layer, respectively, is laminated thereon; and transdermal therapeutic systems of the desired size are cut out or punched out. 13. The transdermal therapeutic system obtained according to the method according to claim 12. 14. The transdermal therapeutic system according to 13 to alleviate pain during an intended wearing time of optionally 3 to 7 days, wherein the matrix layer after application to the skin for the duration of the intended wearing time has a residual content of active ingredient below 35% of the initial content of active ingredient. 15. The transdermal therapeutic system according to claim 14, wherein the transdermal therapeutic system has a delivery rate of the active ingredient that corresponds to that of a transdermal therapeutic system approved by at least one medicine agency. 16. A used transdermal therapeutic system obtained by removing the transdermal therapeutic system according to claim 1 that was applied to the skin for the duration of an intended wearing time of optionally 3 to 7 days. 17. A method of utilizing of the pressure-sensitive adhesive matrix layer in the transdermal therapeutic system as defined in claim 1 for the preparation of the transdermal therapeutic system alleviating pain during an intended wearing time of optionally 3 to 7 days, wherein the matrix layer of the transdermal therapeutic system to be prepared after application to the skin for the intended wearing time has a residual content of active ingredient below 35 of the initial content of active ingredient. 18. A method of utilizing of the pressure-sensitive adhesive matrix layer in the transdermal therapeutic system as defined in claim 1 for the preparation of the transdermal therapeutic system protected from abuse or misuse. 19. A method of utilizing of the pressure-sensitive adhesive matrix layer in the transdermal therapeutic system as defined in claim 1 to reduce the size of the transdermal therapeutic system at a substantially constant release profile. 20. The method according to claim 19, wherein the transdermal therapeutic system is or was a commercially available system, optionally Matrifen® and Durogesic DTrans®. 21. A method of utilizing of the pressure-sensitive adhesive matrix layer in the transdermal therapeutic system as defined in claim 1 for providing the transdermal therapeutic system having a delivery rate of more than 100 μg/h. 22. The method according to claim 21, wherein the transdermal therapeutic system has a size of less than 50 cm2.

The present invention is directed to novel polymorphs and salts of a compound which is an inhibitor of kinase activity.

1-15. (canceled) 16. A salt of N-[5-[4-(5-{[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl}-1,3-oxazol-2-yl)-1H-indazol-6-yl]-2-(methyloxy)-3-pyridinyl]methanesulfonamide which is selected from the group consisting of from sodium, tosylate, maleate, hemi pamoate, hemi naphthalenedisulfonate, mesitylenesulfonate, hemi biphenyldisulfonate, 2-naphthalenesulfonate (napsylate), hemi cinnamate, hemi sebacate, hemi pyromellitate and hemi benzenediacrylate. 17. A pharmaceutical composition comprising a salt according to claim 16 and one or more pharmaceutically acceptable excipients.

The present invention is directed to novel polymorphs and salts of a compound which is an inhibitor of kinase activity.1-15. (canceled) 16. A salt of N-[5-[4-(5-{[(2R,6S)-2,6-dimethyl-4-morpholinyl]methyl}-1,3-oxazol-2-yl)-1H-indazol-6-yl]-2-(methyloxy)-3-pyridinyl]methanesulfonamide which is selected from the group consisting of from sodium, tosylate, maleate, hemi pamoate, hemi naphthalenedisulfonate, mesitylenesulfonate, hemi biphenyldisulfonate, 2-naphthalenesulfonate (napsylate), hemi cinnamate, hemi sebacate, hemi pyromellitate and hemi benzenediacrylate. 17. A pharmaceutical composition comprising a salt according to claim 16 and one or more pharmaceutically acceptable excipients.

An implantable medical device for controlling the rate of release of a therapeutic agent is provided. The implantable medical device features at least one non-bioresorbable polymer, and/or at least one bioresorbable polymer, where the bioresorbable polymers have different degrees of hydrophilicity. Further, the implantable medical device incorporates at least one therapeutic agent contained in said at least one of non-bioresorbable polymer and/or said at least one of bioresorbable polymer. Preferably, the therapeutic agent is at least one anesthetic.

1. An implantable medical device which is capable of repairing soft tissue, and means for controlling the rate of release of a therapeutic agent contained therein comprising: at least one non-bioresorbable polymer; a plurality of bioresorbable polymers, wherein at least one of said plurality of bioresorbable polymers has a degree of hydrophilicity different from another of said plurality of bioresorbable polymers; and at least one therapeutic agent contained in said plurality of bioresorbable polymers; and further comprising a mesh structure constructed from a plurality of non-bioresorbable fibers constructed from said at least one non-bioresorbable polymer, and a plurality of bioresorbable fibers constructed from said plurality of bioresorbable polymers containing said at least one therapeutic agent. 2. The implantable medical device of claim 1, wherein at least one of said plurality of bioresorbable polymers has monomer components that are different from another of said plurality of bioresorbable polymers, or has the same monomer components, but in different proportions. 3. (canceled) 4. The implantable medical device of claim 1, wherein said plurality of bioresorbable fibers are each constructed out of a plurality of bioresorbable filaments. 5. The implantable medical device of claim 1, wherein said plurality of non-bioresorbable fibers are each constructed from a single non-bioresorbable filament, and said plurality of bioresorbable fibers are each constructed from a plurality of bioresorbable filaments. 6. The implantable device of claim 1, wherein at least one of said plurality of bioresorbable fibers is comprised of a bioresorbable polymer with monomer components that are different from the monomer components in the bioresorbable polymer comprising another of said plurality of bioresorbable fibers. 7. The implantable medical device of claim 1, wherein at least one of said plurality of bioresorbable fibers is comprised of a bioresorbable polymer that is constructed out of the same monomer components as the bioresorbable polymer comprising another of said plurality of bioresorbable fibers, but in different proportions. 8. The implantable medical device of claim 4, wherein said plurality of bioresorbable fibers and said plurality of bioresorbable filaments have a range of diameters. 9. The implantable medical device of claim 1, wherein said at least one therapeutic agent comprises at least one anesthetic. 10. The implantable medical device of claim 1, wherein said plurality of bioresorbable polymers with different degrees of hydrophilicity have different moisture resorption rates and different release rates for said at least on therapeutic agent contained therein, that give said implantable medical device the capability to retain and release said at least one therapeutic agent for 3-10 days after being implanted in a human body. 11. The implantable medical device of claim 1, wherein said plurality of bioresorbable polymers with different degrees of hydrophilicity have different moisture resorption rates and different release rates for said at least on therapeutic agent contained therein that is an at least one anesthetic which gives said implantable medical device the capability to release said at least one anesthetic at a sustained rate sufficient to maintain anesthesia for at least 2 days after implantation into the human body. 12. (canceled) 13. An implantable medical device for the treatment of post-operative pain and for the repair of soft tissue comprised of: a surgical mesh structure incorporating a plurality of non-bioresorbable fibers; and at least one anesthetic contained in a plurality of bioresorbable fibers with different degrees of hydrophilicity and lipid solubility; and wherein said implantable medical device is capable of maintaining local anesthesia for at least 2 days after implantation into the human body. 14. (canceled) 15. (canceled) 16. (canceled) 17. An implantable medical device in the form of a knitted mesh structure capable of repairing soft tissue comprising: a plurality of non-bioresorbable fibers comprised of at least one non-bioresorbable polymer; a plurality of bioresorbable fibers comprised of a plurality of bioresorbable polymers wherein one of said plurality of bioresorbable fibers has a hydrophilicity that is different from another of said plurality of bioresorbable fibers; and at least one therapeutic agent contained in said of plurality bioresorbable fibers, and wherein said implantable medical device is capable of releasing said at least one therapeutic agent at a sustained rate sufficient to maintain local therapeutic effect for at least 2 days, after implantation into the human body. 18. (canceled) 19. The medical device of claim 17, wherein said plurality of bioresorbable fibers are comprised of a plurality of bioresorbable filaments having different diameters which require different amounts of time for body fluids to completely permeate resulting in different release rates for said at least one therapeutic agent, and wherein said implantable medical device has the capability to achieve and maintain local therapeutic effect for at least 2 days after implantation into the human body. 20. The implantable medical device of claim 17, wherein said plurality of non-bioresorbable fibers in said knitted mesh structure are constructed from at least one non-bioresorbable polymer capable of integrating into soft tissue that is comprised of polypropylene. 21. The medical device of claim 17, wherein said plurality of bioresorbable fibers comprised of said plurality of bioresorbable polymers having different degrees of hydrophilicity have different rates of moisture resorption and different release rates for said at least one therapeutic agent contained therein; wherein said implantable medical device is capable of maintaining a therapeutic effect for at least 2 days after implantation into the human body. 22. (canceled) 23. An implantable medical device comprising: a plurality of non-bioresorbable fibers comprised of polypropylene; a plurality of bioresorbable fibers comprised of a plurality of bioresorbable polymers composed of monomers including at least one of lactide, glycolide, caprolactone, and trimethylene carbonate; and at least one therapeutic agent that is a incorporated within said plurality of bioresorbable fibers. 24. An implantable medical device in the form of a surgical mesh structure, comprising: a plurality of non-bioresorbable fibers comprised of at least one non-bioresorbable polymer that provide mechanical properties of said implantable medical device, but do not contain a therapeutic agent, and therefore do not contribute to the therapeutic effect of said implantable medical device; and a plurality of bioresorbable fibers comprised of a plurality of bioresorbable polymers capable of containing and controlling the release of at least one therapeutic agent contained therein, and wherein said plurality of bioresorbable fibers are incorporated into said surgical mesh structure utilizing large loops that will not exert any force when said implantable medical device is stressed 25. (canceled) 26. (canceled)

An implantable medical device for controlling the rate of release of a therapeutic agent is provided. The implantable medical device features at least one non-bioresorbable polymer, and/or at least one bioresorbable polymer, where the bioresorbable polymers have different degrees of hydrophilicity. Further, the implantable medical device incorporates at least one therapeutic agent contained in said at least one of non-bioresorbable polymer and/or said at least one of bioresorbable polymer. Preferably, the therapeutic agent is at least one anesthetic.1. An implantable medical device which is capable of repairing soft tissue, and means for controlling the rate of release of a therapeutic agent contained therein comprising: at least one non-bioresorbable polymer; a plurality of bioresorbable polymers, wherein at least one of said plurality of bioresorbable polymers has a degree of hydrophilicity different from another of said plurality of bioresorbable polymers; and at least one therapeutic agent contained in said plurality of bioresorbable polymers; and further comprising a mesh structure constructed from a plurality of non-bioresorbable fibers constructed from said at least one non-bioresorbable polymer, and a plurality of bioresorbable fibers constructed from said plurality of bioresorbable polymers containing said at least one therapeutic agent. 2. The implantable medical device of claim 1, wherein at least one of said plurality of bioresorbable polymers has monomer components that are different from another of said plurality of bioresorbable polymers, or has the same monomer components, but in different proportions. 3. (canceled) 4. The implantable medical device of claim 1, wherein said plurality of bioresorbable fibers are each constructed out of a plurality of bioresorbable filaments. 5. The implantable medical device of claim 1, wherein said plurality of non-bioresorbable fibers are each constructed from a single non-bioresorbable filament, and said plurality of bioresorbable fibers are each constructed from a plurality of bioresorbable filaments. 6. The implantable device of claim 1, wherein at least one of said plurality of bioresorbable fibers is comprised of a bioresorbable polymer with monomer components that are different from the monomer components in the bioresorbable polymer comprising another of said plurality of bioresorbable fibers. 7. The implantable medical device of claim 1, wherein at least one of said plurality of bioresorbable fibers is comprised of a bioresorbable polymer that is constructed out of the same monomer components as the bioresorbable polymer comprising another of said plurality of bioresorbable fibers, but in different proportions. 8. The implantable medical device of claim 4, wherein said plurality of bioresorbable fibers and said plurality of bioresorbable filaments have a range of diameters. 9. The implantable medical device of claim 1, wherein said at least one therapeutic agent comprises at least one anesthetic. 10. The implantable medical device of claim 1, wherein said plurality of bioresorbable polymers with different degrees of hydrophilicity have different moisture resorption rates and different release rates for said at least on therapeutic agent contained therein, that give said implantable medical device the capability to retain and release said at least one therapeutic agent for 3-10 days after being implanted in a human body. 11. The implantable medical device of claim 1, wherein said plurality of bioresorbable polymers with different degrees of hydrophilicity have different moisture resorption rates and different release rates for said at least on therapeutic agent contained therein that is an at least one anesthetic which gives said implantable medical device the capability to release said at least one anesthetic at a sustained rate sufficient to maintain anesthesia for at least 2 days after implantation into the human body. 12. (canceled) 13. An implantable medical device for the treatment of post-operative pain and for the repair of soft tissue comprised of: a surgical mesh structure incorporating a plurality of non-bioresorbable fibers; and at least one anesthetic contained in a plurality of bioresorbable fibers with different degrees of hydrophilicity and lipid solubility; and wherein said implantable medical device is capable of maintaining local anesthesia for at least 2 days after implantation into the human body. 14. (canceled) 15. (canceled) 16. (canceled) 17. An implantable medical device in the form of a knitted mesh structure capable of repairing soft tissue comprising: a plurality of non-bioresorbable fibers comprised of at least one non-bioresorbable polymer; a plurality of bioresorbable fibers comprised of a plurality of bioresorbable polymers wherein one of said plurality of bioresorbable fibers has a hydrophilicity that is different from another of said plurality of bioresorbable fibers; and at least one therapeutic agent contained in said of plurality bioresorbable fibers, and wherein said implantable medical device is capable of releasing said at least one therapeutic agent at a sustained rate sufficient to maintain local therapeutic effect for at least 2 days, after implantation into the human body. 18. (canceled) 19. The medical device of claim 17, wherein said plurality of bioresorbable fibers are comprised of a plurality of bioresorbable filaments having different diameters which require different amounts of time for body fluids to completely permeate resulting in different release rates for said at least one therapeutic agent, and wherein said implantable medical device has the capability to achieve and maintain local therapeutic effect for at least 2 days after implantation into the human body. 20. The implantable medical device of claim 17, wherein said plurality of non-bioresorbable fibers in said knitted mesh structure are constructed from at least one non-bioresorbable polymer capable of integrating into soft tissue that is comprised of polypropylene. 21. The medical device of claim 17, wherein said plurality of bioresorbable fibers comprised of said plurality of bioresorbable polymers having different degrees of hydrophilicity have different rates of moisture resorption and different release rates for said at least one therapeutic agent contained therein; wherein said implantable medical device is capable of maintaining a therapeutic effect for at least 2 days after implantation into the human body. 22. (canceled) 23. An implantable medical device comprising: a plurality of non-bioresorbable fibers comprised of polypropylene; a plurality of bioresorbable fibers comprised of a plurality of bioresorbable polymers composed of monomers including at least one of lactide, glycolide, caprolactone, and trimethylene carbonate; and at least one therapeutic agent that is a incorporated within said plurality of bioresorbable fibers. 24. An implantable medical device in the form of a surgical mesh structure, comprising: a plurality of non-bioresorbable fibers comprised of at least one non-bioresorbable polymer that provide mechanical properties of said implantable medical device, but do not contain a therapeutic agent, and therefore do not contribute to the therapeutic effect of said implantable medical device; and a plurality of bioresorbable fibers comprised of a plurality of bioresorbable polymers capable of containing and controlling the release of at least one therapeutic agent contained therein, and wherein said plurality of bioresorbable fibers are incorporated into said surgical mesh structure utilizing large loops that will not exert any force when said implantable medical device is stressed 25. (canceled) 26. (canceled)

A method for the treatment of recurrent herpes labialis in mammals, including humans, which method comprises administering to the mammal in need of such treatment, and effective amount of penciclovir or famciclovir, or a pharmaceutically acceptable salt thereof for a period of one day.

1. A method for the treatment of recurrent herpes labialis in a human in need thereof, which method comprises administering to said human, an effective amount of the compound 9-(4-acetoxy-3-acetoxymethylbut-1-yl)-2-aminopurine (famciclovir), or a pharmaceutically acceptable salt thereof for a treatment period of one day. 2. A method according to claim 1, wherein treatment is commenced within one hour of onset of prodrome. 3. A method according to claim 2, wherein treatment is commenced within 24 hours of prodrome. 4. A method according to claim 1, where the treatment period is one day. 5. A method according to claim 1, wherein the treatment is carried out on immunocompetent humans with recurrent herpes labialis. 6. A method according to claim 1, wherein famciclovir is administered at a dose of 1,500 mg for a period of one day. 7. A method according to claim 6, wherein famciclovir is administered at a dose of 250 mg six times in one day. 8. A method according to claim 6, wherein famciclovir is administered at a dose of 500 mg three times in one day. 9. A method according to claim 6, wherein famciclovir is administered at a dose of 750 mg twice in one day. 10. A method according to claim 1, wherein the treatment is carried out on immunocompetent humans with recurrent herpes labialis. 11. A method according to claim 1, wherein famciclovir is administered orally. 12. A method according to claim 1, wherein the compound is administered parenterally.

A method for the treatment of recurrent herpes labialis in mammals, including humans, which method comprises administering to the mammal in need of such treatment, and effective amount of penciclovir or famciclovir, or a pharmaceutically acceptable salt thereof for a period of one day.1. A method for the treatment of recurrent herpes labialis in a human in need thereof, which method comprises administering to said human, an effective amount of the compound 9-(4-acetoxy-3-acetoxymethylbut-1-yl)-2-aminopurine (famciclovir), or a pharmaceutically acceptable salt thereof for a treatment period of one day. 2. A method according to claim 1, wherein treatment is commenced within one hour of onset of prodrome. 3. A method according to claim 2, wherein treatment is commenced within 24 hours of prodrome. 4. A method according to claim 1, where the treatment period is one day. 5. A method according to claim 1, wherein the treatment is carried out on immunocompetent humans with recurrent herpes labialis. 6. A method according to claim 1, wherein famciclovir is administered at a dose of 1,500 mg for a period of one day. 7. A method according to claim 6, wherein famciclovir is administered at a dose of 250 mg six times in one day. 8. A method according to claim 6, wherein famciclovir is administered at a dose of 500 mg three times in one day. 9. A method according to claim 6, wherein famciclovir is administered at a dose of 750 mg twice in one day. 10. A method according to claim 1, wherein the treatment is carried out on immunocompetent humans with recurrent herpes labialis. 11. A method according to claim 1, wherein famciclovir is administered orally. 12. A method according to claim 1, wherein the compound is administered parenterally.

This disclosure relates to asparagine endopeptidase inhibitors for managing cancer and compositions related thereto. In certain embodiments, the asparagine endopeptidase inhibitors are substituted 3,7-dihydropurine-2,6-dione derivatives useful for treating or preventing metastasis, tumor growth, and/or cancer. In certain embodiments, the disclosure relates to pharmaceutical compositions comprising an asparagine endopeptidase inhibitor and a pharmaceutically acceptable excipient. In certain embodiments, the disclosure relates to methods of treating a cancer comprising administering an effective amount of pharmaceutical composition a asparagine endopeptidase inhibitor disclosed herein to a subject in need thereof.

1. A compound of the following formula: prodrugs, derivatives, or salts thereof wherein, R1 is selected from hydrogen, alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R1 is optionally substituted with one or more, the same or different, R10; R10 is selected from alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R10 is optionally substituted with one or more, the same or different, R11; R11 is selected from halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, propyl, tert-butyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, and heterocyclyl; R2 is selected from hydrogen, alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R2 is optionally substituted with one or more, the same or different, R20; R20 is selected from alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R20 is optionally substituted with one or more, the same or different, R21; R21 is selected from halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, propyl, tert-butyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, and heterocyclyl; R6 is selected from hydrogen, alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R6 is optionally substituted with one or more, the same or different, R60; R60 is selected from alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R60 is optionally substituted with one or more, the same or different, R61; R61 is selected from halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, propyl, tert-butyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, and heterocyclyl; R7 is selected from hydrogen, alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R7 is optionally substituted with one or more, the same or different, R70; R70 is selected from alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R70 is optionally substituted with one or more, the same or different, R71; and R71 is selected from halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, propyl, tert-butyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, and heterocyclyl. 2. The compound of claim 1, wherein R1 is alkyl. 3. The compound of claim 1, wherein R2 is alkyl. 4. The compound of claim 1, wherein R6 is mercapto. 5. The compound of claim 1, wherein R7 is alkyl. 6. The compound of claim 1 selected from 1-ethyl-8-mercapto-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione; 8-mercapto-1,3,7-trimethyl-3,7-dihydro-1H-purine-2, 6-dione; and 1-benzyl-8-mercapto-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione. 7. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable excipient. 8. The pharmaceutical composition of claim 7 in the form of a pill, capsule, tablet, or saline aqueous buffer. 9. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable excipient is selected from a saccharide, disaccharide, sucrose, lactose, glucose, mannitol, sorbitol, polysaccharides, starch, cellulose, microcrystalline cellulose, cellulose ether, hydroxypropyl cellulose (HPC), xylitol, sorbitol, maltito, gelatin, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), hydroxypropyl methylcellulose (HPMC), crosslinked sodium carboxymethyl cellulose, dibasic calcium phosphate, calcium carbonate, stearic acid, magnesium stearate, talc, magnesium carbonate, silica, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, and sodium citrate, methyl paraben, propyl paraben, and combinations thereof. 10. A method of treating a cancer comprising administering an effective amount of pharmaceutical composition comprising an asparagine endopeptidase inhibitors to a subject in need thereof. 11. The method of claim 10, wherein the subject is at risk of, exhibiting symptoms of, or diagnosed with breast cancer, prostate cancer, colorectal cancer, gastric cancer, lung cancer, skin cancer, bladder cancer, brain cancer, kidney cancer, endometrial cancer, pancreatic cancer, and thyroid cancer. 12. The method of claim 10, wherein a second anti-cancer agent is administered. 13. The method of claim 12, wherein the second anti-cancer agent is bevacizumab, gefitinib, erlotinib, temazolamide, docetaxel, cis-platin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, vincristine, vinblastine, vindesine, vinorelbine taxol, taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin, bortezomib, anegrilide, tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorazole, exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, combretastatin, thalidomide, and/or lenalidomide or combinations thereof.

This disclosure relates to asparagine endopeptidase inhibitors for managing cancer and compositions related thereto. In certain embodiments, the asparagine endopeptidase inhibitors are substituted 3,7-dihydropurine-2,6-dione derivatives useful for treating or preventing metastasis, tumor growth, and/or cancer. In certain embodiments, the disclosure relates to pharmaceutical compositions comprising an asparagine endopeptidase inhibitor and a pharmaceutically acceptable excipient. In certain embodiments, the disclosure relates to methods of treating a cancer comprising administering an effective amount of pharmaceutical composition a asparagine endopeptidase inhibitor disclosed herein to a subject in need thereof.1. A compound of the following formula: prodrugs, derivatives, or salts thereof wherein, R1 is selected from hydrogen, alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R1 is optionally substituted with one or more, the same or different, R10; R10 is selected from alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R10 is optionally substituted with one or more, the same or different, R11; R11 is selected from halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, propyl, tert-butyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, and heterocyclyl; R2 is selected from hydrogen, alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R2 is optionally substituted with one or more, the same or different, R20; R20 is selected from alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R20 is optionally substituted with one or more, the same or different, R21; R21 is selected from halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, propyl, tert-butyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, and heterocyclyl; R6 is selected from hydrogen, alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R6 is optionally substituted with one or more, the same or different, R60; R60 is selected from alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R60 is optionally substituted with one or more, the same or different, R61; R61 is selected from halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, propyl, tert-butyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, and heterocyclyl; R7 is selected from hydrogen, alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R7 is optionally substituted with one or more, the same or different, R70; R70 is selected from alkyl, alkenyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, and heterocyclyl wherein R70 is optionally substituted with one or more, the same or different, R71; and R71 is selected from halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, propyl, tert-butyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, and heterocyclyl. 2. The compound of claim 1, wherein R1 is alkyl. 3. The compound of claim 1, wherein R2 is alkyl. 4. The compound of claim 1, wherein R6 is mercapto. 5. The compound of claim 1, wherein R7 is alkyl. 6. The compound of claim 1 selected from 1-ethyl-8-mercapto-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione; 8-mercapto-1,3,7-trimethyl-3,7-dihydro-1H-purine-2, 6-dione; and 1-benzyl-8-mercapto-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione. 7. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable excipient. 8. The pharmaceutical composition of claim 7 in the form of a pill, capsule, tablet, or saline aqueous buffer. 9. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable excipient is selected from a saccharide, disaccharide, sucrose, lactose, glucose, mannitol, sorbitol, polysaccharides, starch, cellulose, microcrystalline cellulose, cellulose ether, hydroxypropyl cellulose (HPC), xylitol, sorbitol, maltito, gelatin, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), hydroxypropyl methylcellulose (HPMC), crosslinked sodium carboxymethyl cellulose, dibasic calcium phosphate, calcium carbonate, stearic acid, magnesium stearate, talc, magnesium carbonate, silica, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, and sodium citrate, methyl paraben, propyl paraben, and combinations thereof. 10. A method of treating a cancer comprising administering an effective amount of pharmaceutical composition comprising an asparagine endopeptidase inhibitors to a subject in need thereof. 11. The method of claim 10, wherein the subject is at risk of, exhibiting symptoms of, or diagnosed with breast cancer, prostate cancer, colorectal cancer, gastric cancer, lung cancer, skin cancer, bladder cancer, brain cancer, kidney cancer, endometrial cancer, pancreatic cancer, and thyroid cancer. 12. The method of claim 10, wherein a second anti-cancer agent is administered. 13. The method of claim 12, wherein the second anti-cancer agent is bevacizumab, gefitinib, erlotinib, temazolamide, docetaxel, cis-platin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, vincristine, vinblastine, vindesine, vinorelbine taxol, taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin, bortezomib, anegrilide, tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorazole, exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, combretastatin, thalidomide, and/or lenalidomide or combinations thereof.

The invention relates to a proteolytic enzyme which is capable of forming fibrin when it reacts with fibrinogen, a fibrin-glue kit and a fibrin-glue formulation comprising an enzymatically-permissive concentration of a visualization agent and to their use in methods for prevention and/or reduction of adhesions and/or methods for promotion of blood coagulation sealing or filling body surfaces.

1. A fibrin glue kit for application to a surface of a body part of a patient comprising: (i) at least two separate components required to form a fibrin glue, the at least one separated component comprises fibrinogen, and the at least second separated component comprises a proteolytic enzyme which is capable of forming fibrin when it reacts with fibrinogen; and (ii) a visualization agent in an enzymatically-permissive concentration in the range of from 0.0025 to 0.1%, or of from 0.0025 to 0.01% in the generated clot. 2. The kit according to claim 1, wherein the fibrinogen comprising component is a cryoprecipitate. 3. The kit according to claim 1, further comprising a catalyst capable of inducing cross-linking of fibrin. 4. The kit according to claim 1, wherein the visualization agent is incorporated in the component comprising the proteolytic enzyme. 5. The kit according to claim 1, wherein the visualization agent is methylene blue. 6. The kit according to claim 1, wherein the component comprising the visualization agent is protected from light. 7. The kit according to claim 1, wherein the visualization agent is indigo carmine. 8. A fibrin glue formulation for application to a surface of a body part of a patient comprising fibrinogen, a proteolytic enzyme which is capable of forming fibrin when it reacts with fibrinogen; and a visualization agent in an enzymatically-permissive concentration in the range of from 0.0025 to 0.1%, or from 0.0025 to 0.01% in the generated glue. 9. The formulation according to claim 8, wherein the visualization agent is methylene blue. 10. The formulation according to claim 8, wherein the visualization agent is indigo carmine. 11. A solution for application to a surface of a body part of a patient comprising a proteolytic enzyme which is capable of forming fibrin when it reacts with fibrinogen, and a visualization agent in an enzymatically-permissive concentration in the range of from 0.005 to 0.2%, or from 0.005 to 0.02%. 12. The solution according to claim 11, wherein the visualization agent is methylene blue. 13. The solution according to claim 11, wherein the solution is protected from light. 14. The solution according to claim 11, wherein the visualization agent is indigo carmine. 15. A method of preparing a fibrin glue at a surface comprising: providing a solution A—comprising fibrinogen; providing a solution B—comprising a proteolytic enzyme which is capable of forming fibrin when it reacts with fibrinogen and an enzymatically-permissive concentration of a visualization agent; applying the solutions to said surface so as to cause clotting of the fibrin, wherein the concentration of the visualization agent in the generated glue is in the range of from 0.0025 to 0.1%, or from 0.0025 to 0.01%. 16. The method according to claim 15, wherein the visualization agent is methylene blue. 17. The method according to claim 15, wherein solution B is protected from light. 18. The method according to claim 15, wherein the visualization agent is indigo carmine. 19. A method for preventing or treating bleeding, for preventing or treating adhesions and/or for filling or sealing surfaces comprising application of a kit according to claim 1, a formulation according to claim 8, or a solution according to claim 11. 20. A fibrin glue kit according to claim 1, a formulation according to claim 8, or a solution according to claim 11, for use in laparoscopic surgery.

The invention relates to a proteolytic enzyme which is capable of forming fibrin when it reacts with fibrinogen, a fibrin-glue kit and a fibrin-glue formulation comprising an enzymatically-permissive concentration of a visualization agent and to their use in methods for prevention and/or reduction of adhesions and/or methods for promotion of blood coagulation sealing or filling body surfaces.1. A fibrin glue kit for application to a surface of a body part of a patient comprising: (i) at least two separate components required to form a fibrin glue, the at least one separated component comprises fibrinogen, and the at least second separated component comprises a proteolytic enzyme which is capable of forming fibrin when it reacts with fibrinogen; and (ii) a visualization agent in an enzymatically-permissive concentration in the range of from 0.0025 to 0.1%, or of from 0.0025 to 0.01% in the generated clot. 2. The kit according to claim 1, wherein the fibrinogen comprising component is a cryoprecipitate. 3. The kit according to claim 1, further comprising a catalyst capable of inducing cross-linking of fibrin. 4. The kit according to claim 1, wherein the visualization agent is incorporated in the component comprising the proteolytic enzyme. 5. The kit according to claim 1, wherein the visualization agent is methylene blue. 6. The kit according to claim 1, wherein the component comprising the visualization agent is protected from light. 7. The kit according to claim 1, wherein the visualization agent is indigo carmine. 8. A fibrin glue formulation for application to a surface of a body part of a patient comprising fibrinogen, a proteolytic enzyme which is capable of forming fibrin when it reacts with fibrinogen; and a visualization agent in an enzymatically-permissive concentration in the range of from 0.0025 to 0.1%, or from 0.0025 to 0.01% in the generated glue. 9. The formulation according to claim 8, wherein the visualization agent is methylene blue. 10. The formulation according to claim 8, wherein the visualization agent is indigo carmine. 11. A solution for application to a surface of a body part of a patient comprising a proteolytic enzyme which is capable of forming fibrin when it reacts with fibrinogen, and a visualization agent in an enzymatically-permissive concentration in the range of from 0.005 to 0.2%, or from 0.005 to 0.02%. 12. The solution according to claim 11, wherein the visualization agent is methylene blue. 13. The solution according to claim 11, wherein the solution is protected from light. 14. The solution according to claim 11, wherein the visualization agent is indigo carmine. 15. A method of preparing a fibrin glue at a surface comprising: providing a solution A—comprising fibrinogen; providing a solution B—comprising a proteolytic enzyme which is capable of forming fibrin when it reacts with fibrinogen and an enzymatically-permissive concentration of a visualization agent; applying the solutions to said surface so as to cause clotting of the fibrin, wherein the concentration of the visualization agent in the generated glue is in the range of from 0.0025 to 0.1%, or from 0.0025 to 0.01%. 16. The method according to claim 15, wherein the visualization agent is methylene blue. 17. The method according to claim 15, wherein solution B is protected from light. 18. The method according to claim 15, wherein the visualization agent is indigo carmine. 19. A method for preventing or treating bleeding, for preventing or treating adhesions and/or for filling or sealing surfaces comprising application of a kit according to claim 1, a formulation according to claim 8, or a solution according to claim 11. 20. A fibrin glue kit according to claim 1, a formulation according to claim 8, or a solution according to claim 11, for use in laparoscopic surgery.

A method for a pre-symptomatic diagnosis of a viral illness includes obtaining a biological sample with a peripheral blood mononuclear cell from a subject. The method further includes stimulating the biological sample, where the stimulation of the biological sample includes adding to the biological sample a predetermined amount of antigen configured to trigger a T-cell IFN-γ response in the biological sample and a predetermined amount of reagent configured to capture the T-cell IFN-γ response. The method may even further include comparing the level of the T-cell IFN-γ response detected in the biological sample to a control sample level, the control sample level including a control sample T-cell IFN-γ response level detected in a control sample of the subject, where if the level of the T-cell IFN-γ response in the biological sample is at a predetermined amount greater than the control sample level, the subject is diagnosed with the viral illness.

1. A method for a pre-symptomatic diagnosis of a viral illness in a subject, the method comprising: (a) obtaining a biological sample comprising at least one peripheral blood mononuclear cell from a subject; (b) stimulating the biological sample, wherein the stimulation of the biological sample comprises adding to the biological sample a predetermined amount of antigen configured to trigger a T-cell IFN-γ response in the biological sample and a predetermined amount of reagent configured to capture the T-cell IFN-γ response; (c) detecting and determining a level of the T-cell IFN-γ response produced by the biological sample; and (d) comparing the level of the T-cell IFN-γ response detected in the biological sample to a control sample level, the control sample level comprising a control sample T-cell IFN-γ response level detected in a control sample of the subject, wherein if the level of the T-cell IFN-γ response in the biological sample is at a predetermined amount greater than the control sample level, the subject is diagnosed with the viral illness. 2. The method of claim 1, wherein the detecting of the level of the T-cell IFN-γ response produced by the biological sample comprises detecting the level of the T-cell IFN-γ response produced by the biological sample using an enzyme-linked immunosorbent spot (ELISPOT) assay. 3. The method of claim 1, wherein the reagent configured to capture the T-cell IFN-γ response is an antibody. 4. The method of claim 1, wherein obtaining the biological sample comprises isolating the at least one PBMC from other components of the biological sample. 5. The method of claim 1, wherein the viral illness is Ebola Virus Disease, and wherein the antigen is an Ebola Virus Disease antigen. 6. The method of claim 1, wherein the at least one PBMC comprises at least one CD3+ T cell. 7. The method of claim 1, wherein the predetermined amount greater than the control sample level is greater than 2.5 times above a standard deviation. 8. The method of claim 7, wherein the predetermined amount greater than the control sample level is greater than 5 times above the standard deviation. 9. The method of claim 1, wherein the stimulation of the biological sample further comprises incubating the biological sample, the reagent, and the antigen for a predetermined time. 10. The method of claim 9, wherein the predetermined time for incubating the biological sample is for a period of 1-5 hours. 11. The method of claim 10, wherein the predetermined time for incubating the biological sample is for a period of 1-2 hours. 12. The method of claim 1, wherein the predetermined amount of antigen is 0.1 ng to 10,000 ug. 13. The method of claim 1, wherein obtaining the biological sample further comprises obtaining the biological sample within five days from suspected exposure to the viral illness. 14. A diagnostic kit for a pre-symptomatic diagnosis of a viral illness in a subject, the diagnostic kit comprising: a) a predetermined amount of antigen associated with the viral illness; and b) a predetermined amount of reagent for capturing IFN-γ produced by a biological sample from the subject. 15. The diagnostic kit of claim 14, wherein the diagnostic kit further comprises a well, the well being pre-coated with the predetermined amount of reagent for capturing the IFN-γ produced by the biological sample from the subject. 16. The diagnostic kit of claim 14, wherein the reagent configured for capturing the T-cell IFN-γ response is an antibody. 17. The diagnostic kit of claim 14, wherein the predetermined amount of antigen is 0.1 ng to 10,000 ug. 18. The diagnostic kit of claim 14, wherein the biological sample from the subject is obtained from the subject within five days of suspected exposure to the viral illness. 19. The diagnostic kit of claim 14, wherein the viral illness is Ebola Virus Disease, and wherein the antigen is an Ebola Virus Disease antigen. 20. The diagnostic kit of claim 14, wherein the diagnostic kit further comprises a receptacle for collecting the biological sample from the subject.

A method for a pre-symptomatic diagnosis of a viral illness includes obtaining a biological sample with a peripheral blood mononuclear cell from a subject. The method further includes stimulating the biological sample, where the stimulation of the biological sample includes adding to the biological sample a predetermined amount of antigen configured to trigger a T-cell IFN-γ response in the biological sample and a predetermined amount of reagent configured to capture the T-cell IFN-γ response. The method may even further include comparing the level of the T-cell IFN-γ response detected in the biological sample to a control sample level, the control sample level including a control sample T-cell IFN-γ response level detected in a control sample of the subject, where if the level of the T-cell IFN-γ response in the biological sample is at a predetermined amount greater than the control sample level, the subject is diagnosed with the viral illness.1. A method for a pre-symptomatic diagnosis of a viral illness in a subject, the method comprising: (a) obtaining a biological sample comprising at least one peripheral blood mononuclear cell from a subject; (b) stimulating the biological sample, wherein the stimulation of the biological sample comprises adding to the biological sample a predetermined amount of antigen configured to trigger a T-cell IFN-γ response in the biological sample and a predetermined amount of reagent configured to capture the T-cell IFN-γ response; (c) detecting and determining a level of the T-cell IFN-γ response produced by the biological sample; and (d) comparing the level of the T-cell IFN-γ response detected in the biological sample to a control sample level, the control sample level comprising a control sample T-cell IFN-γ response level detected in a control sample of the subject, wherein if the level of the T-cell IFN-γ response in the biological sample is at a predetermined amount greater than the control sample level, the subject is diagnosed with the viral illness. 2. The method of claim 1, wherein the detecting of the level of the T-cell IFN-γ response produced by the biological sample comprises detecting the level of the T-cell IFN-γ response produced by the biological sample using an enzyme-linked immunosorbent spot (ELISPOT) assay. 3. The method of claim 1, wherein the reagent configured to capture the T-cell IFN-γ response is an antibody. 4. The method of claim 1, wherein obtaining the biological sample comprises isolating the at least one PBMC from other components of the biological sample. 5. The method of claim 1, wherein the viral illness is Ebola Virus Disease, and wherein the antigen is an Ebola Virus Disease antigen. 6. The method of claim 1, wherein the at least one PBMC comprises at least one CD3+ T cell. 7. The method of claim 1, wherein the predetermined amount greater than the control sample level is greater than 2.5 times above a standard deviation. 8. The method of claim 7, wherein the predetermined amount greater than the control sample level is greater than 5 times above the standard deviation. 9. The method of claim 1, wherein the stimulation of the biological sample further comprises incubating the biological sample, the reagent, and the antigen for a predetermined time. 10. The method of claim 9, wherein the predetermined time for incubating the biological sample is for a period of 1-5 hours. 11. The method of claim 10, wherein the predetermined time for incubating the biological sample is for a period of 1-2 hours. 12. The method of claim 1, wherein the predetermined amount of antigen is 0.1 ng to 10,000 ug. 13. The method of claim 1, wherein obtaining the biological sample further comprises obtaining the biological sample within five days from suspected exposure to the viral illness. 14. A diagnostic kit for a pre-symptomatic diagnosis of a viral illness in a subject, the diagnostic kit comprising: a) a predetermined amount of antigen associated with the viral illness; and b) a predetermined amount of reagent for capturing IFN-γ produced by a biological sample from the subject. 15. The diagnostic kit of claim 14, wherein the diagnostic kit further comprises a well, the well being pre-coated with the predetermined amount of reagent for capturing the IFN-γ produced by the biological sample from the subject. 16. The diagnostic kit of claim 14, wherein the reagent configured for capturing the T-cell IFN-γ response is an antibody. 17. The diagnostic kit of claim 14, wherein the predetermined amount of antigen is 0.1 ng to 10,000 ug. 18. The diagnostic kit of claim 14, wherein the biological sample from the subject is obtained from the subject within five days of suspected exposure to the viral illness. 19. The diagnostic kit of claim 14, wherein the viral illness is Ebola Virus Disease, and wherein the antigen is an Ebola Virus Disease antigen. 20. The diagnostic kit of claim 14, wherein the diagnostic kit further comprises a receptacle for collecting the biological sample from the subject.

This invention relates to the use of the phytocannabinoid cannabidivarin (CBDV) and combinations of the phytocannabinoid CBDV with tetrahydrocannabivarin (THCV) and cannabidiol (CBD) in the treatment of epilepsy. The invention further relates to the use of the phytocannabinoid CBDV in combination with standard anti-epileptic drugs (SAEDs). Preferably the SAED is one of ethosuximide, valproate or phenobarbital.

1-20. (canceled) 21. A method for the treatment of epileptic seizures, which comprises administering to a subject in need thereof a therapeutically effective amount of the phytocannabinoid CBDV, wherein the CBDV is in the form of a botanical drug substance. 22. The method of claim 21, wherein the type of epileptic seizure to be treated is a generalised seizure or a temporal lobe seizure. 23. The method of claim 21, wherein the CBDV is used in combination with a standard anti-epileptic drug. 24. The method of claim 23, wherein the standard anti-epileptic drug has a mechanism of action which acts via sodium or calcium channels, or enhances GABAergic inhibition. 25. The method of claim 24, wherein the standard anti-epileptic drug that enhances GABAergic inhibition is phenobarbital. 26. The method of claim 24, wherein the standard anti-epileptic drug having a mechanism of action which acts via sodium or calcium channels either: modifies low-threshold or transient neuronal calcium currents; or reduces high-frequency neuronal firing and sodium-dependent action potentials and may additionally enhance GABA effects. 27. The method of claim 26, wherein the standard anti-epileptic drug that modifies low-threshold or transient neuronal calcium currents is ethosuximide or the standard anti-epileptic drug that reduces high-frequency neuronal firing and sodium-dependent action potentials and may additionally enhance GABA effects is valproate.

This invention relates to the use of the phytocannabinoid cannabidivarin (CBDV) and combinations of the phytocannabinoid CBDV with tetrahydrocannabivarin (THCV) and cannabidiol (CBD) in the treatment of epilepsy. The invention further relates to the use of the phytocannabinoid CBDV in combination with standard anti-epileptic drugs (SAEDs). Preferably the SAED is one of ethosuximide, valproate or phenobarbital.1-20. (canceled) 21. A method for the treatment of epileptic seizures, which comprises administering to a subject in need thereof a therapeutically effective amount of the phytocannabinoid CBDV, wherein the CBDV is in the form of a botanical drug substance. 22. The method of claim 21, wherein the type of epileptic seizure to be treated is a generalised seizure or a temporal lobe seizure. 23. The method of claim 21, wherein the CBDV is used in combination with a standard anti-epileptic drug. 24. The method of claim 23, wherein the standard anti-epileptic drug has a mechanism of action which acts via sodium or calcium channels, or enhances GABAergic inhibition. 25. The method of claim 24, wherein the standard anti-epileptic drug that enhances GABAergic inhibition is phenobarbital. 26. The method of claim 24, wherein the standard anti-epileptic drug having a mechanism of action which acts via sodium or calcium channels either: modifies low-threshold or transient neuronal calcium currents; or reduces high-frequency neuronal firing and sodium-dependent action potentials and may additionally enhance GABA effects. 27. The method of claim 26, wherein the standard anti-epileptic drug that modifies low-threshold or transient neuronal calcium currents is ethosuximide or the standard anti-epileptic drug that reduces high-frequency neuronal firing and sodium-dependent action potentials and may additionally enhance GABA effects is valproate.

Disclosed are nanoparticles for the delivery of a therapeutic agent or a diagnostic agent to a subject that include a chitosan and a polyphosphate, wherein the weight ratio of the chitosan to the polyphosphate is about 1.0 or greater and the weight ratio of the polyphosphate to the therapeutic agent or diagnostic agent is about 15.0 or less. Also disclosed are nanoparticles that include a chitosan and an inhibitor of enhancer of Zeste homologue 2 (EZH2). Methods of delivering a therapeutic agent or a diagnostic agent to a subject for the treatment or prevention of a disease and methods of predicting prognosis of ovarian cancer in a subject that involve determining the expression and/or function of EZH2 in the subject are also disclosed.

1.-38. (canceled) 39. A method of treating a subject with ovarian cancer, comprising administering to a subject with ovarian cancer a pharmaceutically effective amount of a composition comprising: (a) a chitosan; and (b) a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes EZH2. 40. The method of claim 39, wherein the nucleic acid component comprises a siRNA or a nucleic acid encoding a siRNA, wherein the siRNA inhibits the expression of a gene that encodes EZH2 in the subject. 41. The method of claim 39, wherein the composition further comprises a lipid. 42. The method of claim 41, wherein the lipid is cholesterol, phosphatidylcholine, or phosphatidylethanolamine. 43. The method of claim 39, wherein the composition further comprises a polyphosphate anion of formula (I): wherein n is an integer ranging from 2-10. 44. The method of claim 39, wherein the subject is a human subject. 45. The method of claim 39, further comprising administering an additional anticancer therapy to the subject. 46. The method of claim 45, wherein the additional anticancer therapy is chemotherapy, radiation therapy, surgical therapy, immunotherapy, gene therapy, or a combination thereof. 47. The method of claim 45, wherein the additional anticancer therapy is a VEGF inhibitor. 48. The method of claim 39, wherein the composition is administered to the patient intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. 49. A method to inhibit angiogenesis of an ovarian cancer, comprising contacting said cancer with a composition comprising: (a) a chitosan; and (b) a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes EZH2, wherein angiogenesis of the ovarian cancer is inhibited. 50. The method of claim 49, wherein the ovarian cancer is in a human subject. 51. (canceled) 52. The method of claim 49, wherein the composition further comprises cholesterol. 53. The method of claim 49, wherein the composition further comprises tripolyphosphate. 54.-60. (canceled) 61. The method of claim 49, wherein the nucleic acid component comprises a siRNA or a nucleic acid encoding a siRNA, wherein the siRNA inhibits the expression of a gene that encodes EZH2 in the subject.

Disclosed are nanoparticles for the delivery of a therapeutic agent or a diagnostic agent to a subject that include a chitosan and a polyphosphate, wherein the weight ratio of the chitosan to the polyphosphate is about 1.0 or greater and the weight ratio of the polyphosphate to the therapeutic agent or diagnostic agent is about 15.0 or less. Also disclosed are nanoparticles that include a chitosan and an inhibitor of enhancer of Zeste homologue 2 (EZH2). Methods of delivering a therapeutic agent or a diagnostic agent to a subject for the treatment or prevention of a disease and methods of predicting prognosis of ovarian cancer in a subject that involve determining the expression and/or function of EZH2 in the subject are also disclosed.1.-38. (canceled) 39. A method of treating a subject with ovarian cancer, comprising administering to a subject with ovarian cancer a pharmaceutically effective amount of a composition comprising: (a) a chitosan; and (b) a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes EZH2. 40. The method of claim 39, wherein the nucleic acid component comprises a siRNA or a nucleic acid encoding a siRNA, wherein the siRNA inhibits the expression of a gene that encodes EZH2 in the subject. 41. The method of claim 39, wherein the composition further comprises a lipid. 42. The method of claim 41, wherein the lipid is cholesterol, phosphatidylcholine, or phosphatidylethanolamine. 43. The method of claim 39, wherein the composition further comprises a polyphosphate anion of formula (I): wherein n is an integer ranging from 2-10. 44. The method of claim 39, wherein the subject is a human subject. 45. The method of claim 39, further comprising administering an additional anticancer therapy to the subject. 46. The method of claim 45, wherein the additional anticancer therapy is chemotherapy, radiation therapy, surgical therapy, immunotherapy, gene therapy, or a combination thereof. 47. The method of claim 45, wherein the additional anticancer therapy is a VEGF inhibitor. 48. The method of claim 39, wherein the composition is administered to the patient intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. 49. A method to inhibit angiogenesis of an ovarian cancer, comprising contacting said cancer with a composition comprising: (a) a chitosan; and (b) a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes EZH2, wherein angiogenesis of the ovarian cancer is inhibited. 50. The method of claim 49, wherein the ovarian cancer is in a human subject. 51. (canceled) 52. The method of claim 49, wherein the composition further comprises cholesterol. 53. The method of claim 49, wherein the composition further comprises tripolyphosphate. 54.-60. (canceled) 61. The method of claim 49, wherein the nucleic acid component comprises a siRNA or a nucleic acid encoding a siRNA, wherein the siRNA inhibits the expression of a gene that encodes EZH2 in the subject.

The present invention relates to a pharmaceutical composition comprising a modified mRNA that is stabilised by sequence modifications and optimised for translation. The pharmaceutical composition according to the invention is particularly well suited for use as an inoculating agent, as well as a therapeutic agent for tissue regeneration. In addition, a process is described for determining sequence modifications that promote stabilisation and translational efficiency of modified mRNA of the invention.

1.-28. (canceled) 29. A method for producing a stabilized mRNA comprising synthesizing an mRNA encoding a native polypeptide sequence, wherein the mRNA encoding the polypeptide comprises a nucleic acid sequence that has an increased GuanineCytosine (G/C) content relative to the native nucleic acid sequence encoding the polypeptide, said relative G/C content being increased by at least 7 percentage points compared to native nucleic acid sequence encoding the polypeptide, to thereby produce a stabilized mRNA. 30. The method of claim 29, wherein the sequence encoding the polypeptide has a G/C content increased by at least 15 percentage points compared to native nucleic acid sequence encoding the polypeptide. 31. The method of claim 30, wherein the sequence encoding the polypeptide has a G/C content increased by at least 20 percentage points compared to native nucleic acid sequence encoding the polypeptide. 32. The method of claim 29, wherein the sequence encoding the polypeptide has a G/C content increased sufficiently to reduce the susceptibility of the mRNA to exonuclease digestion compared to native nucleic acid sequence encoding the polypeptide. 33. The method of claim 29, wherein the stabilized mRNA comprises a nucleic acid sequence encoding the polypeptide that has at least one codon recognized by a rare cellular tRNA replaced with a codon recognized by an abundant tRNA relative to the native nucleic acid sequence encoding the polypeptide. 34. The method of claim 29, wherein the stabilized mRNA comprises a nucleic acid sequence having at least one destabilizing sequence element (DSE) removed relative to the native mRNA encoding the polypeptide. 35. The method of claim 34, wherein the DSE is an AU-rich sequence in the 3′-UTR of the native mRNA encoding the polypeptide. 36. The method of claim 29, wherein the stabilized mRNA comprises a 5′ cap. 37. The method of claim 29, wherein synthesizing the stabilized mRNA comprises producing a DNA molecule encoding the stabilized mRNA. 38. The method of claim 37, wherein synthesizing the stabilized mRNA further comprises transcribing the stabilized mRNA from the DNA molecule. 39. The method of claim 38, wherein the transcription is in vitro transcription. 40. The method of claim 29, wherein the native polypeptide sequence is the polypeptide sequence of a virus, bacterium, protozoan or tumour antigen. 41. The method of claim 29, wherein the native polypeptide sequence is the polypeptide sequence of a biologically active polypeptide. 42. The method according to claim 29, wherein synthesizing a stabilized mRNA comprises using a computer to determine the nucleic acid sequence encoding the polypeptide that has an increased GuanineCytosine (G/C) content. 43. The method of claim 42, wherein using the computer comprises using a software program comprising a source code of Appendix I. 44. The method of claim 29, further comprising the formulating the stabilized mRNA into a pharmaceutically acceptable carrier. 45. The method of claim 29, further comprising synthesizing a stabilized mRNA comprising at least one nucleotide position replaced with a nucleotide analogue selected from the group consisting of phosphorus amidates, phosphorus thioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.

The present invention relates to a pharmaceutical composition comprising a modified mRNA that is stabilised by sequence modifications and optimised for translation. The pharmaceutical composition according to the invention is particularly well suited for use as an inoculating agent, as well as a therapeutic agent for tissue regeneration. In addition, a process is described for determining sequence modifications that promote stabilisation and translational efficiency of modified mRNA of the invention.1.-28. (canceled) 29. A method for producing a stabilized mRNA comprising synthesizing an mRNA encoding a native polypeptide sequence, wherein the mRNA encoding the polypeptide comprises a nucleic acid sequence that has an increased GuanineCytosine (G/C) content relative to the native nucleic acid sequence encoding the polypeptide, said relative G/C content being increased by at least 7 percentage points compared to native nucleic acid sequence encoding the polypeptide, to thereby produce a stabilized mRNA. 30. The method of claim 29, wherein the sequence encoding the polypeptide has a G/C content increased by at least 15 percentage points compared to native nucleic acid sequence encoding the polypeptide. 31. The method of claim 30, wherein the sequence encoding the polypeptide has a G/C content increased by at least 20 percentage points compared to native nucleic acid sequence encoding the polypeptide. 32. The method of claim 29, wherein the sequence encoding the polypeptide has a G/C content increased sufficiently to reduce the susceptibility of the mRNA to exonuclease digestion compared to native nucleic acid sequence encoding the polypeptide. 33. The method of claim 29, wherein the stabilized mRNA comprises a nucleic acid sequence encoding the polypeptide that has at least one codon recognized by a rare cellular tRNA replaced with a codon recognized by an abundant tRNA relative to the native nucleic acid sequence encoding the polypeptide. 34. The method of claim 29, wherein the stabilized mRNA comprises a nucleic acid sequence having at least one destabilizing sequence element (DSE) removed relative to the native mRNA encoding the polypeptide. 35. The method of claim 34, wherein the DSE is an AU-rich sequence in the 3′-UTR of the native mRNA encoding the polypeptide. 36. The method of claim 29, wherein the stabilized mRNA comprises a 5′ cap. 37. The method of claim 29, wherein synthesizing the stabilized mRNA comprises producing a DNA molecule encoding the stabilized mRNA. 38. The method of claim 37, wherein synthesizing the stabilized mRNA further comprises transcribing the stabilized mRNA from the DNA molecule. 39. The method of claim 38, wherein the transcription is in vitro transcription. 40. The method of claim 29, wherein the native polypeptide sequence is the polypeptide sequence of a virus, bacterium, protozoan or tumour antigen. 41. The method of claim 29, wherein the native polypeptide sequence is the polypeptide sequence of a biologically active polypeptide. 42. The method according to claim 29, wherein synthesizing a stabilized mRNA comprises using a computer to determine the nucleic acid sequence encoding the polypeptide that has an increased GuanineCytosine (G/C) content. 43. The method of claim 42, wherein using the computer comprises using a software program comprising a source code of Appendix I. 44. The method of claim 29, further comprising the formulating the stabilized mRNA into a pharmaceutically acceptable carrier. 45. The method of claim 29, further comprising synthesizing a stabilized mRNA comprising at least one nucleotide position replaced with a nucleotide analogue selected from the group consisting of phosphorus amidates, phosphorus thioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.

Provided herein are monoclonal antibodies against Olfml-3. In some aspects, methods for treating angiogenesis-related conditions, such as cancer, are provided comprising administering an Olfml-3-binding antibody of the embodiments.

1. An isolated monoclonal antibody, wherein the antibody comprises: (a) a first VH CDR having the sequence of VH CDR1 of 46A9BO (SEQ ID NO: 7), 9F8BO (SEQ ID NO: 21), or Z14A7 (SEQ ID NO: 13); (b) a second VH CDR having the sequence of VH CDR2 of 46A9BO (SEQ ID NO: 8), 9F8BO (SEQ ID NO: 22), or Z14A7 (SEQ ID NO: 14); (c) a third VH CDR having the sequence of VH CDR3 of 46A9BO (SEQ ID NO: 9), 9F8BO (SEQ ID NO: 23), or Z14A7 (SEQ ID NO: 15); (d) a first VL CDR having the sequence of VL CDR1 of 46A9BO (SEQ ID NO: 10), 9F8BO (SEQ ID NO: 24), or Z14A7 (SEQ ID NO: 16); (e) a second VL CDR having the sequence of VL CDR2 of 46A9BO (SEQ ID NO: 11), 9F8BO (SEQ ID NO: 25), or Z14A7 (SEQ ID NO: 17); and (f) a third VL CDR having the sequence of VL CDR3 of 46A9BO (SEQ ID NO: 12), 9F8BO (SEQ ID NO: 26), or Z14A7 (SEQ ID NO: 18). 2. The isolated antibody of claim 1, wherein the antibody comprises: (a) a first VH CDR is identical to SEQ ID NO: 7; (b) a second VH CDR is identical to SEQ ID NO: 8; (c) a third VH CDR is identical to SEQ ID NO: 9; (d) a first VL CDR is identical to SEQ ID NO: 10; (e) a second VL CDR is identical to SEQ ID NO: 11; and (f) a third VL CDR is identical to SEQ ID NO: 12. 3. The isolated antibody of claim 1, wherein the antibody comprises: (a) a first VH CDR is identical to SEQ ID NO: 21; (b) a second VH CDR is identical to SEQ ID NO: 22; (c) a third VH CDR is identical to SEQ ID NO: 23; (d) a first VL CDR is identical to SEQ ID NO: 24; (e) a second VL CDR is identical to SEQ ID NO: 25; and (f) a third VL CDR is identical to SEQ ID NO: 26. 4. The isolated antibody of claim 1, wherein the antibody comprises: (a) a first VH CDR is identical to SEQ ID NO: 13; (b) a second VH CDR is identical to SEQ ID NO: 14; (c) a third VH CDR is identical to SEQ ID NO: 15; (d) a first VL CDR is identical to SEQ ID NO: 16; (e) a second VL CDR is identical to SEQ ID NO: 17; and (f) a third VL CDR is identical to SEQ ID NO: 18. 5. The antibody of claim 1, wherein the antibody comprises: (i) a VH domain at least about 80% identical to the VH domain of 46A9BO (SEQ ID NO: 1) and a VL domain at least about 80% identical to the VL domain of 46A9BO (SEQ ID NO: 2); (i) a VH domain at least about 80% identical to the VH domain of 9F8BO (SEQ ID NO: 19) and a VL domain at least about 80% identical to the VL domain of 9F8BO (SEQ ID NO: 20); or (iii) a VH domain at least about 80% identical to the VH domain of Z14A7 (SEQ ID NO: 3) and a VL domain at least about 80% identical to the VL domain of Z14A7 (SEQ ID NO: 4). 6. The antibody of claim 5, wherein the antibody comprises a VH domain identical to the VH domain of 46A9BO (SEQ ID NO: 1) and a VL domain identical to the VL domain of 46A9BO (SEQ ID NO: 2). 7. The antibody of claim 5, wherein the antibody comprises a VH domain identical to the VH domain of 9F8BO (SEQ ID NO: 19) and a VL domain identical to the VL domain of 9F8BO (SEQ ID NO: 20). 8. The antibody of claim 5, wherein the antibody comprises a VH domain identical to the VH domain of Z14A7 (SEQ ID NO: 3) and a VL domain identical to the VL domain of Z14A7 (SEQ ID NO: 4). 9. The antibody of claim 5, wherein the antibody is the 46A9BO, 9F8BO or Z14A7 antibody. 10. The antibody of claim 1, wherein the antibody is recombinant. 11. The antibody of claim 1, wherein the antibody is an IgG, IgM, IgA or an antigen binding fragment thereof. 12. The antibody of claim 1, wherein the antibody is a Fab′, a F(ab′)2, a F(ab′)3, a monovalent scFv, a bivalent scFv, or a single domain antibody. 13. The antibody of claim 1, wherein the antibody is a human, humanized antibody or de-immunized antibody. 14. The antibody of claim 1, wherein the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin or a radionuclide. 15. A composition comprising an antibody of claim 1 in a pharmaceutically acceptable carrier. 16. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding an antibody of claim 1. 17. A host cell comprising one or more polynucleotide molecule(s) encoding an antibody of claim 1. 18. A method of manufacturing an antibody comprising: (a) expressing one or more polynucleotide molecule(s) encoding a VL and VH chain of an antibody of claim 1 in a cell; and (b) purifying the antibody from the cell. 19. A method of treating an angiogenesis-related condition in a subject comprising administering to the subject an amount of an antibody in accordance with of claim 1 that is effective to treat the angiogenesis-related condition. 20. The method of claim 19, wherein the angiogenesis-related condition comprises cancer.

Provided herein are monoclonal antibodies against Olfml-3. In some aspects, methods for treating angiogenesis-related conditions, such as cancer, are provided comprising administering an Olfml-3-binding antibody of the embodiments.1. An isolated monoclonal antibody, wherein the antibody comprises: (a) a first VH CDR having the sequence of VH CDR1 of 46A9BO (SEQ ID NO: 7), 9F8BO (SEQ ID NO: 21), or Z14A7 (SEQ ID NO: 13); (b) a second VH CDR having the sequence of VH CDR2 of 46A9BO (SEQ ID NO: 8), 9F8BO (SEQ ID NO: 22), or Z14A7 (SEQ ID NO: 14); (c) a third VH CDR having the sequence of VH CDR3 of 46A9BO (SEQ ID NO: 9), 9F8BO (SEQ ID NO: 23), or Z14A7 (SEQ ID NO: 15); (d) a first VL CDR having the sequence of VL CDR1 of 46A9BO (SEQ ID NO: 10), 9F8BO (SEQ ID NO: 24), or Z14A7 (SEQ ID NO: 16); (e) a second VL CDR having the sequence of VL CDR2 of 46A9BO (SEQ ID NO: 11), 9F8BO (SEQ ID NO: 25), or Z14A7 (SEQ ID NO: 17); and (f) a third VL CDR having the sequence of VL CDR3 of 46A9BO (SEQ ID NO: 12), 9F8BO (SEQ ID NO: 26), or Z14A7 (SEQ ID NO: 18). 2. The isolated antibody of claim 1, wherein the antibody comprises: (a) a first VH CDR is identical to SEQ ID NO: 7; (b) a second VH CDR is identical to SEQ ID NO: 8; (c) a third VH CDR is identical to SEQ ID NO: 9; (d) a first VL CDR is identical to SEQ ID NO: 10; (e) a second VL CDR is identical to SEQ ID NO: 11; and (f) a third VL CDR is identical to SEQ ID NO: 12. 3. The isolated antibody of claim 1, wherein the antibody comprises: (a) a first VH CDR is identical to SEQ ID NO: 21; (b) a second VH CDR is identical to SEQ ID NO: 22; (c) a third VH CDR is identical to SEQ ID NO: 23; (d) a first VL CDR is identical to SEQ ID NO: 24; (e) a second VL CDR is identical to SEQ ID NO: 25; and (f) a third VL CDR is identical to SEQ ID NO: 26. 4. The isolated antibody of claim 1, wherein the antibody comprises: (a) a first VH CDR is identical to SEQ ID NO: 13; (b) a second VH CDR is identical to SEQ ID NO: 14; (c) a third VH CDR is identical to SEQ ID NO: 15; (d) a first VL CDR is identical to SEQ ID NO: 16; (e) a second VL CDR is identical to SEQ ID NO: 17; and (f) a third VL CDR is identical to SEQ ID NO: 18. 5. The antibody of claim 1, wherein the antibody comprises: (i) a VH domain at least about 80% identical to the VH domain of 46A9BO (SEQ ID NO: 1) and a VL domain at least about 80% identical to the VL domain of 46A9BO (SEQ ID NO: 2); (i) a VH domain at least about 80% identical to the VH domain of 9F8BO (SEQ ID NO: 19) and a VL domain at least about 80% identical to the VL domain of 9F8BO (SEQ ID NO: 20); or (iii) a VH domain at least about 80% identical to the VH domain of Z14A7 (SEQ ID NO: 3) and a VL domain at least about 80% identical to the VL domain of Z14A7 (SEQ ID NO: 4). 6. The antibody of claim 5, wherein the antibody comprises a VH domain identical to the VH domain of 46A9BO (SEQ ID NO: 1) and a VL domain identical to the VL domain of 46A9BO (SEQ ID NO: 2). 7. The antibody of claim 5, wherein the antibody comprises a VH domain identical to the VH domain of 9F8BO (SEQ ID NO: 19) and a VL domain identical to the VL domain of 9F8BO (SEQ ID NO: 20). 8. The antibody of claim 5, wherein the antibody comprises a VH domain identical to the VH domain of Z14A7 (SEQ ID NO: 3) and a VL domain identical to the VL domain of Z14A7 (SEQ ID NO: 4). 9. The antibody of claim 5, wherein the antibody is the 46A9BO, 9F8BO or Z14A7 antibody. 10. The antibody of claim 1, wherein the antibody is recombinant. 11. The antibody of claim 1, wherein the antibody is an IgG, IgM, IgA or an antigen binding fragment thereof. 12. The antibody of claim 1, wherein the antibody is a Fab′, a F(ab′)2, a F(ab′)3, a monovalent scFv, a bivalent scFv, or a single domain antibody. 13. The antibody of claim 1, wherein the antibody is a human, humanized antibody or de-immunized antibody. 14. The antibody of claim 1, wherein the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin or a radionuclide. 15. A composition comprising an antibody of claim 1 in a pharmaceutically acceptable carrier. 16. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding an antibody of claim 1. 17. A host cell comprising one or more polynucleotide molecule(s) encoding an antibody of claim 1. 18. A method of manufacturing an antibody comprising: (a) expressing one or more polynucleotide molecule(s) encoding a VL and VH chain of an antibody of claim 1 in a cell; and (b) purifying the antibody from the cell. 19. A method of treating an angiogenesis-related condition in a subject comprising administering to the subject an amount of an antibody in accordance with of claim 1 that is effective to treat the angiogenesis-related condition. 20. The method of claim 19, wherein the angiogenesis-related condition comprises cancer.

A methodology of producing and utilizing antibodies that recognize peptides associated with a tumorigenic or disease state, wherein the peptides are displayed in the context of HLA molecules, is disclosed. These antibodies may be utilized in therapeutic methods of mediating cell lysis.

1. A method of directly mediating lysis of tumorigenic cells expressing at least one specific peptide/MHC complex on a surface thereof, wherein the specific peptide of the at least one specific peptide/MHC complex is associated with a tumorigenic state, the method comprising the steps of: contacting tumorigenic cells expressing at least one specific peptide/MHC complex on a surface thereof with a therapeutic T cell receptor mimic, such that the therapeutic T cell receptor mimic directly mediates lysis of the tumor cells expressing the at least one specific peptide/MHC complex on a surface thereof by induction of apoptosis, wherein the therapeutic T cell receptor mimic comprises a therapeutic antibody or antibody fragment reactive against a specific peptide/MHC complex, and wherein the antibody or antibody fragment can differentiate the specific peptide/MHC complex from the MHC molecule alone, the specific peptide alone, and a complex of MHC and an irrelevant peptide. 2. The method of claim 1, wherein the specific peptide is associated with at least one of breast cancer, ovarian cancer, prostate cancer, lung cancer, multiple myeloma, biliary cancer, and pancreatic cancer. 3. The method of claim 1 wherein, in the step of providing a T cell receptor mimic, the therapeutic T cell receptor mimic has a binding affinity of about 10 nanomolar or greater. 4. The method of claim 1, wherein the therapeutic T cell receptor mimic is produced by immunizing a host with an effective amount of an immunogen comprising a multimer of two or more specific peptide/MHC complexes.

A methodology of producing and utilizing antibodies that recognize peptides associated with a tumorigenic or disease state, wherein the peptides are displayed in the context of HLA molecules, is disclosed. These antibodies may be utilized in therapeutic methods of mediating cell lysis.1. A method of directly mediating lysis of tumorigenic cells expressing at least one specific peptide/MHC complex on a surface thereof, wherein the specific peptide of the at least one specific peptide/MHC complex is associated with a tumorigenic state, the method comprising the steps of: contacting tumorigenic cells expressing at least one specific peptide/MHC complex on a surface thereof with a therapeutic T cell receptor mimic, such that the therapeutic T cell receptor mimic directly mediates lysis of the tumor cells expressing the at least one specific peptide/MHC complex on a surface thereof by induction of apoptosis, wherein the therapeutic T cell receptor mimic comprises a therapeutic antibody or antibody fragment reactive against a specific peptide/MHC complex, and wherein the antibody or antibody fragment can differentiate the specific peptide/MHC complex from the MHC molecule alone, the specific peptide alone, and a complex of MHC and an irrelevant peptide. 2. The method of claim 1, wherein the specific peptide is associated with at least one of breast cancer, ovarian cancer, prostate cancer, lung cancer, multiple myeloma, biliary cancer, and pancreatic cancer. 3. The method of claim 1 wherein, in the step of providing a T cell receptor mimic, the therapeutic T cell receptor mimic has a binding affinity of about 10 nanomolar or greater. 4. The method of claim 1, wherein the therapeutic T cell receptor mimic is produced by immunizing a host with an effective amount of an immunogen comprising a multimer of two or more specific peptide/MHC complexes.

The present invention is in the field of drug delivery, and specifically, cationic liposome-based drug delivery. In embodiments, this invention provides methods of making ligand-targeted (e.g., antibody- or antibody fragment-targeted) liposomes useful for the delivery of liposomes to tumors, including brain tumors. In embodiments, the liposomes deliver temozolomide across the blood-brain barrier for treatment of primary or metastatic brain tumors. Additional cancers that can be treated with the liposomes include neuroendocrine tumors, melanoma, prostate, head and neck, ovarian, lung, liver, kidney, breast, urogenital, gastric, colorectal, cervical, vaginal, angiosarcoma, liposarcoma, rhabdomyosarcoma, choriocarcinoma, pancreatic, retinoblastoma and other types of cancer. In another embodiment the liposomes deliver melphalan for the treatment of multiple myeloma, other tumors of the blood or other solid tumors. In still other embodiments the liposomes can deliver other drugs such as pemetrexed or irinotecan for treatment of cancer or drugs including atropine for treatment of organophosphate poisoning.

1. A method of preparing a targeted active agent cationic liposome complex, comprising: (a) preparing a lipid solution comprising one or more cationic lipids in ethanol; (b) preparing a solution of an active agent selected from temozolomide, melphalan and atropine; (c) mixing the lipid solution with the solution of active agent; (d) injecting the mixture of lipid and active anent into an aqueous solution, thereby forming an active agent cationic liposome; (e) mixing the active agent cationic liposome with a ligand to form the targeted active agent cationic liposome, wherein the ligand is directly complexed with, but not chemically conjugated to, the cationic liposome. 2. The method of claim 1, wherein the ligand is an antibody, an antibody fragment or a protein. 3. The method of claim 2, wherein the ligand is a single chain Fv antibody fragment. 4. (canceled) 5. (canceled) 6. The method of claim 1, wherein the lipid solution comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). 7. The method of claim 1, wherein the solution of active agent is prepared at a concentration of about 1 mM to about 200 mM. 8. (canceled) 9. The method of claim 1, wherein the molar ratio of lipid:active agent is about 0.1:1 to about 5:1. 10. (canceled) 11. (canceled) 12. The method of claim 1, wherein the weight ratio of ligand:lipid is about 0.01:1 to about 0.5:10. 13-24. (canceled) 25. A method of treating cancer in a patient, comprising administering to the patient a targeted active agent cationic liposome complex, wherein the targeted active agent cationic liposome complex comprises: (a) a cationic liposome comprising 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (b) an active agent selected from temozolomide and melphalan; and (c) a ligand directly complexed with, but not chemically conjugated to, the cationic liposome. 26. The method of claim 25, wherein the ligand is an antibody, an antibody fragment or a protein. 27. The method of claim 26, wherein the ligand is a single chain Fv antibody fragment. 28. (canceled) 29. The method of claim 25, wherein the active agent is administered to the patient at a dose of about 10 mg/m2 to about 500 mg/m2. 30. (canceled) 31. The method of claim 25, wherein the molar ratio of lipid:active agent in the cationic liposomes is about 0.1:1 to about 5:1. 32. (canceled) 33. (canceled) 34. The method of claim 25, wherein the weight ratio of ligand:lipid in the cationic liposome is about 0.01:1 to about 0.5:10. 35. (canceled) 36. (canceled) 37. The method of claim 25, wherein the administration is intravenous (IV), intratumoral (IT), intralesional (IL), sublingual (SL), aerosal, percutaneous, oral, endoscopic, topical, intramuscular (IM), intradermal (ID), intraocular (IO), intraperitoneal (IP), transdermal (TD), intranasal (IN), intracereberal (IC), intraorgan (e.g. intrahepatic), slow release implant, or subcutaneous administration, or via administration using an osmotic or mechanical pump 38. The method of claim 25, wherein the cancer is head and neck cancer, breast cancer, prostate cancer, pancreatic cancer, brain cancer, neuroendocrine cancer, cervical cancer, lung cancer, liver cancer, kidney cancer, liposarcoma, angiosarcoma, rhabdomyosarcoma, choriocarcinoma, melanoma, retinoblastoma, ovarian cancer, vaginal cancer, urogenital cancer, gastric cancer, colorectal cancer, multiple myeloma or a cancer of the blood. 39. The method of claim 38, wherein the brain cancer is a glioma, astrocytoma or a glioblastoma. 40. The method of claim 25, further comprising administering an additional different therapy to the patient in combination with the targeted active agent cationic liposome complex. 41. The method of claim 40, wherein the additional different therapy comprises administration of a chemotherapeutic agent, a small molecule, radiation therapy or a nucleic acid-based therapy. 42. The method of claim 41, wherein the nucleic acid-based therapy comprises administration of a cationic liposome complex comprising an antisense oligonucleotide, an siRNA, an miRNA, a plasmid DNA or an shRNA 43-54. (canceled) 55. A method of treating cancer in a patient, comprising administering to the patient a targeted active agent cationic liposome complex prepared by the method of claim 1. 56. The method of claim 55, wherein the ligand is an antibody, an antibody fragment or a protein. 57. The method of claim 56, wherein the ligand is a single chain Fv antibody fragment. 58. (canceled) 59. The method of claim 55, wherein active agent is administered to the patient at a dose of about 10 mg/m2 to about 500 mg/m2. 60. (canceled) 61. The method of claim 55, wherein the molar ratio of lipid:active agent is about 0.1:1 to about 5:1. 62. (canceled) 63. (canceled) 64. The method of claim 55, wherein the weight ratio of ligand:lipid is about 0.01:1 to about 0.5:10. 65. (canceled) 66. (canceled) 67. The method of claim 55, wherein the administration is intravenous (IV), intratumoral (IT), intralesional (IL), aerosal, percutaneous, oral, endoscopic, topical, intramuscular (IM), intradermal (ID), sublingual (SL), intraocular (IO), intraperitoneal (IP), transdermal (TD), intranasal (IN), intracereberal (IC), intraorgan (e.g. intrahepatic), slow release implant, or subcutaneous administration, or via administration using an osmotic or mechanical pump 68. The method of claim 55, wherein the cancer is head and neck cancer, breast cancer, prostate cancer, pancreatic cancer, brain cancer, neuroendocrine cancer, cervical cancer, lung cancer, liver cancer, kidney cancer, liposarcoma, angiosarcoma, rhabdomyosarcoma, choriocarcinoma, melanoma, retinoblastoma, ovarian cancer, vaginal cancer, urogenital cancer, gastric cancer, colorectal cancer, multiple myeloma or a cancer of the blood. 69. (canceled) 70. The method of claim 55, further comprising administering an additional different therapy to the patient in combination with the targeted active agent cationic liposome complex. 71. The method of claim 70, wherein the additional different therapy comprises administration of a chemotherapeutic agent, a small molecule, radiation therapy or a nucleic acid-based therapy. 72. The method of claim 71, wherein the nucleic acid-based therapy comprises administration of a cationic liposome complex comprising an antisense oligonucleotide, an siRNA, an miRNA, a plasmid DNA or an shRNA 73-153. (canceled) 154. A method of treating organophosphate poisoning in a patient, comprising administering to the patient a targeted atropine cationic liposome complex, wherein the targeted atropine cationic liposome complex comprises: (a) a cationic liposome comprising 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (b) atropine; and (c) a ligand directly complexed with, but not chemically conjugated to, the cationic liposome. 155. The method of claim 154, wherein the ligand is an antibody, an antibody fragment or a protein. 156. The method of claim 155, wherein the ligand is a single chain Fv antibody fragment. 157. (canceled) 158. The method of claim 154, wherein the atropine is administered to the patient at a dose of about 1 mg to about 10 mg. 159. (canceled) 160. The method of claim 154, wherein the molar ratio of lipid:atropine in the cationic liposome: atropine is about 0.1:1 to about 5:1. 161. (canceled) 162. (canceled) 163. The method of claim 154, wherein the weight ratio of ligand:lipid in the cationic liposome is about 0.01:1 to about 0.5:10. 164. (canceled) 165. (canceled) 166. The method of claim 154, wherein the administration is intravenous (IV), intratumoral (IT), intralesional (IL), sublingual (SL), aerosal, percutaneous, oral, endoscopic, topical, intramuscular (IM), intradermal (ID), intraocular (IO), intraperitoneal (IP), transdermal (TD), intranasal (IN), intracereberal (IC), intraorgan (e.g. intrahepatic), slow release implant, or subcutaneous administration, or via administration using an osmotic or mechanical pump. 167. The method of claim 154, wherein the liposome crosses the blood-brain barrier. 168-240. (canceled) 241. The method of claim 25, wherein the liposome crosses the blood-brain barrier.

The present invention is in the field of drug delivery, and specifically, cationic liposome-based drug delivery. In embodiments, this invention provides methods of making ligand-targeted (e.g., antibody- or antibody fragment-targeted) liposomes useful for the delivery of liposomes to tumors, including brain tumors. In embodiments, the liposomes deliver temozolomide across the blood-brain barrier for treatment of primary or metastatic brain tumors. Additional cancers that can be treated with the liposomes include neuroendocrine tumors, melanoma, prostate, head and neck, ovarian, lung, liver, kidney, breast, urogenital, gastric, colorectal, cervical, vaginal, angiosarcoma, liposarcoma, rhabdomyosarcoma, choriocarcinoma, pancreatic, retinoblastoma and other types of cancer. In another embodiment the liposomes deliver melphalan for the treatment of multiple myeloma, other tumors of the blood or other solid tumors. In still other embodiments the liposomes can deliver other drugs such as pemetrexed or irinotecan for treatment of cancer or drugs including atropine for treatment of organophosphate poisoning.1. A method of preparing a targeted active agent cationic liposome complex, comprising: (a) preparing a lipid solution comprising one or more cationic lipids in ethanol; (b) preparing a solution of an active agent selected from temozolomide, melphalan and atropine; (c) mixing the lipid solution with the solution of active agent; (d) injecting the mixture of lipid and active anent into an aqueous solution, thereby forming an active agent cationic liposome; (e) mixing the active agent cationic liposome with a ligand to form the targeted active agent cationic liposome, wherein the ligand is directly complexed with, but not chemically conjugated to, the cationic liposome. 2. The method of claim 1, wherein the ligand is an antibody, an antibody fragment or a protein. 3. The method of claim 2, wherein the ligand is a single chain Fv antibody fragment. 4. (canceled) 5. (canceled) 6. The method of claim 1, wherein the lipid solution comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). 7. The method of claim 1, wherein the solution of active agent is prepared at a concentration of about 1 mM to about 200 mM. 8. (canceled) 9. The method of claim 1, wherein the molar ratio of lipid:active agent is about 0.1:1 to about 5:1. 10. (canceled) 11. (canceled) 12. The method of claim 1, wherein the weight ratio of ligand:lipid is about 0.01:1 to about 0.5:10. 13-24. (canceled) 25. A method of treating cancer in a patient, comprising administering to the patient a targeted active agent cationic liposome complex, wherein the targeted active agent cationic liposome complex comprises: (a) a cationic liposome comprising 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (b) an active agent selected from temozolomide and melphalan; and (c) a ligand directly complexed with, but not chemically conjugated to, the cationic liposome. 26. The method of claim 25, wherein the ligand is an antibody, an antibody fragment or a protein. 27. The method of claim 26, wherein the ligand is a single chain Fv antibody fragment. 28. (canceled) 29. The method of claim 25, wherein the active agent is administered to the patient at a dose of about 10 mg/m2 to about 500 mg/m2. 30. (canceled) 31. The method of claim 25, wherein the molar ratio of lipid:active agent in the cationic liposomes is about 0.1:1 to about 5:1. 32. (canceled) 33. (canceled) 34. The method of claim 25, wherein the weight ratio of ligand:lipid in the cationic liposome is about 0.01:1 to about 0.5:10. 35. (canceled) 36. (canceled) 37. The method of claim 25, wherein the administration is intravenous (IV), intratumoral (IT), intralesional (IL), sublingual (SL), aerosal, percutaneous, oral, endoscopic, topical, intramuscular (IM), intradermal (ID), intraocular (IO), intraperitoneal (IP), transdermal (TD), intranasal (IN), intracereberal (IC), intraorgan (e.g. intrahepatic), slow release implant, or subcutaneous administration, or via administration using an osmotic or mechanical pump 38. The method of claim 25, wherein the cancer is head and neck cancer, breast cancer, prostate cancer, pancreatic cancer, brain cancer, neuroendocrine cancer, cervical cancer, lung cancer, liver cancer, kidney cancer, liposarcoma, angiosarcoma, rhabdomyosarcoma, choriocarcinoma, melanoma, retinoblastoma, ovarian cancer, vaginal cancer, urogenital cancer, gastric cancer, colorectal cancer, multiple myeloma or a cancer of the blood. 39. The method of claim 38, wherein the brain cancer is a glioma, astrocytoma or a glioblastoma. 40. The method of claim 25, further comprising administering an additional different therapy to the patient in combination with the targeted active agent cationic liposome complex. 41. The method of claim 40, wherein the additional different therapy comprises administration of a chemotherapeutic agent, a small molecule, radiation therapy or a nucleic acid-based therapy. 42. The method of claim 41, wherein the nucleic acid-based therapy comprises administration of a cationic liposome complex comprising an antisense oligonucleotide, an siRNA, an miRNA, a plasmid DNA or an shRNA 43-54. (canceled) 55. A method of treating cancer in a patient, comprising administering to the patient a targeted active agent cationic liposome complex prepared by the method of claim 1. 56. The method of claim 55, wherein the ligand is an antibody, an antibody fragment or a protein. 57. The method of claim 56, wherein the ligand is a single chain Fv antibody fragment. 58. (canceled) 59. The method of claim 55, wherein active agent is administered to the patient at a dose of about 10 mg/m2 to about 500 mg/m2. 60. (canceled) 61. The method of claim 55, wherein the molar ratio of lipid:active agent is about 0.1:1 to about 5:1. 62. (canceled) 63. (canceled) 64. The method of claim 55, wherein the weight ratio of ligand:lipid is about 0.01:1 to about 0.5:10. 65. (canceled) 66. (canceled) 67. The method of claim 55, wherein the administration is intravenous (IV), intratumoral (IT), intralesional (IL), aerosal, percutaneous, oral, endoscopic, topical, intramuscular (IM), intradermal (ID), sublingual (SL), intraocular (IO), intraperitoneal (IP), transdermal (TD), intranasal (IN), intracereberal (IC), intraorgan (e.g. intrahepatic), slow release implant, or subcutaneous administration, or via administration using an osmotic or mechanical pump 68. The method of claim 55, wherein the cancer is head and neck cancer, breast cancer, prostate cancer, pancreatic cancer, brain cancer, neuroendocrine cancer, cervical cancer, lung cancer, liver cancer, kidney cancer, liposarcoma, angiosarcoma, rhabdomyosarcoma, choriocarcinoma, melanoma, retinoblastoma, ovarian cancer, vaginal cancer, urogenital cancer, gastric cancer, colorectal cancer, multiple myeloma or a cancer of the blood. 69. (canceled) 70. The method of claim 55, further comprising administering an additional different therapy to the patient in combination with the targeted active agent cationic liposome complex. 71. The method of claim 70, wherein the additional different therapy comprises administration of a chemotherapeutic agent, a small molecule, radiation therapy or a nucleic acid-based therapy. 72. The method of claim 71, wherein the nucleic acid-based therapy comprises administration of a cationic liposome complex comprising an antisense oligonucleotide, an siRNA, an miRNA, a plasmid DNA or an shRNA 73-153. (canceled) 154. A method of treating organophosphate poisoning in a patient, comprising administering to the patient a targeted atropine cationic liposome complex, wherein the targeted atropine cationic liposome complex comprises: (a) a cationic liposome comprising 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (b) atropine; and (c) a ligand directly complexed with, but not chemically conjugated to, the cationic liposome. 155. The method of claim 154, wherein the ligand is an antibody, an antibody fragment or a protein. 156. The method of claim 155, wherein the ligand is a single chain Fv antibody fragment. 157. (canceled) 158. The method of claim 154, wherein the atropine is administered to the patient at a dose of about 1 mg to about 10 mg. 159. (canceled) 160. The method of claim 154, wherein the molar ratio of lipid:atropine in the cationic liposome: atropine is about 0.1:1 to about 5:1. 161. (canceled) 162. (canceled) 163. The method of claim 154, wherein the weight ratio of ligand:lipid in the cationic liposome is about 0.01:1 to about 0.5:10. 164. (canceled) 165. (canceled) 166. The method of claim 154, wherein the administration is intravenous (IV), intratumoral (IT), intralesional (IL), sublingual (SL), aerosal, percutaneous, oral, endoscopic, topical, intramuscular (IM), intradermal (ID), intraocular (IO), intraperitoneal (IP), transdermal (TD), intranasal (IN), intracereberal (IC), intraorgan (e.g. intrahepatic), slow release implant, or subcutaneous administration, or via administration using an osmotic or mechanical pump. 167. The method of claim 154, wherein the liposome crosses the blood-brain barrier. 168-240. (canceled) 241. The method of claim 25, wherein the liposome crosses the blood-brain barrier.

A method of making hydrolyzed marine Type II collagen includes the mixing of marine cartilage, water, an enzyme and a protease enzyme for an extended period of time. Once mixed, the mixture is heated for a period of time at 150° F. Once heated, the enzymes are deactivated, the bone sediment separated, and the fat removed. Next, maltodextrin is added to the mixture and finally the mixture is spray dried to form a collagen powder.

1. A method of making hydrolyzed marine Type I collagen, comprising the steps of: combining water, marine skin, and sodium hydroxide to form a mixture in a tank; mixing the tank for approximately 40 minutes; rinsing the mixture; adding water and sulfuric acid to the tank and mixing for approximately 40 minutes; draining liquid from the tank while retaining the marine skin; and adding citric acid and water to the tank and mixing for approximately 40 minutes.

A method of making hydrolyzed marine Type II collagen includes the mixing of marine cartilage, water, an enzyme and a protease enzyme for an extended period of time. Once mixed, the mixture is heated for a period of time at 150° F. Once heated, the enzymes are deactivated, the bone sediment separated, and the fat removed. Next, maltodextrin is added to the mixture and finally the mixture is spray dried to form a collagen powder.1. A method of making hydrolyzed marine Type I collagen, comprising the steps of: combining water, marine skin, and sodium hydroxide to form a mixture in a tank; mixing the tank for approximately 40 minutes; rinsing the mixture; adding water and sulfuric acid to the tank and mixing for approximately 40 minutes; draining liquid from the tank while retaining the marine skin; and adding citric acid and water to the tank and mixing for approximately 40 minutes.

Devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore are provided. The devices and methods also determine (˜>50 Hz) the nucleotide base sequence of a polynucleotide under feedback control or using signals generated by the interactions between the polynucleotide and the nanopore. The invention is of particular use in the fields of molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.

1. A method for nucleic acid sequencing, comprising: (a) providing a chip comprising a plurality of individually addressable nanopores, an individually addressable nanopore of said plurality of individually addressable nanopores containing at least one nanopore formed in a membrane disposed adjacent to an electrode, wherein each individually addressable nanopore is adapted to detect a nucleic acid molecule or a portion thereof; (b) directing a plurality of nucleic acid molecules to said individually addressable nanopores; and (c) characterizing, with the aid of a processor coupled to said chip, a nucleic acid sequence of each of said nucleic molecules based on electrical signals received from said plurality of individually addressable nanopores. 2. The method of claim 1, wherein said plurality of nucleic acid molecules are derived from a nucleic acid sample. 3. The method of claim 2, wherein each of said plurality of nucleic acid molecules has a shorter nucleic acid sequence than said nucleic acid sample. 4. The method of claim 3, further comprising, prior to (b), fragmenting said nucleic acid sample to provide said plurality of nucleic acid molecules. 5. The method of claim 2, further comprising characterizing a nucleic acid sequence of said nucleic acid sample based upon a characterization of a nucleic acid sequence of each of said nucleic molecules. 6. The method of claim 1, wherein said electrode is adapted to supply an electrical stimulus across said membrane, which stimulus enables the generation of a detectable signal upon molecular flow of said nucleic acid molecule or portion thereof. 7. The method of claim 1, wherein said membrane has a capacitance greater than about 5 fF/μm2 as measured across said membrane. 8. The method of claim 1, wherein said membrane has a resistance greater than or equal to about 500 MΩ as measured across said membrane. 9. The method of claim 8, wherein said resistance is measured with the aid of opposing electrodes disposed adjacent to said membrane. 10. The method of claim 1, wherein said membrane has a resistance less than or equal to about 1 GΩ across said membrane. 11. The method of claim 10, wherein said resistance is measured with the aid of opposing electrodes disposed adjacent to said membrane. 12. The method of claim 1, wherein each individually addressable nanopore is adapted to regulate molecular flow. 13. The method of claim 12, wherein each individually addressable nanopore is adapted to regulate molecular flow with the aid of an electrical stimulus applied to said nanopore. 14. The method of claim 13, wherein said electrical stimulus comprises one or more voltage pulses. 15. The method of claim 1, wherein each individually addressable nanopore is adapted to regulate molecular flow adjacent to said at least one nanopore. 16. The method of claim 1, wherein each individually addressable nanopore is adapted to regulate molecular flow through said at least one nanopore. 17. The method of claim 1, wherein each individually addressable nanopore is adapted to detect said nucleic acid molecule or a portion thereof upon molecular flow of said nucleic acid molecule or portion thereof through or adjacent to said at least one nanopore. 18. The method of claim 1, wherein said nucleic acid sequence is characterized upon movement of each of said plurality of nucleic acid molecules or portions thereof. 19. The method of claim 1, wherein said electrode is coupled to an integrated circuit that processes a signal detected with the aid of said electrode. 20. A system for nucleic acid sequencing, comprising: (a) a chip comprising a plurality of individually addressable nanopores, an individually addressable nanopore of said plurality of individually addressable nanopores containing at least one nanopore formed in a membrane disposed adjacent to an electrode, wherein each individually addressable nanopore is adapted to aid in the detection of said nucleic acid molecule or a portion; and (b) a processor coupled to said chip, wherein said processor is programmed to aid in characterizing a nucleic acid sequence of said nucleic acid molecule based on electrical signals received from said plurality of individually addressable nanopores. 21. The system of claim 20, wherein said electrode is adapted to supply an electrical stimulus across said membrane, which stimulus enables the generation of a detectable signal upon molecular flow of said nucleic acid molecule or portion thereof. 22. The system of claim 20, wherein said membrane has a capacitance greater than about 5 fF/μm2 as measured across said membrane. 23. The system of claim 20, wherein said membrane has a resistance greater than or equal to about 500 MΩ as measured across said membrane. 24. The system of claim 23, wherein said resistance is measured with the aid of opposing electrodes disposed adjacent to said membrane. 25. The system of claim 20, wherein said membrane has a resistance less than or equal to about 1 GΩ across said membrane. 26. The system of claim 25, wherein said resistance is measured with the aid of opposing electrodes disposed adjacent to said membrane. 27. The system of claim 20, wherein each individually addressable nanopore is adapted to regulate molecular flow. 28. The system of claim 27, wherein each individually addressable nanopore is adapted to regulate molecular flow with the aid of an electrical stimulus applied to said nanopore. 29. The system of claim 28, wherein said electrical stimulus comprises one or more voltage pulses. 30. The system of claim 20, wherein each individually addressable nanopore is adapted to regulate molecular flow adjacent to said at least one nanopore. 31. The system of claim 20, wherein each individually addressable nanopore is adapted to regulate molecular flow through said at least one nanopore. 32. The system of claim 20, wherein each individually addressable nanopore is adapted to detect said nucleic acid molecule or a portion thereof upon molecular flow of said nucleic acid molecule or portion thereof through or adjacent to said at least one nanopore. 33. The system of claim 20, wherein said processor is in a workstation that is in proximity to said chip. 34. The system of claim 20, wherein said electrode is coupled to an integrated circuit that processes a signal detected with the aid of said electrode.

Devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore are provided. The devices and methods also determine (˜>50 Hz) the nucleotide base sequence of a polynucleotide under feedback control or using signals generated by the interactions between the polynucleotide and the nanopore. The invention is of particular use in the fields of molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.1. A method for nucleic acid sequencing, comprising: (a) providing a chip comprising a plurality of individually addressable nanopores, an individually addressable nanopore of said plurality of individually addressable nanopores containing at least one nanopore formed in a membrane disposed adjacent to an electrode, wherein each individually addressable nanopore is adapted to detect a nucleic acid molecule or a portion thereof; (b) directing a plurality of nucleic acid molecules to said individually addressable nanopores; and (c) characterizing, with the aid of a processor coupled to said chip, a nucleic acid sequence of each of said nucleic molecules based on electrical signals received from said plurality of individually addressable nanopores. 2. The method of claim 1, wherein said plurality of nucleic acid molecules are derived from a nucleic acid sample. 3. The method of claim 2, wherein each of said plurality of nucleic acid molecules has a shorter nucleic acid sequence than said nucleic acid sample. 4. The method of claim 3, further comprising, prior to (b), fragmenting said nucleic acid sample to provide said plurality of nucleic acid molecules. 5. The method of claim 2, further comprising characterizing a nucleic acid sequence of said nucleic acid sample based upon a characterization of a nucleic acid sequence of each of said nucleic molecules. 6. The method of claim 1, wherein said electrode is adapted to supply an electrical stimulus across said membrane, which stimulus enables the generation of a detectable signal upon molecular flow of said nucleic acid molecule or portion thereof. 7. The method of claim 1, wherein said membrane has a capacitance greater than about 5 fF/μm2 as measured across said membrane. 8. The method of claim 1, wherein said membrane has a resistance greater than or equal to about 500 MΩ as measured across said membrane. 9. The method of claim 8, wherein said resistance is measured with the aid of opposing electrodes disposed adjacent to said membrane. 10. The method of claim 1, wherein said membrane has a resistance less than or equal to about 1 GΩ across said membrane. 11. The method of claim 10, wherein said resistance is measured with the aid of opposing electrodes disposed adjacent to said membrane. 12. The method of claim 1, wherein each individually addressable nanopore is adapted to regulate molecular flow. 13. The method of claim 12, wherein each individually addressable nanopore is adapted to regulate molecular flow with the aid of an electrical stimulus applied to said nanopore. 14. The method of claim 13, wherein said electrical stimulus comprises one or more voltage pulses. 15. The method of claim 1, wherein each individually addressable nanopore is adapted to regulate molecular flow adjacent to said at least one nanopore. 16. The method of claim 1, wherein each individually addressable nanopore is adapted to regulate molecular flow through said at least one nanopore. 17. The method of claim 1, wherein each individually addressable nanopore is adapted to detect said nucleic acid molecule or a portion thereof upon molecular flow of said nucleic acid molecule or portion thereof through or adjacent to said at least one nanopore. 18. The method of claim 1, wherein said nucleic acid sequence is characterized upon movement of each of said plurality of nucleic acid molecules or portions thereof. 19. The method of claim 1, wherein said electrode is coupled to an integrated circuit that processes a signal detected with the aid of said electrode. 20. A system for nucleic acid sequencing, comprising: (a) a chip comprising a plurality of individually addressable nanopores, an individually addressable nanopore of said plurality of individually addressable nanopores containing at least one nanopore formed in a membrane disposed adjacent to an electrode, wherein each individually addressable nanopore is adapted to aid in the detection of said nucleic acid molecule or a portion; and (b) a processor coupled to said chip, wherein said processor is programmed to aid in characterizing a nucleic acid sequence of said nucleic acid molecule based on electrical signals received from said plurality of individually addressable nanopores. 21. The system of claim 20, wherein said electrode is adapted to supply an electrical stimulus across said membrane, which stimulus enables the generation of a detectable signal upon molecular flow of said nucleic acid molecule or portion thereof. 22. The system of claim 20, wherein said membrane has a capacitance greater than about 5 fF/μm2 as measured across said membrane. 23. The system of claim 20, wherein said membrane has a resistance greater than or equal to about 500 MΩ as measured across said membrane. 24. The system of claim 23, wherein said resistance is measured with the aid of opposing electrodes disposed adjacent to said membrane. 25. The system of claim 20, wherein said membrane has a resistance less than or equal to about 1 GΩ across said membrane. 26. The system of claim 25, wherein said resistance is measured with the aid of opposing electrodes disposed adjacent to said membrane. 27. The system of claim 20, wherein each individually addressable nanopore is adapted to regulate molecular flow. 28. The system of claim 27, wherein each individually addressable nanopore is adapted to regulate molecular flow with the aid of an electrical stimulus applied to said nanopore. 29. The system of claim 28, wherein said electrical stimulus comprises one or more voltage pulses. 30. The system of claim 20, wherein each individually addressable nanopore is adapted to regulate molecular flow adjacent to said at least one nanopore. 31. The system of claim 20, wherein each individually addressable nanopore is adapted to regulate molecular flow through said at least one nanopore. 32. The system of claim 20, wherein each individually addressable nanopore is adapted to detect said nucleic acid molecule or a portion thereof upon molecular flow of said nucleic acid molecule or portion thereof through or adjacent to said at least one nanopore. 33. The system of claim 20, wherein said processor is in a workstation that is in proximity to said chip. 34. The system of claim 20, wherein said electrode is coupled to an integrated circuit that processes a signal detected with the aid of said electrode.

The present invention generally relates to liquid compositions and methods for treating oral inflammation by administering a liquid composition to the oral cavity. The liquid composition is prepared from a powder containing calcium glycerophosphate, one or more sodium phosphate salts, sodium chloride, and optionally sodium bicarbonate and silica. The powder is mixed with a quality of water to form a liquid that is supersaturated with calcium ions and phosphate ions and is essentially free of visible particles and precipitate.

1. A powder adapted for producing a liquid composition for preventing or treating oral injury, oral inflammation, oral pain, chronic hyposalivation or complications therefrom, the powder comprising: calcium glycerophosphate or calcium lactate gluconate; a sodium phosphate; and sodium chloride. 2. The powder of claim 1, wherein the powder comprises calcium glycerophosphate in an amount at least about 5% w/w and at most 25% w/w of the powder. 3. The powder of claim 1, wherein the sodium phosphate comprises dibasic sodium phosphate anhydrous, present in an amount at least about 0.5% w/w and at most about 10% w/w of the powder, and monobasic sodium phosphate anhydrous, present in an amount at least about 0.3% w/w and at most about 6% w/w of the powder. 4. The powder of claim 3, wherein the dibasic sodium phosphate anhydrous is present in an amount at most about 4.5% w/w of the powder, and the monobasic sodium phosphate anhydrous is present in an amount at most about 2.5% w/w of the powder. 5. The powder of claim 1, further comprising a pH buffering agent. 6. The powder of claim 5, wherein the pH buffering agent is sodium bicarbonate, present in an amount at least about 1% w/w and at most about 7.5% w/w of the powder. 7. The powder of claim 6, wherein the liquid composition has a pH between about 6.5 and about 7.5. 8. The powder of claim 1, further comprising silica. 9. The powder of claim 1, wherein the powder comprises a concentration of calcium that is at least about 20,000 ppm, calculated based on weight percentages of solid components in the powder. 10. The powder of claim 1, wherein the composition comprises a concentration of phosphate that is at least about 60,000 ppm, calculated based on weight percentages of solid components in the powder. 11. The powder of claim 1, wherein the powder further comprises one or more of a fluoride salt, an analgesic, an antihistamine, a corticosteroid, an anti-microbial agent, an anti-fungal agent, a flavoring or a preservative. 12. A method for treating a subject having an oral cavity, the method comprising: mixing a powder to form a liquid composition, the powder comprising: calcium glycerophosphate or calcium lactate gluconate, a sodium phosphate, and sodium chloride; admitting the liquid composition into the oral cavity of the subject, wherein the subject is in need of treatment for one or more of oral injury, oral inflammation, oral pain, xerostomia, chronic hyposalivation or complications therefrom; moving the composition within the oral cavity; expelling the composition from the oral cavity of the subject. 13. The method of claim 12, wherein the subject is in need of treatment for xerostomia or chronic hyposalivation. 14. A liquid composition supersaturated in calcium ions and phosphate ions, the liquid composition being the product of mixing a powder with water, in a weight ratio of powder to water from about 0.005:1 about 0.1:1, wherein the powder comprises: calcium glycerophosphate or calcium lactate gluconate, a sodium phosphate, and sodium chloride. 15. The liquid composition of claim 14, wherein the liquid composition is essentially free of visible particles or precipitates.

The present invention generally relates to liquid compositions and methods for treating oral inflammation by administering a liquid composition to the oral cavity. The liquid composition is prepared from a powder containing calcium glycerophosphate, one or more sodium phosphate salts, sodium chloride, and optionally sodium bicarbonate and silica. The powder is mixed with a quality of water to form a liquid that is supersaturated with calcium ions and phosphate ions and is essentially free of visible particles and precipitate.1. A powder adapted for producing a liquid composition for preventing or treating oral injury, oral inflammation, oral pain, chronic hyposalivation or complications therefrom, the powder comprising: calcium glycerophosphate or calcium lactate gluconate; a sodium phosphate; and sodium chloride. 2. The powder of claim 1, wherein the powder comprises calcium glycerophosphate in an amount at least about 5% w/w and at most 25% w/w of the powder. 3. The powder of claim 1, wherein the sodium phosphate comprises dibasic sodium phosphate anhydrous, present in an amount at least about 0.5% w/w and at most about 10% w/w of the powder, and monobasic sodium phosphate anhydrous, present in an amount at least about 0.3% w/w and at most about 6% w/w of the powder. 4. The powder of claim 3, wherein the dibasic sodium phosphate anhydrous is present in an amount at most about 4.5% w/w of the powder, and the monobasic sodium phosphate anhydrous is present in an amount at most about 2.5% w/w of the powder. 5. The powder of claim 1, further comprising a pH buffering agent. 6. The powder of claim 5, wherein the pH buffering agent is sodium bicarbonate, present in an amount at least about 1% w/w and at most about 7.5% w/w of the powder. 7. The powder of claim 6, wherein the liquid composition has a pH between about 6.5 and about 7.5. 8. The powder of claim 1, further comprising silica. 9. The powder of claim 1, wherein the powder comprises a concentration of calcium that is at least about 20,000 ppm, calculated based on weight percentages of solid components in the powder. 10. The powder of claim 1, wherein the composition comprises a concentration of phosphate that is at least about 60,000 ppm, calculated based on weight percentages of solid components in the powder. 11. The powder of claim 1, wherein the powder further comprises one or more of a fluoride salt, an analgesic, an antihistamine, a corticosteroid, an anti-microbial agent, an anti-fungal agent, a flavoring or a preservative. 12. A method for treating a subject having an oral cavity, the method comprising: mixing a powder to form a liquid composition, the powder comprising: calcium glycerophosphate or calcium lactate gluconate, a sodium phosphate, and sodium chloride; admitting the liquid composition into the oral cavity of the subject, wherein the subject is in need of treatment for one or more of oral injury, oral inflammation, oral pain, xerostomia, chronic hyposalivation or complications therefrom; moving the composition within the oral cavity; expelling the composition from the oral cavity of the subject. 13. The method of claim 12, wherein the subject is in need of treatment for xerostomia or chronic hyposalivation. 14. A liquid composition supersaturated in calcium ions and phosphate ions, the liquid composition being the product of mixing a powder with water, in a weight ratio of powder to water from about 0.005:1 about 0.1:1, wherein the powder comprises: calcium glycerophosphate or calcium lactate gluconate, a sodium phosphate, and sodium chloride. 15. The liquid composition of claim 14, wherein the liquid composition is essentially free of visible particles or precipitates.

The sagging problem in the manufacture of thick walled pipes is solved using a polyethylene molding composition having a multimodal molecular mass distribution and comprising from 45 to 55% by weight of a first low molecular weight ethylene homopolymer A, from 20 to 40% by weight of a second high molecular weight copolymer B comprising ethylene and another olefin having from 4 to 8 carbon atoms and from 15 to 30% by weight of a third ultrahigh molecular weight ethylene copolymer C prepared in the presence of a Ziegler catalyst in a three-stage polymerization process comprising additionally an organic polyoxy compound or an organic polyhydroxy compound in an amount of from 0.01 to 0.5% by weight.

1. A polyethylene molding composition having a multimodal molecular mass distribution for producing pipes, which comprises from 45 to 55% by weight of a first ethylene homopolymer A, from 20 to 40% by weight of a second copolymer B comprising ethylene and another olefin having from 4 to 8 carbon atoms and from 15 to 30% by weight of a third ethylene copolymer C, where all percentages are based on the total weight of the molding composition and comprising additionally an amount of from 0.01 to 0.5% by weight, based on the total weight of the molding composition, of an organic polyoxy compound having the general chemical formula: R—[(CH2)n—O]m—H where n is an integer in the range from 1 to 10, m is an integer in the range from 3 to 500 and R is a hydrogen atom or an OH group or an alkyl group which has from 1 to 10 carbon atoms and may bear further substituents such as —OH, —COOH, —COOR, —OCH3 or —OC2H5, or an organic polyhydroxy compound having the general chemical formula: RO—CH2—C—(CH2—OR)3 where R can be a hydrogen atom or an alkyl group which has from 1 to 5 carbon atoms and may bear further substituents such as —OH, —COOH, —COOR, —OCH3 or —OC2H5, or a combination of the two. 2. The polyethylene molding composition according to claim 1 which has a density at a temperature of 23° C. in the range from 0.945 to 0.957 g/cm3. 3. The polyethylene molding composition according to claim 1, wherein the second copolymer B comprises from 1 to 8% by weight, based on the weight of the second copolymer B, of further olefin monomer units having from 4 to 8 carbon atoms. 4. The polyethylene molding composition according to claim 1, wherein the third ethylene copolymer C comprises from 1 to 8% by weight, based on the weight of the third ethylene copolymer C, of one or more comonomers having from 4 to 8 carbon atoms. 5. The polyethylene molding composition according to claim 1 which has a melt flow index in accordance with ISO 1133, expressed as MFI190/5, in the range from 0.1 to 0.8 dg/min, preferably from 0.1 to 0.5 dg/min. 6. The polyethylene molding composition according to claim 1 which has a viscosity number VNtot, measured in accordance with ISO/R 1191 in decalin at a temperature of 135° C., in the range from 200 to 600 cm3/g, preferably from 250 to 550 cm3/g, particularly preferably from 350 to 490 cm3/g. 7. The polyethylene molding composition according to claim 1 comprising as organic polyoxy compounds polyethylene glycol, methoxypolyethylene glycol or polypropylene glycol, preferably having a mean molar mass in the range from 400 to 9000 g/mol, in an amount in the range of from 0.02 to 0.4% by weight, particularly preferably from 0.1 to 0.3% by weight. 8. The polyethylene molding composition according to claim 1 comprising as organic polyhydroxy compounds pentaerythritol, trimethylolpropane, glycerol, mannitol or sorbitol in an amount in the range of from 0.02 to 0.4% by weight, particularly preferably from 0.1 to 0.3% by weight. 9. A process for preparing a polyethylene molding composition according to claim 1 which comprises carrying out the polymerization of the monomers in suspension at temperatures in the range from 70 to 100° C., preferably from 75 to 90° C., under a pressure in the range from 2 to 10 bar and in the presence of a highly active Ziegler catalyst which is composed of a transition metal compound and an organoaluminum compound and carrying out the polymerization in three stages in three reactors connected in series, with the molar mass of the polyethylene prepared in the respective stage being set in each case by means of hydrogen. 10. A pipe comprising a polyethylene molding composition according to claim 1 which has an environmental stress cracking resistance, expressed as the FNCT value, of greater than 1500 h, preferably greater than 2000 h, particularly preferably greater than 2500 h, and has a notched impact toughness in accordance with DIN 53453 at −30° C. of greater than 12.5 kJ/m2.

The sagging problem in the manufacture of thick walled pipes is solved using a polyethylene molding composition having a multimodal molecular mass distribution and comprising from 45 to 55% by weight of a first low molecular weight ethylene homopolymer A, from 20 to 40% by weight of a second high molecular weight copolymer B comprising ethylene and another olefin having from 4 to 8 carbon atoms and from 15 to 30% by weight of a third ultrahigh molecular weight ethylene copolymer C prepared in the presence of a Ziegler catalyst in a three-stage polymerization process comprising additionally an organic polyoxy compound or an organic polyhydroxy compound in an amount of from 0.01 to 0.5% by weight.1. A polyethylene molding composition having a multimodal molecular mass distribution for producing pipes, which comprises from 45 to 55% by weight of a first ethylene homopolymer A, from 20 to 40% by weight of a second copolymer B comprising ethylene and another olefin having from 4 to 8 carbon atoms and from 15 to 30% by weight of a third ethylene copolymer C, where all percentages are based on the total weight of the molding composition and comprising additionally an amount of from 0.01 to 0.5% by weight, based on the total weight of the molding composition, of an organic polyoxy compound having the general chemical formula: R—[(CH2)n—O]m—H where n is an integer in the range from 1 to 10, m is an integer in the range from 3 to 500 and R is a hydrogen atom or an OH group or an alkyl group which has from 1 to 10 carbon atoms and may bear further substituents such as —OH, —COOH, —COOR, —OCH3 or —OC2H5, or an organic polyhydroxy compound having the general chemical formula: RO—CH2—C—(CH2—OR)3 where R can be a hydrogen atom or an alkyl group which has from 1 to 5 carbon atoms and may bear further substituents such as —OH, —COOH, —COOR, —OCH3 or —OC2H5, or a combination of the two. 2. The polyethylene molding composition according to claim 1 which has a density at a temperature of 23° C. in the range from 0.945 to 0.957 g/cm3. 3. The polyethylene molding composition according to claim 1, wherein the second copolymer B comprises from 1 to 8% by weight, based on the weight of the second copolymer B, of further olefin monomer units having from 4 to 8 carbon atoms. 4. The polyethylene molding composition according to claim 1, wherein the third ethylene copolymer C comprises from 1 to 8% by weight, based on the weight of the third ethylene copolymer C, of one or more comonomers having from 4 to 8 carbon atoms. 5. The polyethylene molding composition according to claim 1 which has a melt flow index in accordance with ISO 1133, expressed as MFI190/5, in the range from 0.1 to 0.8 dg/min, preferably from 0.1 to 0.5 dg/min. 6. The polyethylene molding composition according to claim 1 which has a viscosity number VNtot, measured in accordance with ISO/R 1191 in decalin at a temperature of 135° C., in the range from 200 to 600 cm3/g, preferably from 250 to 550 cm3/g, particularly preferably from 350 to 490 cm3/g. 7. The polyethylene molding composition according to claim 1 comprising as organic polyoxy compounds polyethylene glycol, methoxypolyethylene glycol or polypropylene glycol, preferably having a mean molar mass in the range from 400 to 9000 g/mol, in an amount in the range of from 0.02 to 0.4% by weight, particularly preferably from 0.1 to 0.3% by weight. 8. The polyethylene molding composition according to claim 1 comprising as organic polyhydroxy compounds pentaerythritol, trimethylolpropane, glycerol, mannitol or sorbitol in an amount in the range of from 0.02 to 0.4% by weight, particularly preferably from 0.1 to 0.3% by weight. 9. A process for preparing a polyethylene molding composition according to claim 1 which comprises carrying out the polymerization of the monomers in suspension at temperatures in the range from 70 to 100° C., preferably from 75 to 90° C., under a pressure in the range from 2 to 10 bar and in the presence of a highly active Ziegler catalyst which is composed of a transition metal compound and an organoaluminum compound and carrying out the polymerization in three stages in three reactors connected in series, with the molar mass of the polyethylene prepared in the respective stage being set in each case by means of hydrogen. 10. A pipe comprising a polyethylene molding composition according to claim 1 which has an environmental stress cracking resistance, expressed as the FNCT value, of greater than 1500 h, preferably greater than 2000 h, particularly preferably greater than 2500 h, and has a notched impact toughness in accordance with DIN 53453 at −30° C. of greater than 12.5 kJ/m2.

TIM1 antagonists reduce GVHD by symptom score and show statistically significant improved survival.

1. A method for treating a graft versus host disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an antagonist of TIM-1. 2. The method of claim 1, wherein the antagonist is an antibody. 3. The method of claim 2, wherein the graft versus host disease is selected from the group consisting of acute graft-versus-host-disease (aGVHD) and chronic graft-versus-host-disease (cGVHD). 4. The method of claim 1, wherein graft cells are contacted with the TIM-1 antagonist prior to transplantation. 5. The method of claim 1, wherein the TIM-1 antagonist is administered following transplantation of the graft.

TIM1 antagonists reduce GVHD by symptom score and show statistically significant improved survival.1. A method for treating a graft versus host disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an antagonist of TIM-1. 2. The method of claim 1, wherein the antagonist is an antibody. 3. The method of claim 2, wherein the graft versus host disease is selected from the group consisting of acute graft-versus-host-disease (aGVHD) and chronic graft-versus-host-disease (cGVHD). 4. The method of claim 1, wherein graft cells are contacted with the TIM-1 antagonist prior to transplantation. 5. The method of claim 1, wherein the TIM-1 antagonist is administered following transplantation of the graft.

Disclosed herein are methods and compositions for treating learning and memory deficits associated with Noonan Syndrome.

1. A method of treating a cognitive deficit in a subject having Noonan syndrome, comprising: administering an effective amount of one or more hydroxymethylglutaryl CoA (HMG CoA) reductase inhibitors to a subject having the cognitive deficit and Noonan syndrome. 2-3. (canceled) 4. The method of claim 1, wherein the one or more HMG CoA inhibitors comprises a statin. 5. The method of claim 4, wherein the statin is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, pravastatin, pitavastatin, rovustatin, simvastatin, and mixtures thereof. 6. The method according to claim 1, wherein the effective amount does not significantly lower total serum cholesterol level in the subject. 7. The method according to claim 1, which further comprises administering a supplementary compound such as a farnesyl transferase inhibitor, a geranylgeranyl transferase inhibitor, an inhibitor of γ-aminobutyric acid (GABA) mediated inhibition, an inhibitor of GABA receptor activity and/or a modulator of Ras-ERK signaling. 8. The method according to claim 1, wherein the supplementary compound is administered before, during, and/or after the inhibitor. 9. The method according to claim 1, wherein the administering is by adjunctive administration. 10. The method of claim 9, wherein the adjunctive administration is simultaneous administration. 11. The method of claim 9, wherein the adjunctive administration is sequential administration. 12. The method of claim 1, wherein said subject has a normal cholesterol level. 13. The method of claim 1, which further comprises administering an effective amount of an inhibitor of MEK to the subject. 14-16. (canceled) 17. The method of claim 13, wherein the inhibitor of MEK is a specific inhibitor of MEK1 and/or MEK2. 18. The method of claim 13, wherein the inhibitor of MEK is SL327. 19. The method of claim 13, wherein the one or more hydroxymethylglutaryl CoA (HMG CoA) reductase inhibitors are administered before, during, and/or after administration of the inhibitor of MEK.

Disclosed herein are methods and compositions for treating learning and memory deficits associated with Noonan Syndrome.1. A method of treating a cognitive deficit in a subject having Noonan syndrome, comprising: administering an effective amount of one or more hydroxymethylglutaryl CoA (HMG CoA) reductase inhibitors to a subject having the cognitive deficit and Noonan syndrome. 2-3. (canceled) 4. The method of claim 1, wherein the one or more HMG CoA inhibitors comprises a statin. 5. The method of claim 4, wherein the statin is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, pravastatin, pitavastatin, rovustatin, simvastatin, and mixtures thereof. 6. The method according to claim 1, wherein the effective amount does not significantly lower total serum cholesterol level in the subject. 7. The method according to claim 1, which further comprises administering a supplementary compound such as a farnesyl transferase inhibitor, a geranylgeranyl transferase inhibitor, an inhibitor of γ-aminobutyric acid (GABA) mediated inhibition, an inhibitor of GABA receptor activity and/or a modulator of Ras-ERK signaling. 8. The method according to claim 1, wherein the supplementary compound is administered before, during, and/or after the inhibitor. 9. The method according to claim 1, wherein the administering is by adjunctive administration. 10. The method of claim 9, wherein the adjunctive administration is simultaneous administration. 11. The method of claim 9, wherein the adjunctive administration is sequential administration. 12. The method of claim 1, wherein said subject has a normal cholesterol level. 13. The method of claim 1, which further comprises administering an effective amount of an inhibitor of MEK to the subject. 14-16. (canceled) 17. The method of claim 13, wherein the inhibitor of MEK is a specific inhibitor of MEK1 and/or MEK2. 18. The method of claim 13, wherein the inhibitor of MEK is SL327. 19. The method of claim 13, wherein the one or more hydroxymethylglutaryl CoA (HMG CoA) reductase inhibitors are administered before, during, and/or after administration of the inhibitor of MEK.

The present invention relates to apparatus, methods, and applications for treating wastewater, and more particularly to biological processes for removing pollutants from wastewater. This invention further relates to apparatus and methods for growing microbes on-site at a wastewater treatment facility, and for economically inoculating sufficient microbes to solve various treatment problems rapidly

1-72. (canceled) 73. A method of removing contaminants from soil, comprising: providing an on-site system for growing of microbes at the location of the contaminated soil; depositing inoculum, nutrient, and water into the on-site system; growing the inoculum in the on-site system to provide a treatment batch comprising an increased number of microbes, the growing comprising heating and mixing the inoculum in the on-site system; and directly applying at least a portion of the treatment batch, said portion containing microbes, to a contaminated soil surface. 74-95. (canceled) 96. The method of claim 73, wherein the microbes are in gelatin capsules. 97. The method of claim 73, wherein the nutrient is in gelatin capsules. 98. The method of claim 73, wherein the nutrient is in water soluble bags. 99. The method of claim 73, wherein growing the inoculum is for about 8 to 24 hours. 100. The method of claim 73, wherein the on-site system comprises at least one growth tank, aeration, and a controller. 101. The method of claim 100, wherein the on-site system comprises at least two growth tanks. 102. The method of claim 100, wherein the growth tank holds from about 250 gallons to about 1000 gallons. 103. The method of claim 100, wherein the growth tank holds from about 0.25 gallons to about 250 gallons. 104. The method of claim 100, wherein the growth tank holds about 1000 gallons. 105. The method of claim 73, wherein the step of applying comprises releasing the portion of the treatment batch from the on-site system directly onto the contaminated soil surface. 106. The method of claim 73, wherein the step of growing further comprises aerating the inoculum. 107. The method of claim 73, comprising depositing a defoamer. 108. A kit for bioremediation of contaminated soil comprising: at least one growth tank; an inoculum comprising microbes; and nutrients. 109. The kit of claim 108, comprising a heater. 110. The kit of claim 108, comprising an aeration pump. 111. The kit of claim 110, wherein the aeration pump is exterior to the at least one growth tank. 112. The kit of claim 108, comprising a diffuser. 113. The kit of claim 108, wherein the kit is manual. 114. The kit of claim 108, wherein the microbes are in capsules. 115. The kit of claim 114, wherein the capsules are gelatin capsules. 116. The kit of claim 108, wherein the microbes are custom blended. 117. The kit of claim 108, wherein the nutrients are in gelatin capsules. 118. The kit of claim 108, wherein the nutrients are in water-soluble bags. 119. The kit of claim 108, wherein the growth tank holds from about 0.25 gallons to about 250 gallons. 120. The kit of claim 108, wherein the growth tank comprises an about 5-gallon container. 121. The kit of claim 108, wherein the growth tank comprises at least two growth tanks. 122. The kit of claim 108, comprising a cleaning agent. 123. A kit for bioremediation of contaminated soil comprising: at least one growth tank holding about 5 gallons; an inoculum comprising microbes, wherein the microbes are in gelatin capsules; nutrients in gelatin capsules; a heater; and an aeration pump.

The present invention relates to apparatus, methods, and applications for treating wastewater, and more particularly to biological processes for removing pollutants from wastewater. This invention further relates to apparatus and methods for growing microbes on-site at a wastewater treatment facility, and for economically inoculating sufficient microbes to solve various treatment problems rapidly1-72. (canceled) 73. A method of removing contaminants from soil, comprising: providing an on-site system for growing of microbes at the location of the contaminated soil; depositing inoculum, nutrient, and water into the on-site system; growing the inoculum in the on-site system to provide a treatment batch comprising an increased number of microbes, the growing comprising heating and mixing the inoculum in the on-site system; and directly applying at least a portion of the treatment batch, said portion containing microbes, to a contaminated soil surface. 74-95. (canceled) 96. The method of claim 73, wherein the microbes are in gelatin capsules. 97. The method of claim 73, wherein the nutrient is in gelatin capsules. 98. The method of claim 73, wherein the nutrient is in water soluble bags. 99. The method of claim 73, wherein growing the inoculum is for about 8 to 24 hours. 100. The method of claim 73, wherein the on-site system comprises at least one growth tank, aeration, and a controller. 101. The method of claim 100, wherein the on-site system comprises at least two growth tanks. 102. The method of claim 100, wherein the growth tank holds from about 250 gallons to about 1000 gallons. 103. The method of claim 100, wherein the growth tank holds from about 0.25 gallons to about 250 gallons. 104. The method of claim 100, wherein the growth tank holds about 1000 gallons. 105. The method of claim 73, wherein the step of applying comprises releasing the portion of the treatment batch from the on-site system directly onto the contaminated soil surface. 106. The method of claim 73, wherein the step of growing further comprises aerating the inoculum. 107. The method of claim 73, comprising depositing a defoamer. 108. A kit for bioremediation of contaminated soil comprising: at least one growth tank; an inoculum comprising microbes; and nutrients. 109. The kit of claim 108, comprising a heater. 110. The kit of claim 108, comprising an aeration pump. 111. The kit of claim 110, wherein the aeration pump is exterior to the at least one growth tank. 112. The kit of claim 108, comprising a diffuser. 113. The kit of claim 108, wherein the kit is manual. 114. The kit of claim 108, wherein the microbes are in capsules. 115. The kit of claim 114, wherein the capsules are gelatin capsules. 116. The kit of claim 108, wherein the microbes are custom blended. 117. The kit of claim 108, wherein the nutrients are in gelatin capsules. 118. The kit of claim 108, wherein the nutrients are in water-soluble bags. 119. The kit of claim 108, wherein the growth tank holds from about 0.25 gallons to about 250 gallons. 120. The kit of claim 108, wherein the growth tank comprises an about 5-gallon container. 121. The kit of claim 108, wherein the growth tank comprises at least two growth tanks. 122. The kit of claim 108, comprising a cleaning agent. 123. A kit for bioremediation of contaminated soil comprising: at least one growth tank holding about 5 gallons; an inoculum comprising microbes, wherein the microbes are in gelatin capsules; nutrients in gelatin capsules; a heater; and an aeration pump.

The invention provides Tagetes patula ray florets comprising cyanidin-3-rutinoside, cyanidin-3-glucoside, petunidin-3-glucoside, and cyanidin in the lower epidermal layers. The invention also provides Tagetes plants comprising a mutant prdr1-1 allele and methods for producing a plant produced by crossing such plants with themselves or with another plant, such as a plant of another genotype. The invention further relates to seeds and plants produced by crossing Tagetes plants comprising a mutant prdr1-1 allele. The invention further relates to parts of such plants.

1. A Tagetes patula ray floret comprising cyanidin-3-rutinoside, cyanidin-3-glucoside, petunidin-3-glucoside, and cyanidin in the lower epidermal layers of the ray floret. 2. The ray floret of claim 1, further comprising a prdr1-1 allele. 3. The ray floret of claim 2, wherein said prdr1-1 allele confers a red color in the lower epidermal layers of the ray floret. 4. A plant comprising the ray floret of claim 3. 5. A seed that produces the plant of claim 4. 6. The ray floret of claim 1, further comprising a transgene. 7. The ray floret of claim 1, further comprising a single locus conversion. 8. A Tagetes patula plant comprising a prdr1-1 allele that confers to the plant an anthocyanin pigment in the lower epidermal layers of the ray floret, wherein a representative deposit of seed comprising said allele has been deposited under ATCC Accession No. PTA-121614. 9. The plant of claim 8, wherein the plant is homozygous for the allele. 10. The plant of claim 8, wherein the plant is heterozygous for the allele. 11. The plant of claim 8, wherein the plant is hybrid. 12. The plant of claim 8, wherein the plant is inbred. 13. The plant of claim 8, wherein said anthocyanin pigment confers a red color in the lower epidermal layers of the ray floret. 14. The plant of claim 13, wherein the anthocyanin conferring the red color is petunidin-3-glucoside. 15. The plant of claim 8, wherein the plant comprises a transgene. 16. The plant of claim 8, wherein the plant comprises a single locus conversion. 17. A plant part comprising a cell of the plant of claim 8. 18. The plant part of claim 17, further defined as a cutting, leaf, a floret, an ovule, pollen, or a flower. 19. A seed that produces the plant of claim 8. 20. A tissue culture of regenerable cells of the plant of claim 8. 21. The tissue culture according to claim 20, comprising cells or protoplasts from a plant part selected from the group consisting of embryos, meristems, cotyledons, pollen, leaves, anthers, roots, root tips, pistil, flower, florets, seed, stems, and protoplasts or callus derived therefrom. 22. A plant regenerated from the tissue culture of claim 20, wherein the regenerated plant comprises the prdr1-1 allele. 23. A method of introducing a desired trait into a plant comprising: (a) crossing a plant according to claim 2 with a second plant that comprises a desired trait to produce F1 progeny; (b) selecting an F1 progeny that comprises the desired trait; (c) crossing the selected F1 progeny with a plant of the same variety as said plant according to claim 2 to produce backcross progeny; and (d) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait. 24. A plant produced by the method of claim 23, wherein the plant comprises said prdr1-1 allele. 25. A method of producing a plant comprising an added desired trait, the method comprising introducing a transgene or single locus conversion conferring the desired trait into a plant according to claim 8. 26. A method for producing Tagetes seed comprising the steps of: (a) crossing a plant according to claim 2 with itself or a second plant capable of being crossed thereto; and (b) collecting resulting seed. 27. The method of claim 26, further comprising the steps of: (c) crossing a plant grown from said seed of step (b) with itself or a different plant at least one additional time to yield additional seed. 28. The method of claim 26, wherein the second plant is a Tagetes erecta plant. 29. The method of claim 26, wherein the plant according to claim 2 is a plant of Tagetes variety TAS1146954′, a sample of seed of said Tagetes variety having been deposited under ATCC Accession Number PTA-121614. 30. A method of producing a Tagetes plant with a prdr1-1 allele that confers the presence of anthocyanin pigment in the lower epidermal layers of the ray floret, said method comprising introgressing the prdr1-1 allele from a plant according to claim 8 into a plant of a different genotype. 31. The method of claim 30, wherein said prdr1-1 allele has been inherited from Tagetes variety TAS1146954′ or a progeny of any generation thereof comprising said allele, a sample of seed comprising the allele having been deposited under ATCC Accession Number PTA-121614. 32. An F1 hybrid seed having a plant of claim 8 as one parent. 33. The F1 hybrid seed of claim 32, wherein said plant is a male parent. 34. The F1 hybrid seed of claim 32, wherein said plant is a female parent. 35. A plant produced by growing the seed of claim 32, wherein the plant comprises the prdr1-1 allele. 36. A plant part comprising a cell of the plant of claim 35. 37. The plant part of claim 36, further defined as a cutting, leaf, a floret, an ovule, pollen, or a flower.

The invention provides Tagetes patula ray florets comprising cyanidin-3-rutinoside, cyanidin-3-glucoside, petunidin-3-glucoside, and cyanidin in the lower epidermal layers. The invention also provides Tagetes plants comprising a mutant prdr1-1 allele and methods for producing a plant produced by crossing such plants with themselves or with another plant, such as a plant of another genotype. The invention further relates to seeds and plants produced by crossing Tagetes plants comprising a mutant prdr1-1 allele. The invention further relates to parts of such plants.1. A Tagetes patula ray floret comprising cyanidin-3-rutinoside, cyanidin-3-glucoside, petunidin-3-glucoside, and cyanidin in the lower epidermal layers of the ray floret. 2. The ray floret of claim 1, further comprising a prdr1-1 allele. 3. The ray floret of claim 2, wherein said prdr1-1 allele confers a red color in the lower epidermal layers of the ray floret. 4. A plant comprising the ray floret of claim 3. 5. A seed that produces the plant of claim 4. 6. The ray floret of claim 1, further comprising a transgene. 7. The ray floret of claim 1, further comprising a single locus conversion. 8. A Tagetes patula plant comprising a prdr1-1 allele that confers to the plant an anthocyanin pigment in the lower epidermal layers of the ray floret, wherein a representative deposit of seed comprising said allele has been deposited under ATCC Accession No. PTA-121614. 9. The plant of claim 8, wherein the plant is homozygous for the allele. 10. The plant of claim 8, wherein the plant is heterozygous for the allele. 11. The plant of claim 8, wherein the plant is hybrid. 12. The plant of claim 8, wherein the plant is inbred. 13. The plant of claim 8, wherein said anthocyanin pigment confers a red color in the lower epidermal layers of the ray floret. 14. The plant of claim 13, wherein the anthocyanin conferring the red color is petunidin-3-glucoside. 15. The plant of claim 8, wherein the plant comprises a transgene. 16. The plant of claim 8, wherein the plant comprises a single locus conversion. 17. A plant part comprising a cell of the plant of claim 8. 18. The plant part of claim 17, further defined as a cutting, leaf, a floret, an ovule, pollen, or a flower. 19. A seed that produces the plant of claim 8. 20. A tissue culture of regenerable cells of the plant of claim 8. 21. The tissue culture according to claim 20, comprising cells or protoplasts from a plant part selected from the group consisting of embryos, meristems, cotyledons, pollen, leaves, anthers, roots, root tips, pistil, flower, florets, seed, stems, and protoplasts or callus derived therefrom. 22. A plant regenerated from the tissue culture of claim 20, wherein the regenerated plant comprises the prdr1-1 allele. 23. A method of introducing a desired trait into a plant comprising: (a) crossing a plant according to claim 2 with a second plant that comprises a desired trait to produce F1 progeny; (b) selecting an F1 progeny that comprises the desired trait; (c) crossing the selected F1 progeny with a plant of the same variety as said plant according to claim 2 to produce backcross progeny; and (d) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny that comprise the desired trait. 24. A plant produced by the method of claim 23, wherein the plant comprises said prdr1-1 allele. 25. A method of producing a plant comprising an added desired trait, the method comprising introducing a transgene or single locus conversion conferring the desired trait into a plant according to claim 8. 26. A method for producing Tagetes seed comprising the steps of: (a) crossing a plant according to claim 2 with itself or a second plant capable of being crossed thereto; and (b) collecting resulting seed. 27. The method of claim 26, further comprising the steps of: (c) crossing a plant grown from said seed of step (b) with itself or a different plant at least one additional time to yield additional seed. 28. The method of claim 26, wherein the second plant is a Tagetes erecta plant. 29. The method of claim 26, wherein the plant according to claim 2 is a plant of Tagetes variety TAS1146954′, a sample of seed of said Tagetes variety having been deposited under ATCC Accession Number PTA-121614. 30. A method of producing a Tagetes plant with a prdr1-1 allele that confers the presence of anthocyanin pigment in the lower epidermal layers of the ray floret, said method comprising introgressing the prdr1-1 allele from a plant according to claim 8 into a plant of a different genotype. 31. The method of claim 30, wherein said prdr1-1 allele has been inherited from Tagetes variety TAS1146954′ or a progeny of any generation thereof comprising said allele, a sample of seed comprising the allele having been deposited under ATCC Accession Number PTA-121614. 32. An F1 hybrid seed having a plant of claim 8 as one parent. 33. The F1 hybrid seed of claim 32, wherein said plant is a male parent. 34. The F1 hybrid seed of claim 32, wherein said plant is a female parent. 35. A plant produced by growing the seed of claim 32, wherein the plant comprises the prdr1-1 allele. 36. A plant part comprising a cell of the plant of claim 35. 37. The plant part of claim 36, further defined as a cutting, leaf, a floret, an ovule, pollen, or a flower.

The present invention relates to a method for the detection of a specific nucleic acid. Specifically, the invention provides a method and kits for detecting the presence of a specific nucleic acid using engineered DNA-binding domains such as Transcription Activator Like-Effector (TALE) domain or modular base-per-base binding domains (MBBBD). The method of the invention is particularly useful for in vitro diagnostic application.

1. A method of detecting a nucleic acid sequence of interest comprising: (a) Providing nucleic acids which can comprise the nucleic acid sequence of interest; (b) Providing at least one DNA binding domain capable of binding said nucleic acid sequence of interest; (c) Contacting said nucleic acids with said DNA binding domain; (d) Removing unbound said DNA binding domain and nucleic acids and; (e) Detecting the presence or the absence of the nucleic acid sequence of interest. 2. The method of claim 1 further comprising quantifying said nucleic acid of interest. 3. The method of claim 1 for detecting a single strand nucleic acid of interest (pray) comprising: providing a nucleic acid sequence (bait) comprising a complementary sequence to a part of said pray sequence; hybridizing said pray and bait sequences such that said DNA binding domain can bind the hybridized pray: bait sequence. 4. The method according to claim 1 for detecting a specific nucleic acid wherein said nucleic acid is a DNA, RNA or DNA/RNA heteroduplex. 5. The method according to claim 1 wherein said DNA binding domain is derived from a TALE or MBBBD protein. 6. The method of detecting a nucleic acid sequence according to claim 1, wherein said nucleic acids are in a biological sample. 7. The method according to claim 1 wherein said nucleic acids are within a cell. 8. The method according to claim 1 wherein the DNA binding domain is fused to a reporter molecule. 9. The method of claim 8 wherein the reporter molecule is selected from the group consisting of: fluorescent protein, chemiluminescent molecule, tag and enzyme. 10. The method according to claim 3 wherein the bait sequence is labeled. 11. The method according to claim 3 wherein the DNA-binding domain and/or said bait sequence are immobilized on a solid support. 12. A kit comprising a DNA binding domain specific to a nucleic acid sequence of interest to detect. 13. The kit of claim 12 wherein said DNA binding domain is fused to a reporter molecule. 14. A diagnostic kit of claim 12 comprising a DNA binding domain specific to a genetic abnormality in a subject. 15. A diagnostic kit of claim 12 comprising a DNA binding domain specific to a nucleic acid sequence comprising a single nucleotide polymorphism present in a subject. 16. A diagnostic kit of claim 12 comprising a DNA binding domain specific to a nucleic acid sequence comprising a unique marker for a specific type of cancer.

The present invention relates to a method for the detection of a specific nucleic acid. Specifically, the invention provides a method and kits for detecting the presence of a specific nucleic acid using engineered DNA-binding domains such as Transcription Activator Like-Effector (TALE) domain or modular base-per-base binding domains (MBBBD). The method of the invention is particularly useful for in vitro diagnostic application.1. A method of detecting a nucleic acid sequence of interest comprising: (a) Providing nucleic acids which can comprise the nucleic acid sequence of interest; (b) Providing at least one DNA binding domain capable of binding said nucleic acid sequence of interest; (c) Contacting said nucleic acids with said DNA binding domain; (d) Removing unbound said DNA binding domain and nucleic acids and; (e) Detecting the presence or the absence of the nucleic acid sequence of interest. 2. The method of claim 1 further comprising quantifying said nucleic acid of interest. 3. The method of claim 1 for detecting a single strand nucleic acid of interest (pray) comprising: providing a nucleic acid sequence (bait) comprising a complementary sequence to a part of said pray sequence; hybridizing said pray and bait sequences such that said DNA binding domain can bind the hybridized pray: bait sequence. 4. The method according to claim 1 for detecting a specific nucleic acid wherein said nucleic acid is a DNA, RNA or DNA/RNA heteroduplex. 5. The method according to claim 1 wherein said DNA binding domain is derived from a TALE or MBBBD protein. 6. The method of detecting a nucleic acid sequence according to claim 1, wherein said nucleic acids are in a biological sample. 7. The method according to claim 1 wherein said nucleic acids are within a cell. 8. The method according to claim 1 wherein the DNA binding domain is fused to a reporter molecule. 9. The method of claim 8 wherein the reporter molecule is selected from the group consisting of: fluorescent protein, chemiluminescent molecule, tag and enzyme. 10. The method according to claim 3 wherein the bait sequence is labeled. 11. The method according to claim 3 wherein the DNA-binding domain and/or said bait sequence are immobilized on a solid support. 12. A kit comprising a DNA binding domain specific to a nucleic acid sequence of interest to detect. 13. The kit of claim 12 wherein said DNA binding domain is fused to a reporter molecule. 14. A diagnostic kit of claim 12 comprising a DNA binding domain specific to a genetic abnormality in a subject. 15. A diagnostic kit of claim 12 comprising a DNA binding domain specific to a nucleic acid sequence comprising a single nucleotide polymorphism present in a subject. 16. A diagnostic kit of claim 12 comprising a DNA binding domain specific to a nucleic acid sequence comprising a unique marker for a specific type of cancer.

Described are transdermal drug delivery systems for the transdermal administration of agomelatine. Methods of making and using such systems also are described.

1. A transdermal drug delivery system for the transdermal delivery of agomelatine in the form of a flexible, finite system, comprising a composition comprising agomelatine and an enhancer. 2. The transdermal drug delivery system of claim 1, wherein the enhancer is selected from the group consisting of isopropanol and ethanol. 3. The transdermal drug delivery system of claim 1, wherein the composition comprises an amount of the enhancer effective to achieve substantially complete drug delivery within 12 hours of application or less. 4. The transdermal drug delivery system of claim 1, wherein the composition comprises an amount of the enhancer effective to achieve at least about 60% of the agomelatine delivery within about 4 to about 6 hours of application. 5. The transdermal drug delivery system of claim 1, wherein the composition comprises an amount of the enhancer effective to achieve at least about 75% of the agomelatine delivery within about 4 to about 6 hours of application. 6. The transdermal drug delivery system of claim 1, wherein the composition comprises an amount of the enhancer effective to achieve at least about 80% of the agomelatine delivery within about 6 to about 8 hours of application. 7. The transdermal drug delivery system of claim 1, wherein the composition is a drug-in-solution reservoir-type composition comprising agomelatine and aqueous isopropanol. 8. The transdermal drug delivery system of claim 7, wherein the aqueous isopropanol comprises 50-75% v/v isopropanol. 9. The transdermal drug delivery system of claim 7, wherein the agomelatine is present at its saturation concentration. 10. The transdermal drug delivery system of claim 7, wherein the composition comprises at least 50 mg/mL agomelatine. 11. The transdermal drug delivery system of claim 1, wherein the composition is a drug-in-gel reservoir-type composition comprising agomelatine, aqueous isopropanol, and a gelling agent. 12. The transdermal drug delivery system of claim 11, wherein the aqueous isopropanol comprises 50-75% v/v isopropanol. 13. The transdermal drug delivery system of claim 11, wherein the agomelatine is present at its saturation concentration. 14. The transdermal drug delivery system of claim 11, wherein the composition comprises at least 50 mg/mL agomelatine. 15. The transdermal drug delivery system of claim 1, wherein the composition is a drug-in-polymer matrix composition comprising agomelatine formulated in a polymer matrix comprising isopropanol. 16. The transdermal drug delivery system of claim 15, wherein the polymer matrix comprises a pressure sensitive adhesive polymer. 17. The transdermal drug delivery system of claim 16, wherein the polymer matrix comprises an acrylic polymer, a silicone polymer, or a mixture of two or more thereof. 18. The transdermal drug delivery system of claim 16, wherein the polymer components comprise about 70% (w/w) acrylic polymer and about 30% (w/w) silicone polymer, based on the dry weight of the polymer components. 19. The transdermal drug delivery system of claim 15, wherein the agomelatine is present at its saturation concentration in the polymer matrix. 20. The transdermal drug delivery system of claim 15, wherein the polymer matrix composition comprises about 10.5% (w/w) agomelatine and about 6% (w/w) isopropanol. 21. The transdermal drug delivery system of claim 1, further comprising a backing. 22. The transdermal drug delivery system of claim 1, further comprising a release liner. 23. A method of making a transdermal drug delivery system for the transdermal delivery of agomelatine, comprising preparing a drug-containing composition comprising agomelatine and an enhancer. 24. A method of transdermally delivering agomelatine, comprising applying a transdermal drug delivery system according to claim 1 to the skin or mucosa of a subject in need thereof. 25. A method of treating depression in a subject in need thereof, comprising applying a transdermal drug delivery system according to claim 1 once daily to the skin or mucosa of a subject in need thereof.

Described are transdermal drug delivery systems for the transdermal administration of agomelatine. Methods of making and using such systems also are described.1. A transdermal drug delivery system for the transdermal delivery of agomelatine in the form of a flexible, finite system, comprising a composition comprising agomelatine and an enhancer. 2. The transdermal drug delivery system of claim 1, wherein the enhancer is selected from the group consisting of isopropanol and ethanol. 3. The transdermal drug delivery system of claim 1, wherein the composition comprises an amount of the enhancer effective to achieve substantially complete drug delivery within 12 hours of application or less. 4. The transdermal drug delivery system of claim 1, wherein the composition comprises an amount of the enhancer effective to achieve at least about 60% of the agomelatine delivery within about 4 to about 6 hours of application. 5. The transdermal drug delivery system of claim 1, wherein the composition comprises an amount of the enhancer effective to achieve at least about 75% of the agomelatine delivery within about 4 to about 6 hours of application. 6. The transdermal drug delivery system of claim 1, wherein the composition comprises an amount of the enhancer effective to achieve at least about 80% of the agomelatine delivery within about 6 to about 8 hours of application. 7. The transdermal drug delivery system of claim 1, wherein the composition is a drug-in-solution reservoir-type composition comprising agomelatine and aqueous isopropanol. 8. The transdermal drug delivery system of claim 7, wherein the aqueous isopropanol comprises 50-75% v/v isopropanol. 9. The transdermal drug delivery system of claim 7, wherein the agomelatine is present at its saturation concentration. 10. The transdermal drug delivery system of claim 7, wherein the composition comprises at least 50 mg/mL agomelatine. 11. The transdermal drug delivery system of claim 1, wherein the composition is a drug-in-gel reservoir-type composition comprising agomelatine, aqueous isopropanol, and a gelling agent. 12. The transdermal drug delivery system of claim 11, wherein the aqueous isopropanol comprises 50-75% v/v isopropanol. 13. The transdermal drug delivery system of claim 11, wherein the agomelatine is present at its saturation concentration. 14. The transdermal drug delivery system of claim 11, wherein the composition comprises at least 50 mg/mL agomelatine. 15. The transdermal drug delivery system of claim 1, wherein the composition is a drug-in-polymer matrix composition comprising agomelatine formulated in a polymer matrix comprising isopropanol. 16. The transdermal drug delivery system of claim 15, wherein the polymer matrix comprises a pressure sensitive adhesive polymer. 17. The transdermal drug delivery system of claim 16, wherein the polymer matrix comprises an acrylic polymer, a silicone polymer, or a mixture of two or more thereof. 18. The transdermal drug delivery system of claim 16, wherein the polymer components comprise about 70% (w/w) acrylic polymer and about 30% (w/w) silicone polymer, based on the dry weight of the polymer components. 19. The transdermal drug delivery system of claim 15, wherein the agomelatine is present at its saturation concentration in the polymer matrix. 20. The transdermal drug delivery system of claim 15, wherein the polymer matrix composition comprises about 10.5% (w/w) agomelatine and about 6% (w/w) isopropanol. 21. The transdermal drug delivery system of claim 1, further comprising a backing. 22. The transdermal drug delivery system of claim 1, further comprising a release liner. 23. A method of making a transdermal drug delivery system for the transdermal delivery of agomelatine, comprising preparing a drug-containing composition comprising agomelatine and an enhancer. 24. A method of transdermally delivering agomelatine, comprising applying a transdermal drug delivery system according to claim 1 to the skin or mucosa of a subject in need thereof. 25. A method of treating depression in a subject in need thereof, comprising applying a transdermal drug delivery system according to claim 1 once daily to the skin or mucosa of a subject in need thereof.

Described herein is an oral care composition comprising an orally acceptable vehicle, a fluoride ion and a buffer having a pKa of less than 7.0 wherein the pH of the oral composition is greater than 3.5 and less than 5.0, and wherein the oral composition has an acid number greater than 4.5.

1. An oral care composition comprising an orally acceptable vehicle, a fluoride ion and a buffer having a pKa of less than 7.0 wherein the buffer comprises an aqueous solution of an acid and a salt of the acid, and wherein the ratio of acid:salt is between 2:1 and 1:2, wherein the pH of the oral composition is greater than 3.5 and less than 5.0, and wherein the oral composition has an acid number greater than 4.5. 2. The oral care composition according to claim 1 wherein the acid is selected from α-hydroxy acids, amino acids, dicarboxylic acids, phosphoric acid, diphosphoric acid and combinations thereof. 3. The oral care composition according to claim 1 wherein the acid is selected from succinic acid, tartaric acid, malic acid, fumaric acid, glutamic acid, lactic acid, suberic acid, adipic acid, sebacic acid, glutaric acid, azelaic acid and combinations thereof. 4. The oral care composition according to claim 1 wherein the acid is selected from fumaric acid, salts of fumaric acid, malic acid and combinations thereof. 5. The oral care composition according to claim 1 wherein the ratio of acid:salt is between 1.5:1 and 1:1.5. 6. The oral care composition according to claim 1 wherein the buffer is present at a level of 0.05-5.00 weight % based on the total weight of the composition. 7. The oral care composition according to claim 1 wherein the buffer is present at a level of 0.50-2.00% weight % based on the total weight of the composition. 8. The oral care composition according to claim 1 wherein the fluoride ion is selected from sodium fluoride, potassium fluoride, stannous fluoride, amine fluoride and combinations thereof. 9. The oral care composition according to claim 1 wherein the fluoride ion is present at a level of 100 ppm to 10000 ppm. 10. The oral care composition according to claim 1 wherein the pH of the oral composition is greater than 4.0. 11. The oral care composition according to claim 1 wherein the pH of the oral composition is from 4.2 to 4.8. 12. The oral care composition according to claim 1 wherein the buffer has a pKa from 2.5 to 6.0. 13. The oral care composition according to claim 1 wherein the buffer has a pKa from 3.5 to 5.6. 14. The oral care composition according to claim 1 wherein the buffer has a pKa from 4.0 to 5.6. 15. The oral care composition according to claim 1 wherein the buffer has a pKa between 4.2 and 5.6. 16. The oral care composition according to claim 1 wherein the oral composition has an acid number greater than 6.0. 17. The oral care composition according to claim 1 wherein the acid number is less than 25. 18. The oral care composition according to claim 1 wherein the oral composition is a dentifrice, toothpaste, mouthrinse, mouthwash, strip or a solid or liquid gel. 19. The oral care composition according to claim 1 wherein the oral composition is a dentifrice or mouthrinse. 20. The oral care composition according to claim 1 wherein the buffer is selected from an aqueous solution of succinic acid and the sodium or potassium salt of succinic acid, an aqueous solution of tartaric acid and the sodium or potassium salt of tartaric acid, an aqueous solution of malic acid and the sodium or potassium salt of malic acid, an aqueous solution of fumaric acid and the sodium or potassium salt of fumaric acid, an aqueous solution of glutamic acid and the sodium or potassium salt of glutamic acid, an aqueous solution of lactic acid and the sodium or potassium salt of lactic acid, an aqueous solution of suberic acid and the sodium or potassium salt of suberic acid, an aqueous solution of adipic acid and the sodium or potassium salt of adipic acid, an aqueous solution of sebacic acid and the sodium or potassium salt of sebacic acid, an aqueous solution of glutaric acid and the sodium or potassium salt of glutaric acid, and an aqueous solution of azelaic acid and the sodium or potassium salt of azelaic acid, and combinations thereof, wherein the buffer is present at a level from 0.05 to 5.00 weight % based on the total weight of the composition wherein the pH of the composition is from 4.2 to 4.8 and wherein the oral care composition has an acid number greater than or equal to 6.0. 21. A method of providing fluoride ions to the oral cavity of a mammal comprising contacting a composition according to claim 1 with the oral cavity. 22. A method of treating or preventing a disease or condition of the oral cavity of a mammal comprising applying a composition according to claim 1 to the oral cavity. 23. A method of decreasing dental cavity formation in mammalian teeth comprising applying a composition according to claim 1 to the oral cavity of a mammal. 24. A method of re-mineralizing teeth in a mammal comprising applying a composition according to claim 1 to the oral cavity of a mammal. 25. A composition according to claim 1 for use in a method of providing fluoride ions to the oral cavity of a mammal. 26. A composition according to claim 1 for use in a method of treating or preventing a disease or condition of the oral cavity of a mammal. 27. A composition according to claim 1 for use in a method of decreasing dental cavity formation in mammalian teeth. 28. A composition according to claim 1 for use in a method of re-mineralizing mammalian teeth. 29. (canceled) 30. The oral care composition according to claim 1 wherein the buffer controls the pH of the composition to be greater than 3.5 and less than 5.0, and buffers the composition such that the composition has an acid number greater than or equal to 4.5.

Described herein is an oral care composition comprising an orally acceptable vehicle, a fluoride ion and a buffer having a pKa of less than 7.0 wherein the pH of the oral composition is greater than 3.5 and less than 5.0, and wherein the oral composition has an acid number greater than 4.5.1. An oral care composition comprising an orally acceptable vehicle, a fluoride ion and a buffer having a pKa of less than 7.0 wherein the buffer comprises an aqueous solution of an acid and a salt of the acid, and wherein the ratio of acid:salt is between 2:1 and 1:2, wherein the pH of the oral composition is greater than 3.5 and less than 5.0, and wherein the oral composition has an acid number greater than 4.5. 2. The oral care composition according to claim 1 wherein the acid is selected from α-hydroxy acids, amino acids, dicarboxylic acids, phosphoric acid, diphosphoric acid and combinations thereof. 3. The oral care composition according to claim 1 wherein the acid is selected from succinic acid, tartaric acid, malic acid, fumaric acid, glutamic acid, lactic acid, suberic acid, adipic acid, sebacic acid, glutaric acid, azelaic acid and combinations thereof. 4. The oral care composition according to claim 1 wherein the acid is selected from fumaric acid, salts of fumaric acid, malic acid and combinations thereof. 5. The oral care composition according to claim 1 wherein the ratio of acid:salt is between 1.5:1 and 1:1.5. 6. The oral care composition according to claim 1 wherein the buffer is present at a level of 0.05-5.00 weight % based on the total weight of the composition. 7. The oral care composition according to claim 1 wherein the buffer is present at a level of 0.50-2.00% weight % based on the total weight of the composition. 8. The oral care composition according to claim 1 wherein the fluoride ion is selected from sodium fluoride, potassium fluoride, stannous fluoride, amine fluoride and combinations thereof. 9. The oral care composition according to claim 1 wherein the fluoride ion is present at a level of 100 ppm to 10000 ppm. 10. The oral care composition according to claim 1 wherein the pH of the oral composition is greater than 4.0. 11. The oral care composition according to claim 1 wherein the pH of the oral composition is from 4.2 to 4.8. 12. The oral care composition according to claim 1 wherein the buffer has a pKa from 2.5 to 6.0. 13. The oral care composition according to claim 1 wherein the buffer has a pKa from 3.5 to 5.6. 14. The oral care composition according to claim 1 wherein the buffer has a pKa from 4.0 to 5.6. 15. The oral care composition according to claim 1 wherein the buffer has a pKa between 4.2 and 5.6. 16. The oral care composition according to claim 1 wherein the oral composition has an acid number greater than 6.0. 17. The oral care composition according to claim 1 wherein the acid number is less than 25. 18. The oral care composition according to claim 1 wherein the oral composition is a dentifrice, toothpaste, mouthrinse, mouthwash, strip or a solid or liquid gel. 19. The oral care composition according to claim 1 wherein the oral composition is a dentifrice or mouthrinse. 20. The oral care composition according to claim 1 wherein the buffer is selected from an aqueous solution of succinic acid and the sodium or potassium salt of succinic acid, an aqueous solution of tartaric acid and the sodium or potassium salt of tartaric acid, an aqueous solution of malic acid and the sodium or potassium salt of malic acid, an aqueous solution of fumaric acid and the sodium or potassium salt of fumaric acid, an aqueous solution of glutamic acid and the sodium or potassium salt of glutamic acid, an aqueous solution of lactic acid and the sodium or potassium salt of lactic acid, an aqueous solution of suberic acid and the sodium or potassium salt of suberic acid, an aqueous solution of adipic acid and the sodium or potassium salt of adipic acid, an aqueous solution of sebacic acid and the sodium or potassium salt of sebacic acid, an aqueous solution of glutaric acid and the sodium or potassium salt of glutaric acid, and an aqueous solution of azelaic acid and the sodium or potassium salt of azelaic acid, and combinations thereof, wherein the buffer is present at a level from 0.05 to 5.00 weight % based on the total weight of the composition wherein the pH of the composition is from 4.2 to 4.8 and wherein the oral care composition has an acid number greater than or equal to 6.0. 21. A method of providing fluoride ions to the oral cavity of a mammal comprising contacting a composition according to claim 1 with the oral cavity. 22. A method of treating or preventing a disease or condition of the oral cavity of a mammal comprising applying a composition according to claim 1 to the oral cavity. 23. A method of decreasing dental cavity formation in mammalian teeth comprising applying a composition according to claim 1 to the oral cavity of a mammal. 24. A method of re-mineralizing teeth in a mammal comprising applying a composition according to claim 1 to the oral cavity of a mammal. 25. A composition according to claim 1 for use in a method of providing fluoride ions to the oral cavity of a mammal. 26. A composition according to claim 1 for use in a method of treating or preventing a disease or condition of the oral cavity of a mammal. 27. A composition according to claim 1 for use in a method of decreasing dental cavity formation in mammalian teeth. 28. A composition according to claim 1 for use in a method of re-mineralizing mammalian teeth. 29. (canceled) 30. The oral care composition according to claim 1 wherein the buffer controls the pH of the composition to be greater than 3.5 and less than 5.0, and buffers the composition such that the composition has an acid number greater than or equal to 4.5.

Methods and kits for assessing severity index for alcohol abuse, drug abuse, and other reward deficiency syndromes. It has been discovered that a multifaceted non-specific RDS behaviors should be considered as the true “reward” phenotype (endophenotype) instead of a single subset RDS behavior such as alcoholism. In an embodiment of the present invention, it has been discovered that there are at least eleven risk alleles associated with ten candidate genes. The methods and kits of the present invention satisfy the need to classify patients at genetic risk for drug/alcohol seeking behavior prior to or upon entry to residential and or non-residential chemical dependency and pain programs.

1-11. (canceled) 12. A method comprising a genotypic analysis of a panel of genes to identify a plurality of alleles comprising: (a) allele G of gene DRD1; (b) allele A1 of gene DRD2; (c) allele C of gene DRD3; (d) allele C of gene DRD4; (e) allele 9R of gene DAT1; (f) allele 7-11R of gene DRD4; (g) allele S or L of gene HTTLPR; (h) allele 4R of gene MAOA; (i) allele G of gene COMT; (j) allele G of gene OPRM1; and (k) allele 181 of gene GABRB3. 13. A kit for analyzing a panel of genes comprising primers for sequencing (5′ to 3′) consisting of: (l) allele G of gene DRD1; (m) allele A1 of gene DRD2; (n) allele C of gene DRD3; (o) allele C of gene DRD4; (p) allele 9R of gene DAT1; (q) allele 7-11R of gene DRD4; (r) allele S or L of gene HTTLPR; (s) allele 4R of gene MAOA; (t) allele G of gene COMT; (u) allele G of gene OPRM1; and (v) allele 181 of gene GABRB3. 14-19. (canceled)

Methods and kits for assessing severity index for alcohol abuse, drug abuse, and other reward deficiency syndromes. It has been discovered that a multifaceted non-specific RDS behaviors should be considered as the true “reward” phenotype (endophenotype) instead of a single subset RDS behavior such as alcoholism. In an embodiment of the present invention, it has been discovered that there are at least eleven risk alleles associated with ten candidate genes. The methods and kits of the present invention satisfy the need to classify patients at genetic risk for drug/alcohol seeking behavior prior to or upon entry to residential and or non-residential chemical dependency and pain programs.1-11. (canceled) 12. A method comprising a genotypic analysis of a panel of genes to identify a plurality of alleles comprising: (a) allele G of gene DRD1; (b) allele A1 of gene DRD2; (c) allele C of gene DRD3; (d) allele C of gene DRD4; (e) allele 9R of gene DAT1; (f) allele 7-11R of gene DRD4; (g) allele S or L of gene HTTLPR; (h) allele 4R of gene MAOA; (i) allele G of gene COMT; (j) allele G of gene OPRM1; and (k) allele 181 of gene GABRB3. 13. A kit for analyzing a panel of genes comprising primers for sequencing (5′ to 3′) consisting of: (l) allele G of gene DRD1; (m) allele A1 of gene DRD2; (n) allele C of gene DRD3; (o) allele C of gene DRD4; (p) allele 9R of gene DAT1; (q) allele 7-11R of gene DRD4; (r) allele S or L of gene HTTLPR; (s) allele 4R of gene MAOA; (t) allele G of gene COMT; (u) allele G of gene OPRM1; and (v) allele 181 of gene GABRB3. 14-19. (canceled)

A nutritional plan based on a person's individual genetic APO E genotype, which has been shown to be the number one gene affecting diet, cholesterol, heart disease, vascular dementia, Alzheimer's disease, and chronic illness, possibly autism, Parkinson's disease, and other neurological diseases, and that focuses on the right percentages of macronutrients for each individual's genetic type, is disclosed.

1.-23. (canceled) 24. A method for creating and optimizing a nutritional and fitness regime, comprising: performing a body composition bio-impedance test on an individual to determine the individual's caloric needs for activities of daily living for two basic activity levels, said activity levels comprising both a normal activities of daily living caloric requirement as is necessary to sustain said individual's typical daily activity, and additional calories for daily activity of said individual that requires additional caloric use; collecting a blood sample from said individual and submitting said blood sample for testing to determine said individual's apolipoprotein E (APO E) genotype; analyzing said blood sample to determine said individual's apolipoprotein E (APO E) genotype; based upon both of said individual's APO E genotype and the results of said bio-impedance test: creating a genotype specific nutritional regime and establishing optimal dietary levels of caloric content, fat content and type of fats, carbohydrate content and type of carbohydrates, and protein content and type of proteins; and creating an exercise regime to maintain a genotype specific percentage of body fat mass and lean mass. 25. The method of claim 24, further comprising: additionally establishing said exercise regime based on said individual's height and weight. 26. The method of claim 24, further comprising said nutritional regime establishing dietary levels of: fat content (long term fuel) and type of fats for said genotype selected from among inflammatory fats, comprising fats that come mainly from saturated and trans fat sources, and anti-inflammatory fats, comprising fats that come mainly from monounsaturated and polyunsaturated sources; and carbohydrate content (short term fuel) and type of carbohydrates for said genotype selected from among carbohydrates having various glycemic loads, and protein content and type of proteins for said genotype selected from among proteins coming from plant sources and proteins coming from fish and other animal sources. 27. The method of claim 26, further comprising: establishing a ratio of fat content to carbohydrate content based upon said individual's APO E genotype, said ratio comprising: for APO E 2/2, 35% fat content, 15% protein content, and 50% carbohydrate content; for APO E 2/3, 30% fat content, 15% protein content, and 55% carbohydrate content; for APO E 3/3 and APO E 2/4, 25% fat content, 20% protein content, and 55% carbohydrate content; and for APO E 3/4 and APO E 4/4, 20% fat content, 25% protein content, and 55% carbohydrate content. 28. The method of claim 24, said exercise regime comprising: for APO E 2/2 and APO E 2/3, 45% aerobic exercise and 55% anaerobic exercise; for APO E 3/3 and APO E 2/4, 50% aerobic exercise and 50% anaerobic exercise; and for APO E 3/4 and APO E 4/4, 75% aerobic exercise and 25% anaerobic exercise. 29. The method of claim 24, further comprising: said specific percentage of body fat mass, based on said individual's height and weight, comprising for males: for APO E 2/2 and APO E 2/3, less than 16% body fat mass, with normal lean mass range based on height; for APO E 3/3 and APO E 2/4, less than 16% body fat mass, with lean mass range based on height; and for APO E 3/4 and APO E 4/4, less than 16% body fat mass, with lean mass range based on height; and said specific percentage of body fat mass, based on said individual's height and weight, comprising for females: for APO E 2/2 and APO E 2/3, less than 22% body fat mass, with normal lean mass range based on height; for APO E 3/3 and APO E 2/4, less than 22% body fat mass, with normal lean mass range based on height; and for APO E 3/4 and APO E 4/4, less than 22% body fat mass, with normal lean mass range based on height.

A nutritional plan based on a person's individual genetic APO E genotype, which has been shown to be the number one gene affecting diet, cholesterol, heart disease, vascular dementia, Alzheimer's disease, and chronic illness, possibly autism, Parkinson's disease, and other neurological diseases, and that focuses on the right percentages of macronutrients for each individual's genetic type, is disclosed.1.-23. (canceled) 24. A method for creating and optimizing a nutritional and fitness regime, comprising: performing a body composition bio-impedance test on an individual to determine the individual's caloric needs for activities of daily living for two basic activity levels, said activity levels comprising both a normal activities of daily living caloric requirement as is necessary to sustain said individual's typical daily activity, and additional calories for daily activity of said individual that requires additional caloric use; collecting a blood sample from said individual and submitting said blood sample for testing to determine said individual's apolipoprotein E (APO E) genotype; analyzing said blood sample to determine said individual's apolipoprotein E (APO E) genotype; based upon both of said individual's APO E genotype and the results of said bio-impedance test: creating a genotype specific nutritional regime and establishing optimal dietary levels of caloric content, fat content and type of fats, carbohydrate content and type of carbohydrates, and protein content and type of proteins; and creating an exercise regime to maintain a genotype specific percentage of body fat mass and lean mass. 25. The method of claim 24, further comprising: additionally establishing said exercise regime based on said individual's height and weight. 26. The method of claim 24, further comprising said nutritional regime establishing dietary levels of: fat content (long term fuel) and type of fats for said genotype selected from among inflammatory fats, comprising fats that come mainly from saturated and trans fat sources, and anti-inflammatory fats, comprising fats that come mainly from monounsaturated and polyunsaturated sources; and carbohydrate content (short term fuel) and type of carbohydrates for said genotype selected from among carbohydrates having various glycemic loads, and protein content and type of proteins for said genotype selected from among proteins coming from plant sources and proteins coming from fish and other animal sources. 27. The method of claim 26, further comprising: establishing a ratio of fat content to carbohydrate content based upon said individual's APO E genotype, said ratio comprising: for APO E 2/2, 35% fat content, 15% protein content, and 50% carbohydrate content; for APO E 2/3, 30% fat content, 15% protein content, and 55% carbohydrate content; for APO E 3/3 and APO E 2/4, 25% fat content, 20% protein content, and 55% carbohydrate content; and for APO E 3/4 and APO E 4/4, 20% fat content, 25% protein content, and 55% carbohydrate content. 28. The method of claim 24, said exercise regime comprising: for APO E 2/2 and APO E 2/3, 45% aerobic exercise and 55% anaerobic exercise; for APO E 3/3 and APO E 2/4, 50% aerobic exercise and 50% anaerobic exercise; and for APO E 3/4 and APO E 4/4, 75% aerobic exercise and 25% anaerobic exercise. 29. The method of claim 24, further comprising: said specific percentage of body fat mass, based on said individual's height and weight, comprising for males: for APO E 2/2 and APO E 2/3, less than 16% body fat mass, with normal lean mass range based on height; for APO E 3/3 and APO E 2/4, less than 16% body fat mass, with lean mass range based on height; and for APO E 3/4 and APO E 4/4, less than 16% body fat mass, with lean mass range based on height; and said specific percentage of body fat mass, based on said individual's height and weight, comprising for females: for APO E 2/2 and APO E 2/3, less than 22% body fat mass, with normal lean mass range based on height; for APO E 3/3 and APO E 2/4, less than 22% body fat mass, with normal lean mass range based on height; and for APO E 3/4 and APO E 4/4, less than 22% body fat mass, with normal lean mass range based on height.

The present invention relates to methods of producing a food or malt-based beverage suitable for consumption by a subject with Coeliac's disease. In particular, the present invention relates to methods of producing a food or malt-based beverage with very low levels of hordeins. Also provided are barley plants which produce grain that can be used in the methods of the invention.

1. A method of producing a food or malt-based beverage ingredient, or a food or a malt-based beverage, the method comprising (i) processing barley grain to produce malt, wort, flour or wholemeal, and/or (ii) mixing barley grain, or malt, wort, flour or wholemeal produced from said grain, with at least one other food or beverage ingredient, wherein the barley grain, malt, wort, flour or wholemeal comprises about 50 ppm or less hordeins, thereby producing the food or malt-based beverage ingredient, food or malt-based beverage. 2. The method of claim 1, wherein the grain, malt, wort, flour or wholemeal comprises about 20 ppm or less, about 10 ppm or less, hordeins. 3. The method of claim 1, wherein the average weight of the grain is at least about 35 mg. 4. The method according to claim 1, wherein at least about 80%, of the grain do not pass through a 2.8 mm sieve. 5. The method according to claim 1, wherein the grain is from a plant which has a harvest index of about 40% to about 60%. 6. The method according to claim 1, wherein the grain has a length to thickness ratio of less than about 5. 7. The method according to claim 1, wherein the flour or wholemeal produced from the grain comprises about 10 ppm or less, hordeins. 8. The method according to claim 1, wherein the malt or wort produced from the grain comprises less than about 50 ppm, hordeins. 9. The method according to claim 1, wherein the grain, or malt, wort, flour or wholemeal produced from said grain, has a level of less than 10% of one or more than one of: i) B-hordeins comprising a sequence of amino acids provided as SEQ ID NO:53, ii) B-hordeins comprising a sequence of amino acids provided as SEQ ID NO:54, iii) C-hordeins comprising a sequence of amino acids provided as SEQ ID NO:55, and iv) D-hordeins comprising a sequence of amino acids provided as SEQ ID NO:56, wherein each of the levels of less than 10%, less than 5% or less than 2% is relative to a wild-type barley grain, or malt, wort, flour or wholemeal produced from said grain, of the barley variety Bomi, Sloop, Baudin, Yagan, Hindmarsh, or Commander. 10. (canceled) 11. (canceled) 12. The method according to claim 1, wherein the grain is homozygous for an allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted, or wherein the malt, wort, flour or wholemeal produced from said grain comprises DNA which comprises the allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted. 13. The method according to claim 1, wherein the grain is homozygous for a null allele of the gene encoding D-hordein at the Hor3 locus, or wherein the malt, wort, flour or wholemeal produced from said grain comprises DNA which comprises the null allele of the gene encoding D-hordein, the null allele preferably comprising a stop codon, splice site mutation, frame-shift mutation, insertion, deletion or encoding a truncated D-hordein, or where most or all of the D-hordein encoding gene has been deleted. 14. (canceled) 15. The method according to claim 1, wherein the grain is homozygous for an allele at the Lys3 locus of barley which results in the grain lacking C-hordeins, or wherein the malt, wort, flour or wholemeal produced from said grain comprises DNA which comprises the allele at the Lys3 locus. 16. The method according to claim 1, wherein the grain, malt, wort, flour or wholemeal comprises about 1% or less, of the level of hordeins when compared to grain from a corresponding wild-type barley plant or malt, wort, flour or wholemeal produced in the same manner from grain from a corresponding wild-type barley plant. 17-22. (canceled) 23. The method according to claim 1, wherein the coeliac toxicity of flour produced from the grain is less than about 5%, or less than about 1%, of flour produced from grain of a corresponding wild-type barley plant. 24. The method according to claim 1, wherein the average grain weight is at least 1.05 fold, at least 1.1 fold, or 1.05 to 1.3 fold, higher than a grain which is i) homozygous for an allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted, ii) homozygous for an allele at the Lys3 locus of barley which results in the grain lacking C hordeins, and iii) homozygous for a wild type allele of D hordein encoding a full-length protein, and/or wherein the grain is from a plant which has a grain yield which is least 1.20 fold, or at least 1.35 fold, or 1.2 to 1.5 fold, or 1.2 to 2.0 fold higher than the grain yield from a plant which is i) homozygous for an allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted, ii) homozygous for an allele at the Lys3 locus of barley which results in the grain lacking C hordeins, and iii) homozygous for a wild type allele of D hordein encoding a full-length protein. 25-33. (canceled) 34. The method according to claim 1, wherein at least about 50% of the grain germinates within 3 days following imbibition. 35. The method according to claim 1, wherein the food ingredient or malt-based beverage ingredient is flour, starch, malt, or wort, or wherein the food is leavened or unleavened breads, pasta, noodles, breakfast cereals, snack foods, cakes, pastries or foods containing flour-based sauces. 36. The method according to claim 1, wherein the malt-based beverage is beer or whiskey. 37. (canceled) 38. The method according to claim 1, wherein following consumption of the food or drink at least one symptom of coeliac's disease is not developed by a subject with said disease. 39-56. (canceled) 57. A method of avoiding or reducing the incidence or severity of coeliac's disease in a subject, the method comprising orally administering to the subject a food or malt-based beverage according to claim 46, wherein the reduction of the incidence or severity of coeliac's disease is relative to when the subject is orally administered the same amount of a corresponding food or malt-based beverage made from wild-type barley grain. 58. (canceled)

The present invention relates to methods of producing a food or malt-based beverage suitable for consumption by a subject with Coeliac's disease. In particular, the present invention relates to methods of producing a food or malt-based beverage with very low levels of hordeins. Also provided are barley plants which produce grain that can be used in the methods of the invention.1. A method of producing a food or malt-based beverage ingredient, or a food or a malt-based beverage, the method comprising (i) processing barley grain to produce malt, wort, flour or wholemeal, and/or (ii) mixing barley grain, or malt, wort, flour or wholemeal produced from said grain, with at least one other food or beverage ingredient, wherein the barley grain, malt, wort, flour or wholemeal comprises about 50 ppm or less hordeins, thereby producing the food or malt-based beverage ingredient, food or malt-based beverage. 2. The method of claim 1, wherein the grain, malt, wort, flour or wholemeal comprises about 20 ppm or less, about 10 ppm or less, hordeins. 3. The method of claim 1, wherein the average weight of the grain is at least about 35 mg. 4. The method according to claim 1, wherein at least about 80%, of the grain do not pass through a 2.8 mm sieve. 5. The method according to claim 1, wherein the grain is from a plant which has a harvest index of about 40% to about 60%. 6. The method according to claim 1, wherein the grain has a length to thickness ratio of less than about 5. 7. The method according to claim 1, wherein the flour or wholemeal produced from the grain comprises about 10 ppm or less, hordeins. 8. The method according to claim 1, wherein the malt or wort produced from the grain comprises less than about 50 ppm, hordeins. 9. The method according to claim 1, wherein the grain, or malt, wort, flour or wholemeal produced from said grain, has a level of less than 10% of one or more than one of: i) B-hordeins comprising a sequence of amino acids provided as SEQ ID NO:53, ii) B-hordeins comprising a sequence of amino acids provided as SEQ ID NO:54, iii) C-hordeins comprising a sequence of amino acids provided as SEQ ID NO:55, and iv) D-hordeins comprising a sequence of amino acids provided as SEQ ID NO:56, wherein each of the levels of less than 10%, less than 5% or less than 2% is relative to a wild-type barley grain, or malt, wort, flour or wholemeal produced from said grain, of the barley variety Bomi, Sloop, Baudin, Yagan, Hindmarsh, or Commander. 10. (canceled) 11. (canceled) 12. The method according to claim 1, wherein the grain is homozygous for an allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted, or wherein the malt, wort, flour or wholemeal produced from said grain comprises DNA which comprises the allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted. 13. The method according to claim 1, wherein the grain is homozygous for a null allele of the gene encoding D-hordein at the Hor3 locus, or wherein the malt, wort, flour or wholemeal produced from said grain comprises DNA which comprises the null allele of the gene encoding D-hordein, the null allele preferably comprising a stop codon, splice site mutation, frame-shift mutation, insertion, deletion or encoding a truncated D-hordein, or where most or all of the D-hordein encoding gene has been deleted. 14. (canceled) 15. The method according to claim 1, wherein the grain is homozygous for an allele at the Lys3 locus of barley which results in the grain lacking C-hordeins, or wherein the malt, wort, flour or wholemeal produced from said grain comprises DNA which comprises the allele at the Lys3 locus. 16. The method according to claim 1, wherein the grain, malt, wort, flour or wholemeal comprises about 1% or less, of the level of hordeins when compared to grain from a corresponding wild-type barley plant or malt, wort, flour or wholemeal produced in the same manner from grain from a corresponding wild-type barley plant. 17-22. (canceled) 23. The method according to claim 1, wherein the coeliac toxicity of flour produced from the grain is less than about 5%, or less than about 1%, of flour produced from grain of a corresponding wild-type barley plant. 24. The method according to claim 1, wherein the average grain weight is at least 1.05 fold, at least 1.1 fold, or 1.05 to 1.3 fold, higher than a grain which is i) homozygous for an allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted, ii) homozygous for an allele at the Lys3 locus of barley which results in the grain lacking C hordeins, and iii) homozygous for a wild type allele of D hordein encoding a full-length protein, and/or wherein the grain is from a plant which has a grain yield which is least 1.20 fold, or at least 1.35 fold, or 1.2 to 1.5 fold, or 1.2 to 2.0 fold higher than the grain yield from a plant which is i) homozygous for an allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted, ii) homozygous for an allele at the Lys3 locus of barley which results in the grain lacking C hordeins, and iii) homozygous for a wild type allele of D hordein encoding a full-length protein. 25-33. (canceled) 34. The method according to claim 1, wherein at least about 50% of the grain germinates within 3 days following imbibition. 35. The method according to claim 1, wherein the food ingredient or malt-based beverage ingredient is flour, starch, malt, or wort, or wherein the food is leavened or unleavened breads, pasta, noodles, breakfast cereals, snack foods, cakes, pastries or foods containing flour-based sauces. 36. The method according to claim 1, wherein the malt-based beverage is beer or whiskey. 37. (canceled) 38. The method according to claim 1, wherein following consumption of the food or drink at least one symptom of coeliac's disease is not developed by a subject with said disease. 39-56. (canceled) 57. A method of avoiding or reducing the incidence or severity of coeliac's disease in a subject, the method comprising orally administering to the subject a food or malt-based beverage according to claim 46, wherein the reduction of the incidence or severity of coeliac's disease is relative to when the subject is orally administered the same amount of a corresponding food or malt-based beverage made from wild-type barley grain. 58. (canceled)

The invention relates to pharmaceutical compositions and methods for treating inner ear disorders. In particular, the invention provides a method for treating and/or preventing acute inner ear tinnitus in a subject in need thereof by administering a pharmaceutical composition comprising a peptide inhibitor of c-Jun N-terminal kinase.

1. A method of ameliorating or reducing the occurrence of acute inner ear tinnitus induced by a cochlear insult in a human in need thereof comprising administering to the human a pharmaceutical composition comprising a therapeutically effective amount of a peptide inhibitor of c-Jun N-terminal kinase (JNK) or a pharmaceutically acceptable salt thereof, wherein the peptide inhibitor is no more than 50 amino acids in length and comprises a sequence that is at least 80% identical to the sequence of any one of SEQ ID NOs: 1 to 4 and 13 to 45. 2. The method of claim 1, wherein the peptide inhibitor comprises a sequence that is at least 90% identical to DQSRPVQPFLNLTTPRKPR (SEQ ID NO: 1) or RPKRPTTLNLFPQVPRSQD (SEQ ID NO: 4). 3. The method of claim 1, wherein the peptide inhibitor comprises or consists of the sequence of DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG (SEQ ID NO: 2) or GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD (SEQ ID NO: 3). 4. The method of claim 1, wherein all of the chiral amino acids in the peptide inhibitor are in the D configuration. 5. The method of claim 1, wherein all of the chiral amino acids in the peptide inhibitor are in the L configuration. 6. The method of claim 1, wherein the cochlear insult results from acute acoustic trauma, presbycusis, ischemia, anoxia, barotrauma, otitis media, exposure to ototoxic drugs, conductive hearing loss, or sudden deafness. 7. The method of claim 1, wherein the pharmaceutical composition is administered to the human within four weeks following the cochlear insult. 8. The method of claim 1, wherein the pharmaceutical composition is administered to the human within one week following the cochlear insult. 9. The method of claim 1, wherein the pharmaceutical composition is administered to the human within three days following the cochlear insult. 10. The method of claim 1, wherein the human has or is diagnosed with acute hearing loss of at least 60 dB within 48 hours of onset. 11. The method of claim 1, wherein the human has or is diagnosed with acute hearing loss of at least 40 dB that persists after 48 hours of onset. 12. The method of claim 1, wherein the therapeutically effective amount of the peptide inhibitor is effective to reduce the perception of tinnitus following administration of the composition. 13. The method of claim 1, wherein the therapeutically effective amount of the peptide inhibitor is effective to reduce the loudness of tinnitus following administration of the composition. 14. The method of claim 1, wherein the pharmaceutical composition is administered topically via the round window membrane or the oval window membrane to the inner ear. 15. The method of claim 1, wherein the pharmaceutical composition is administered by an intratympanic injection. 16. The method of claim 1, wherein the pharmaceutical composition is delivered to the middle ear. 17. The method of claim 1, wherein the pharmaceutical composition is a gel. 18. The method of claim 1, wherein the pharmaceutical composition comprises about 0.5% to about 1% of hyaluronic acid. 19. The method of claim 1, wherein the pharmaceutical composition comprises a phosphate buffer which buffers the pH of the composition to 6.0 to 7.4. 20. The method of claim 1, wherein the therapeutically effective amount of the peptide inhibitor is about 0.2 mg to about 2 mg. 21. The method of claim 1, wherein the therapeutically effective amount of the peptide inhibitor is about 0.3 mg to about 0.8 mg. 22. The method of claim 1, wherein the therapeutically effective amount of the peptide inhibitor is administered in multiple doses.

The invention relates to pharmaceutical compositions and methods for treating inner ear disorders. In particular, the invention provides a method for treating and/or preventing acute inner ear tinnitus in a subject in need thereof by administering a pharmaceutical composition comprising a peptide inhibitor of c-Jun N-terminal kinase.1. A method of ameliorating or reducing the occurrence of acute inner ear tinnitus induced by a cochlear insult in a human in need thereof comprising administering to the human a pharmaceutical composition comprising a therapeutically effective amount of a peptide inhibitor of c-Jun N-terminal kinase (JNK) or a pharmaceutically acceptable salt thereof, wherein the peptide inhibitor is no more than 50 amino acids in length and comprises a sequence that is at least 80% identical to the sequence of any one of SEQ ID NOs: 1 to 4 and 13 to 45. 2. The method of claim 1, wherein the peptide inhibitor comprises a sequence that is at least 90% identical to DQSRPVQPFLNLTTPRKPR (SEQ ID NO: 1) or RPKRPTTLNLFPQVPRSQD (SEQ ID NO: 4). 3. The method of claim 1, wherein the peptide inhibitor comprises or consists of the sequence of DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG (SEQ ID NO: 2) or GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD (SEQ ID NO: 3). 4. The method of claim 1, wherein all of the chiral amino acids in the peptide inhibitor are in the D configuration. 5. The method of claim 1, wherein all of the chiral amino acids in the peptide inhibitor are in the L configuration. 6. The method of claim 1, wherein the cochlear insult results from acute acoustic trauma, presbycusis, ischemia, anoxia, barotrauma, otitis media, exposure to ototoxic drugs, conductive hearing loss, or sudden deafness. 7. The method of claim 1, wherein the pharmaceutical composition is administered to the human within four weeks following the cochlear insult. 8. The method of claim 1, wherein the pharmaceutical composition is administered to the human within one week following the cochlear insult. 9. The method of claim 1, wherein the pharmaceutical composition is administered to the human within three days following the cochlear insult. 10. The method of claim 1, wherein the human has or is diagnosed with acute hearing loss of at least 60 dB within 48 hours of onset. 11. The method of claim 1, wherein the human has or is diagnosed with acute hearing loss of at least 40 dB that persists after 48 hours of onset. 12. The method of claim 1, wherein the therapeutically effective amount of the peptide inhibitor is effective to reduce the perception of tinnitus following administration of the composition. 13. The method of claim 1, wherein the therapeutically effective amount of the peptide inhibitor is effective to reduce the loudness of tinnitus following administration of the composition. 14. The method of claim 1, wherein the pharmaceutical composition is administered topically via the round window membrane or the oval window membrane to the inner ear. 15. The method of claim 1, wherein the pharmaceutical composition is administered by an intratympanic injection. 16. The method of claim 1, wherein the pharmaceutical composition is delivered to the middle ear. 17. The method of claim 1, wherein the pharmaceutical composition is a gel. 18. The method of claim 1, wherein the pharmaceutical composition comprises about 0.5% to about 1% of hyaluronic acid. 19. The method of claim 1, wherein the pharmaceutical composition comprises a phosphate buffer which buffers the pH of the composition to 6.0 to 7.4. 20. The method of claim 1, wherein the therapeutically effective amount of the peptide inhibitor is about 0.2 mg to about 2 mg. 21. The method of claim 1, wherein the therapeutically effective amount of the peptide inhibitor is about 0.3 mg to about 0.8 mg. 22. The method of claim 1, wherein the therapeutically effective amount of the peptide inhibitor is administered in multiple doses.

The present invention relates to an immunostimulatory composition comprising a) an adjuvant component, comprising or consisting of at least one (m)RNA, complexed with a cationic or polycationic compound, and b) at least one free mRNA, encoding at least one therapeutically active protein, antigen, allergen and/or antibody, wherein the immunostimulatory composition is capable to elicit or enhance an innate and optionally an adaptive immune response in a mammal. The inventive immunostimulatory composition may be a pharmaceutical composition or a vaccine. The invention furthermore relates to a method of preparation of the inventive immunostimulatory composition. The invention also relates to the use of the inventive immunostimulatory composition or its components (for the preparation of a pharmaceutical composition or a vaccine) for the treatment of various diseases. Finally, the invention relates to kits containing the inventive immunostimulatory composition, its components and/or the pharmaceutical composition or vaccine.

1. A method of stimulating an immune response in a subject in need thereof comprising administering to the subject an effective amount of a composition comprising (a) an adjuvant component, comprising or consisting of at least one RNA, complexed with protamine; and (b) at least one free mRNA, encoding at least one antigen for the prophylaxis and/or treatment of a disease. 2. The method of claim 1, wherein the at least one RNA of the adjuvant component, is selected from a short RNA oligonucleotide, a coding RNA, an mRNA, an immunostimulatory RNA, an siRNA, an antisense RNA, riboswitches, ribozymes or aptamers. 3. (canceled) 4. The method of claim 1, wherein the at least one free mRNA and the at least one RNA of the adjuvant component are identical to each other. 5. The method of claim 1, wherein the at least one free mRNA and the at least one RNA of the adjuvant component are different from each other. 6. The method of claim 1, wherein the N/P ratio of the RNA to the protamine in the adjuvant component is in the range of 0.1-10. 7. The method of claim 1, wherein the molar ratio of the RNA of the adjuvant component to the at least one free mRNA of the second component is 1:1 to 1:4. 8. The method of claim 1, wherein the at least one free mRNA and/or the at least one RNA of the adjuvant component are GC-stabilized. 9. The method of claim 8, wherein the G/C content of the coding region of the GC-stabilized RNA is increased compared with the G/C content of the coding region of the native RNA, the coded amino acid sequence of the GC-stabilized modified RNA not being altered compared with the encoded amino acid sequence of the native modified RNA. 10. (canceled) 11. The method of claim 1, wherein the at least one free mRNA encodes an antigen, selected from 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha5beta1-Integrin, alpha5beta6-Integrin, alpha-methylacyl-coenzyme A racemase, ART-4, B7H4, BAGE-1, BCL-2, BING-4, CA 15-3/CA 27-29, CA 19-9, CA 72-4, CA125, calreticulin, CAMEL, CASP-8, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA, CLCA2, CML28, Coactosin-like protein, Collagen XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10/MAGE-B1, DAM-6/MAGE-B2, EGFR/Her1, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, EZH2, FGF-5, FN, Fra-1, G250/CAIX, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, HAGE, HAST-2, hepsin, Her2/neu/ErbB2, HERV-K-MEL, HNE, homeobox NKX 3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205, KK-LC-1, KM-HN-1, LAGE-1, Livin, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/Melan-A, MART-2, matrix protein 22, MC1R, M-CSF, Mesothelin, MG50/PXDN, MMP 11, MN/CA IX-antigen, MRP-3, MUC1, MUC2, NA88-A, N-acetylglucos-aminyltransferase-V, Neo-PAP, NGEP, NMP22, NPM/ALK, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT, OS-9, osteocalcin, osteopontin, p15, p15, p190 minor bcr-abl, p53, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-Kinase, Pin1, POTE, PRAME, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, RAGE-1, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin, survivin-2B, TA-90, TAG-72, TARP, TGFb, TGFbRII, TGM-4, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP-2/INT2, Trp-p8, Tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, WT1; or is selected from mutant antigens expressed in cancer diseases, including alpha-actinin-4/m, ARTC1/m, bcr/abl, beta-Catenin/m, BRCA1/m, BRCA2/m, CASP-5/m, CASP-8/m, CDC27/m, CDK4/m, CDKN2A/m, CML66, COA-1/m, DEK-CAN, EFTUD2/m, ELF2/m, ETV6-AML1, FN1/m, GPNMB/m, HLA-A*0201-R170I, HLA-A11/m, HLA-A2/m, HSP70-2M, KIAA0205/m, K-Ras/m, LDLR-FUT, MART2/m, ME1/m, MUM-1/m, MUM-2/m, MUM-3/m, Myosin class I/m, neo-PAP/m, NFYC/m, N-Ras/m, OGT/m, OS-9/m, p53/m, Pml/RARa, PRDX5/m, PTPRK/m, RBAF600/m, SIRT2/m, SYT-SSX-1, SYT-SSX-2, TEL-AML1, TGFbRII, and TPI/m. 12. The method of claim 1, wherein the at least one free mRNA encodes: a) at least one, two, three or four different antigens of the following group of antigens: (1) PSA (Prostate-Specific Antigen)=KLK3 (Kallikrein-3), (2) PSMA (Prostate-Specific Membrane Antigen), (3) PSCA (Prostate Stem Cell Antigen), (4) STEAP (Six Transmembrane Epithelial Antigen of the Prostate), or b) at least one, two, three, four, five, six, seven, eight, nine, ten eleven or twelve different antigens of the following group of antigens: hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1, and/or MAGE-C2, or a combination thereof. 13. A pharmaceutical composition, comprising an immunostimulatory composition comprising (a) an adjuvant component, comprising at least one RNA, complexed with protamine; (b) at least one free mRNA, encoding at least one antigen; and (c) a pharmaceutically acceptable carrier, adjuvant, and/or vehicle. 14-15. (canceled) 16. The method of claim 1, wherein the subject has a disease selected from cancers or tumor diseases, autoimmune diseases, infectious diseases, viral, bacterial or protozoological infectious diseases or allergic diseases. 17. A kit comprising the pharmaceutical composition according to claim 13, and technical instructions with information on the administration and dosage of the pharmaceutical composition. 18. The method of claim 1, wherein the weight ratio of the at least one RNA to protamine in the adjuvant component is 2:1 to 3:1. 19. The method of claim 1, wherein the molar ratio of the RNA of the adjuvant component to the at least one free mRNA of the second component b) is between 1:1 and 1:3. 20. The method of claim 1, wherein the molar ratio of the RNA of the adjuvant component to the at least one free mRNA of the second component b) is between 1:2 and 1:4. 21. The method of claim 1, wherein the at least one free mRNA encodes an antigen selected from a cancer cell antigen, an autoimmune antigen, an infectious disease antigen or an allergic antigen. 22. The method of claim 1, wherein the at least one free mRNA encodes an infectious disease antigen selected from a viral antigen, a protozoal antigen, or a bacterial antigen. 23. The method of claim 1, wherein the weight ratio of the at least one RNA to protamine in the adjuvant component is 2:1. 24. The method of claim 1, wherein the N/P ratio of the RNA to the protamine in the adjuvant component is 0.3 to 4. 25. A composition comprising: (a) an adjuvant component, comprising or consisting of at least one RNA complexed with protamine; and (b) at least one free mRNA, encoding at least one therapeutically active protein, antigen and/or antibody.

The present invention relates to an immunostimulatory composition comprising a) an adjuvant component, comprising or consisting of at least one (m)RNA, complexed with a cationic or polycationic compound, and b) at least one free mRNA, encoding at least one therapeutically active protein, antigen, allergen and/or antibody, wherein the immunostimulatory composition is capable to elicit or enhance an innate and optionally an adaptive immune response in a mammal. The inventive immunostimulatory composition may be a pharmaceutical composition or a vaccine. The invention furthermore relates to a method of preparation of the inventive immunostimulatory composition. The invention also relates to the use of the inventive immunostimulatory composition or its components (for the preparation of a pharmaceutical composition or a vaccine) for the treatment of various diseases. Finally, the invention relates to kits containing the inventive immunostimulatory composition, its components and/or the pharmaceutical composition or vaccine.1. A method of stimulating an immune response in a subject in need thereof comprising administering to the subject an effective amount of a composition comprising (a) an adjuvant component, comprising or consisting of at least one RNA, complexed with protamine; and (b) at least one free mRNA, encoding at least one antigen for the prophylaxis and/or treatment of a disease. 2. The method of claim 1, wherein the at least one RNA of the adjuvant component, is selected from a short RNA oligonucleotide, a coding RNA, an mRNA, an immunostimulatory RNA, an siRNA, an antisense RNA, riboswitches, ribozymes or aptamers. 3. (canceled) 4. The method of claim 1, wherein the at least one free mRNA and the at least one RNA of the adjuvant component are identical to each other. 5. The method of claim 1, wherein the at least one free mRNA and the at least one RNA of the adjuvant component are different from each other. 6. The method of claim 1, wherein the N/P ratio of the RNA to the protamine in the adjuvant component is in the range of 0.1-10. 7. The method of claim 1, wherein the molar ratio of the RNA of the adjuvant component to the at least one free mRNA of the second component is 1:1 to 1:4. 8. The method of claim 1, wherein the at least one free mRNA and/or the at least one RNA of the adjuvant component are GC-stabilized. 9. The method of claim 8, wherein the G/C content of the coding region of the GC-stabilized RNA is increased compared with the G/C content of the coding region of the native RNA, the coded amino acid sequence of the GC-stabilized modified RNA not being altered compared with the encoded amino acid sequence of the native modified RNA. 10. (canceled) 11. The method of claim 1, wherein the at least one free mRNA encodes an antigen, selected from 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha5beta1-Integrin, alpha5beta6-Integrin, alpha-methylacyl-coenzyme A racemase, ART-4, B7H4, BAGE-1, BCL-2, BING-4, CA 15-3/CA 27-29, CA 19-9, CA 72-4, CA125, calreticulin, CAMEL, CASP-8, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA, CLCA2, CML28, Coactosin-like protein, Collagen XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10/MAGE-B1, DAM-6/MAGE-B2, EGFR/Her1, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, EZH2, FGF-5, FN, Fra-1, G250/CAIX, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, HAGE, HAST-2, hepsin, Her2/neu/ErbB2, HERV-K-MEL, HNE, homeobox NKX 3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205, KK-LC-1, KM-HN-1, LAGE-1, Livin, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/Melan-A, MART-2, matrix protein 22, MC1R, M-CSF, Mesothelin, MG50/PXDN, MMP 11, MN/CA IX-antigen, MRP-3, MUC1, MUC2, NA88-A, N-acetylglucos-aminyltransferase-V, Neo-PAP, NGEP, NMP22, NPM/ALK, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT, OS-9, osteocalcin, osteopontin, p15, p15, p190 minor bcr-abl, p53, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-Kinase, Pin1, POTE, PRAME, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, RAGE-1, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin, survivin-2B, TA-90, TAG-72, TARP, TGFb, TGFbRII, TGM-4, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP-2/INT2, Trp-p8, Tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, WT1; or is selected from mutant antigens expressed in cancer diseases, including alpha-actinin-4/m, ARTC1/m, bcr/abl, beta-Catenin/m, BRCA1/m, BRCA2/m, CASP-5/m, CASP-8/m, CDC27/m, CDK4/m, CDKN2A/m, CML66, COA-1/m, DEK-CAN, EFTUD2/m, ELF2/m, ETV6-AML1, FN1/m, GPNMB/m, HLA-A*0201-R170I, HLA-A11/m, HLA-A2/m, HSP70-2M, KIAA0205/m, K-Ras/m, LDLR-FUT, MART2/m, ME1/m, MUM-1/m, MUM-2/m, MUM-3/m, Myosin class I/m, neo-PAP/m, NFYC/m, N-Ras/m, OGT/m, OS-9/m, p53/m, Pml/RARa, PRDX5/m, PTPRK/m, RBAF600/m, SIRT2/m, SYT-SSX-1, SYT-SSX-2, TEL-AML1, TGFbRII, and TPI/m. 12. The method of claim 1, wherein the at least one free mRNA encodes: a) at least one, two, three or four different antigens of the following group of antigens: (1) PSA (Prostate-Specific Antigen)=KLK3 (Kallikrein-3), (2) PSMA (Prostate-Specific Membrane Antigen), (3) PSCA (Prostate Stem Cell Antigen), (4) STEAP (Six Transmembrane Epithelial Antigen of the Prostate), or b) at least one, two, three, four, five, six, seven, eight, nine, ten eleven or twelve different antigens of the following group of antigens: hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1, and/or MAGE-C2, or a combination thereof. 13. A pharmaceutical composition, comprising an immunostimulatory composition comprising (a) an adjuvant component, comprising at least one RNA, complexed with protamine; (b) at least one free mRNA, encoding at least one antigen; and (c) a pharmaceutically acceptable carrier, adjuvant, and/or vehicle. 14-15. (canceled) 16. The method of claim 1, wherein the subject has a disease selected from cancers or tumor diseases, autoimmune diseases, infectious diseases, viral, bacterial or protozoological infectious diseases or allergic diseases. 17. A kit comprising the pharmaceutical composition according to claim 13, and technical instructions with information on the administration and dosage of the pharmaceutical composition. 18. The method of claim 1, wherein the weight ratio of the at least one RNA to protamine in the adjuvant component is 2:1 to 3:1. 19. The method of claim 1, wherein the molar ratio of the RNA of the adjuvant component to the at least one free mRNA of the second component b) is between 1:1 and 1:3. 20. The method of claim 1, wherein the molar ratio of the RNA of the adjuvant component to the at least one free mRNA of the second component b) is between 1:2 and 1:4. 21. The method of claim 1, wherein the at least one free mRNA encodes an antigen selected from a cancer cell antigen, an autoimmune antigen, an infectious disease antigen or an allergic antigen. 22. The method of claim 1, wherein the at least one free mRNA encodes an infectious disease antigen selected from a viral antigen, a protozoal antigen, or a bacterial antigen. 23. The method of claim 1, wherein the weight ratio of the at least one RNA to protamine in the adjuvant component is 2:1. 24. The method of claim 1, wherein the N/P ratio of the RNA to the protamine in the adjuvant component is 0.3 to 4. 25. A composition comprising: (a) an adjuvant component, comprising or consisting of at least one RNA complexed with protamine; and (b) at least one free mRNA, encoding at least one therapeutically active protein, antigen and/or antibody.

The present invention is directed to a water treatment composition, containing 50-99.9 wt. % of particulate halogen-releasing compound; and 0.1-10 wt. % of granular fluoropolymer, wherein all weight percentages are based on the total weight of the composition. The present invention is also directed to a water treatment composition, containing 50-99 wt. % of particulate halogen-releasing compound; 1-10 wt. % of aluminum sulfate; 0.5-15 wt. % of a sulfate salt selected from the group consisting of copper sulfate, zinc sulfate, and combinations thereof; and 0.1-10 wt. % of particulate polytetrafluoroethylene polymer, wherein all weight percentages are based on the total weight of the composition. One particular halogen-releasing compound is trichloroisocyanuric acid.

1. A water treatment composition, comprising: 50-99.9 wt. % of a particulate halogen-releasing compound, said halogen-releasing compound comprising a compound selected from the group consisting of chlorinated isocyanuric acids, chlorine containing hydantoins, bromine-containing hydantoins and mixtures thereof; and 0.1-10 wt. % of particulate fluoropolymer, wherein all weight percentages are based on the total weight of said composition. 2. The water treatment composition according to claim 1, wherein the halogen-releasing compound comprises particulate trichloroisocyanuric acid. 3. The water treatment composition according to claim 2, wherein said particulate trichloroisocyanuric acid comprises from about 90 wt. % to about 96 wt. %, based on the total weight of the composition. 4. The water treatment composition according to claim 1, wherein said fluoropolymer is polytetrafluoroethylene polymer. 5. The water treatment composition according to claim 1, wherein said fluoropolymer is present in an amount between about 0.25 wt. % and about 5.0 wt. %, based on the total weight of the composition. 6. The water treatment composition according to claim 5, wherein said fluoropolymer is present in an amount between about 0.75 wt. % and about 2.0 wt. %, based on the total weight of the composition. 7. The water treatment composition according to claim 1, further comprising 1-10 wt. % of aluminum sulfate, based on the total weight of the composition. 8. The water treatment composition according to claim 7, further comprising 0.2-4 wt. % of aluminum sulfate, based on the total weight of the composition. 9. The water treatment composition according to claim 1, further comprising 0.5-15 wt. % of a sulfate salt selected from the group consisting of copper sulfate, zinc sulfate, and combinations thereof, based on the total weight of the composition. 10. The water treatment composition according to claim 9, wherein said sulfate salt is present in an about between 1 wt. % and 4 wt. %, based on the total weight of the composition 11. The water treatment composition according to claim 1, further comprising cyanuric acid, and the cyanuric acid is present in an amount up to about 3 wt. % of cyanuric acid, based on the total weight of the composition. 12. The water treatment composition according to claim 1, wherein said composition is in the form of a unitary structure. 13. The water treatment composition according to claim 1, wherein said composition further comprises zinc oxide, citric acid, boric acid, sodium hexametaphosphate, phosphate reducers, polymeric clarifiers, dichlor, cyanuric acid, boric acid and combinations thereof. 14. The water treatment composition according to claim 1, further comprising 1-10 wt. % of aluminum sulfate; and 0.5-15 wt. % of a sulfate salt selected from the group consisting of copper sulfate, zinc sulfate, and combinations thereof; all weight percent are based on the total weight of said composition. 15. The water treatment composition according to claim 13, wherein said halogen-releasing compound comprises particulate trichloroisocyanuric acid, and the trichloroisocyanuric acid comprises from about 90 wt. % to about 96 wt. %, based on the total weight of the composition. 16. The water treatment composition according to claim 14, wherein said fluoropolymer is present in an amount between about 0.25 wt. % and about 5.0 wt. %, based on the total weight of the composition. 17. The water treatment composition according to claim 16, wherein said fluoropolymer is present in an amount between about 0.5 wt. % and about 2.0 wt. %, based on the total weight of the composition. 18. The water treatment composition according to claim 14, wherein said aluminum sulfate is present between about 0.2 wt. % and about 4 wt. %, and said sulfate salt comprises from about 1 wt. % and about 4 wt. % based on the total weight of the composition. 19. The water treatment composition according to claim 14, further comprising cyanuric acid, and the cyanuric acid is present in an amount up to 3 wt. % of cyanuric acid, based on the total weight of the composition. 20. The water treatment composition according to claim 14, wherein said composition is the form of a unitary solid. 21. The water treatment composition according to claim 14, wherein said composition further comprises zinc oxide, citric acid, boric acid, sodium hexametaphosphate, phosphate reducers, polymeric clarifiers, dichlor, cyanuric acid, and combinations thereof. 22. The water treatment composition according to claim 14, further comprising 1-10 wt. % of sodium hexametaphosphate (SHMP), based on the total weight of said composition. 23. The water treatment composition according to claim 1, further comprising 1-10 wt. % of sodium hexametaphosphate (SHMP) and 0.5-15 wt. % of a sulfate salt selected from the group consisting of copper sulfate, zinc sulfate, and combinations thereof; based on the total weight of said composition. 24. The water treatment composition according to claim 1, wherein the fluoropolymer is in the form of agglomerated particles. 25. The water treatment composition according to claim 1, wherein the fluoropolymer is in the form of essentially non-agglomerated particles. 26. The water treatment composition according to claim 1, wherein the halogen-releasing compound comprises a mixture of particulate trichloroisocyanuric acid and a halogen containing hydantoin.

The present invention is directed to a water treatment composition, containing 50-99.9 wt. % of particulate halogen-releasing compound; and 0.1-10 wt. % of granular fluoropolymer, wherein all weight percentages are based on the total weight of the composition. The present invention is also directed to a water treatment composition, containing 50-99 wt. % of particulate halogen-releasing compound; 1-10 wt. % of aluminum sulfate; 0.5-15 wt. % of a sulfate salt selected from the group consisting of copper sulfate, zinc sulfate, and combinations thereof; and 0.1-10 wt. % of particulate polytetrafluoroethylene polymer, wherein all weight percentages are based on the total weight of the composition. One particular halogen-releasing compound is trichloroisocyanuric acid.1. A water treatment composition, comprising: 50-99.9 wt. % of a particulate halogen-releasing compound, said halogen-releasing compound comprising a compound selected from the group consisting of chlorinated isocyanuric acids, chlorine containing hydantoins, bromine-containing hydantoins and mixtures thereof; and 0.1-10 wt. % of particulate fluoropolymer, wherein all weight percentages are based on the total weight of said composition. 2. The water treatment composition according to claim 1, wherein the halogen-releasing compound comprises particulate trichloroisocyanuric acid. 3. The water treatment composition according to claim 2, wherein said particulate trichloroisocyanuric acid comprises from about 90 wt. % to about 96 wt. %, based on the total weight of the composition. 4. The water treatment composition according to claim 1, wherein said fluoropolymer is polytetrafluoroethylene polymer. 5. The water treatment composition according to claim 1, wherein said fluoropolymer is present in an amount between about 0.25 wt. % and about 5.0 wt. %, based on the total weight of the composition. 6. The water treatment composition according to claim 5, wherein said fluoropolymer is present in an amount between about 0.75 wt. % and about 2.0 wt. %, based on the total weight of the composition. 7. The water treatment composition according to claim 1, further comprising 1-10 wt. % of aluminum sulfate, based on the total weight of the composition. 8. The water treatment composition according to claim 7, further comprising 0.2-4 wt. % of aluminum sulfate, based on the total weight of the composition. 9. The water treatment composition according to claim 1, further comprising 0.5-15 wt. % of a sulfate salt selected from the group consisting of copper sulfate, zinc sulfate, and combinations thereof, based on the total weight of the composition. 10. The water treatment composition according to claim 9, wherein said sulfate salt is present in an about between 1 wt. % and 4 wt. %, based on the total weight of the composition 11. The water treatment composition according to claim 1, further comprising cyanuric acid, and the cyanuric acid is present in an amount up to about 3 wt. % of cyanuric acid, based on the total weight of the composition. 12. The water treatment composition according to claim 1, wherein said composition is in the form of a unitary structure. 13. The water treatment composition according to claim 1, wherein said composition further comprises zinc oxide, citric acid, boric acid, sodium hexametaphosphate, phosphate reducers, polymeric clarifiers, dichlor, cyanuric acid, boric acid and combinations thereof. 14. The water treatment composition according to claim 1, further comprising 1-10 wt. % of aluminum sulfate; and 0.5-15 wt. % of a sulfate salt selected from the group consisting of copper sulfate, zinc sulfate, and combinations thereof; all weight percent are based on the total weight of said composition. 15. The water treatment composition according to claim 13, wherein said halogen-releasing compound comprises particulate trichloroisocyanuric acid, and the trichloroisocyanuric acid comprises from about 90 wt. % to about 96 wt. %, based on the total weight of the composition. 16. The water treatment composition according to claim 14, wherein said fluoropolymer is present in an amount between about 0.25 wt. % and about 5.0 wt. %, based on the total weight of the composition. 17. The water treatment composition according to claim 16, wherein said fluoropolymer is present in an amount between about 0.5 wt. % and about 2.0 wt. %, based on the total weight of the composition. 18. The water treatment composition according to claim 14, wherein said aluminum sulfate is present between about 0.2 wt. % and about 4 wt. %, and said sulfate salt comprises from about 1 wt. % and about 4 wt. % based on the total weight of the composition. 19. The water treatment composition according to claim 14, further comprising cyanuric acid, and the cyanuric acid is present in an amount up to 3 wt. % of cyanuric acid, based on the total weight of the composition. 20. The water treatment composition according to claim 14, wherein said composition is the form of a unitary solid. 21. The water treatment composition according to claim 14, wherein said composition further comprises zinc oxide, citric acid, boric acid, sodium hexametaphosphate, phosphate reducers, polymeric clarifiers, dichlor, cyanuric acid, and combinations thereof. 22. The water treatment composition according to claim 14, further comprising 1-10 wt. % of sodium hexametaphosphate (SHMP), based on the total weight of said composition. 23. The water treatment composition according to claim 1, further comprising 1-10 wt. % of sodium hexametaphosphate (SHMP) and 0.5-15 wt. % of a sulfate salt selected from the group consisting of copper sulfate, zinc sulfate, and combinations thereof; based on the total weight of said composition. 24. The water treatment composition according to claim 1, wherein the fluoropolymer is in the form of agglomerated particles. 25. The water treatment composition according to claim 1, wherein the fluoropolymer is in the form of essentially non-agglomerated particles. 26. The water treatment composition according to claim 1, wherein the halogen-releasing compound comprises a mixture of particulate trichloroisocyanuric acid and a halogen containing hydantoin.

Compositions and methods are provided for tissue constructs that promote wound healing. The composition comprises a dimensionally stable fibrin construct for local administration to a wound site or region. In one embodiment, the fibrin construct is a wound healing composition, including components that promote wound healing, such as platelets, growth factors, white blood cells and fibrin clots. In another embodiment, the tissue treatment composition includes (i) aggregated fibrin, (ii) blood cells, and (iii) optionally, growth factors and/or other proteins.

1. A method of preparing a growth factor enriched construct, comprising: collecting a blood sample comprising unaggregated fibrin; mixing the blood sample in a container with a coagulation activator to initiate aggregation of the fibrin; exposing the blood mixture to a separation force that separates the blood mixture into a gradient of plasma, aggregated fibrin and blood cells; and harvesting the aggregated fibrin and at least a portion of the blood cells to form a dimensionally stable, suturable fibrin construct, wherein the fibrin construct has a growth factor enriched surface concentrated with blood cells and platelets capable of releasing a growth factor and an opposed, growth factor depleted surface. 2. The method of claim 1, wherein the fibrin construct has a resiliency that is measured by an elongation at break strength of at least about 200%. 3. The method of claim 1, wherein the fibrin construct has a strength that is measured by an ultimate strength of at least about 0.15 MPa. 4. The method of claim 3, wherein the strength is further measured by a compression strength of at least about 30 kPa. 5. The method of claim 1, wherein the growth factor depleted surface is substantially lacking in blood cells. 6. The method of claim 1, wherein the growth factor depleted surface is substantially lacking in red blood cells. 7. The method of claim 1, wherein the growth factor depleted surface includes white blood cells. 8. The method of claim 1, wherein the blood cells comprise white blood cells and platelets. 9. The method of claim 1, wherein the step of collecting the blood sample comprises obtaining blood from a single donor. 10. The method of claim 1, wherein the step of collecting the blood sample comprises obtaining blood from multiple donors. 11. The method of claim 1, wherein the container is borosilicate glass container. 12. The method of claim 1, wherein the step of collecting the blood sample comprises exposing the blood sample to an anti-coagulant. 13. The method of claim 12, wherein the anti-coagulant is anticoagulant citrate dextrose solution A and the coagulation activator is calcium chloride. 14. The method of claim 1, wherein the separation force is centrifugation. 15. The method of claim 14, wherein the centrifugation is at a speed of at least about 2000×g. 16. The method of claim 14, further comprising an additional centrifugation at a speed of at least about 2000×g. 17. The method of claim 1, further comprising removing excess liquid from the fibrin construct by blotting the fibrin construct on an absorbent material. 18. The method of claim 1, further comprising folding the fibrin construct upon itself to form a folded construct such that adjacent halves of the growth factor enriched surface contact each other and form an inner portion of the folded construct while the growth factor depleted surface forms an outer portion of the folded construct. 19. The method of claim 1, further comprising forming a multilayered construct by layering a second fibrin construct on top of the first fibrin construct such that the growth factor enriched surfaces of each construct are in contact with each other and the growth factor depleted surfaces of each of the constructs form outer surfaces of the multilayered construct. 20. The method of claim 1, further comprising cross-linking the fibrin construct. 21.-51. (canceled)

Compositions and methods are provided for tissue constructs that promote wound healing. The composition comprises a dimensionally stable fibrin construct for local administration to a wound site or region. In one embodiment, the fibrin construct is a wound healing composition, including components that promote wound healing, such as platelets, growth factors, white blood cells and fibrin clots. In another embodiment, the tissue treatment composition includes (i) aggregated fibrin, (ii) blood cells, and (iii) optionally, growth factors and/or other proteins.1. A method of preparing a growth factor enriched construct, comprising: collecting a blood sample comprising unaggregated fibrin; mixing the blood sample in a container with a coagulation activator to initiate aggregation of the fibrin; exposing the blood mixture to a separation force that separates the blood mixture into a gradient of plasma, aggregated fibrin and blood cells; and harvesting the aggregated fibrin and at least a portion of the blood cells to form a dimensionally stable, suturable fibrin construct, wherein the fibrin construct has a growth factor enriched surface concentrated with blood cells and platelets capable of releasing a growth factor and an opposed, growth factor depleted surface. 2. The method of claim 1, wherein the fibrin construct has a resiliency that is measured by an elongation at break strength of at least about 200%. 3. The method of claim 1, wherein the fibrin construct has a strength that is measured by an ultimate strength of at least about 0.15 MPa. 4. The method of claim 3, wherein the strength is further measured by a compression strength of at least about 30 kPa. 5. The method of claim 1, wherein the growth factor depleted surface is substantially lacking in blood cells. 6. The method of claim 1, wherein the growth factor depleted surface is substantially lacking in red blood cells. 7. The method of claim 1, wherein the growth factor depleted surface includes white blood cells. 8. The method of claim 1, wherein the blood cells comprise white blood cells and platelets. 9. The method of claim 1, wherein the step of collecting the blood sample comprises obtaining blood from a single donor. 10. The method of claim 1, wherein the step of collecting the blood sample comprises obtaining blood from multiple donors. 11. The method of claim 1, wherein the container is borosilicate glass container. 12. The method of claim 1, wherein the step of collecting the blood sample comprises exposing the blood sample to an anti-coagulant. 13. The method of claim 12, wherein the anti-coagulant is anticoagulant citrate dextrose solution A and the coagulation activator is calcium chloride. 14. The method of claim 1, wherein the separation force is centrifugation. 15. The method of claim 14, wherein the centrifugation is at a speed of at least about 2000×g. 16. The method of claim 14, further comprising an additional centrifugation at a speed of at least about 2000×g. 17. The method of claim 1, further comprising removing excess liquid from the fibrin construct by blotting the fibrin construct on an absorbent material. 18. The method of claim 1, further comprising folding the fibrin construct upon itself to form a folded construct such that adjacent halves of the growth factor enriched surface contact each other and form an inner portion of the folded construct while the growth factor depleted surface forms an outer portion of the folded construct. 19. The method of claim 1, further comprising forming a multilayered construct by layering a second fibrin construct on top of the first fibrin construct such that the growth factor enriched surfaces of each construct are in contact with each other and the growth factor depleted surfaces of each of the constructs form outer surfaces of the multilayered construct. 20. The method of claim 1, further comprising cross-linking the fibrin construct. 21.-51. (canceled)

A fragrance sampler is provided which is made from a bottom ply and a top ply of material and an applicator. A cosmetic sample, such as a wet fragrance, is deposited on the bottom ply. An absorbent applicator attaches to the top ply. The applicator collects a portion from the sample and then applies the portion for testing. A well is formed in at least one of the plies and is sized and shaped to surround the sample. An applicator joins to the other ply and fits within the well. The two plies are then adhered together to form a liquid tight seal between the plies and around the well.

1. A sampler for inserting into printed matter such as a magazine or a mass mailing, the sampler including: a bottom ply having a top surface and a bottom surface; a well formed in said bottom ply to receive a cosmetic sample; a top ply having a top surface and a bottom surface; one or more walls formed in at least the bottom ply; a channel formed in the top ply; and, an applicator located within said channel and attached to said top ply; whereby said channel, said walls, and said well when placed adjacent resist compression; whereby said top ply adheres to said bottom ply to form a liquid tight seal to prevent leakage of the cosmetic sample. 2. The sampler of claim 1 wherein said well is a depression; said channel has dimensions at least as large as said well, such that said walls align and said channel fits over said well; and, said top surface of said top ply adheres to said top surface of said bottom ply. 3. The sampler of claim 2 further comprising: a frame upon said top surface of said bottom ply outside of said well; and, a frame upon said top surface of said top ply outside of said channel, whereby said frames adhere together by heat sealing, sonic sealing, or waterproof adhesive, with said channel atop said well. 4. The sampler of claim 2 further comprising: said channel, said well, and said walls having a generally rectangular cross section and radiused corners; and, said well having a texture to retain the cosmetic sample. 5. The sampler of claim 2 wherein said well, said channel, and said applicator have a mutually similar shape in plan view of one of round, oval, ovoid, rectangular, or polygonal. 6. The sampler of claim 2 wherein said applicator is absorbent fabric and said top ply and said bottom ply are a laminate of one of metal foil, polyester, or polyethylene. 7. The sampler of claim 6 further comprising: said applicator having cellulose, organic, or inorganic non-woven material, and said applicator joining to said top ply by one of welding, fusing, pressing, or gluing. 8. The sampler of claim 1 wherein said top ply is symmetric to said bottom ply and said applicator is symmetric to said well, said bottom ply has a pressure sensitive adhesive to connect said sampler to the printed matter, and said top ply and said bottom ply are formed by an embossing/debossing process. 9. The sampler of claim 4 wherein said walls are one of single or double. 10. A sampler for inserting into printed matter such as a magazine or a mass mailing has a bottom ply with a top surface and a bottom surface, the bottom ply having a pressure sensitive adhesive to connect the sampler into printed matter, a well formed in the bottom ply with a texture to store a cosmetic sample, a top ply with a top surface and a bottom surface and position opposite the bottom ply, one or more walls formed in at least the bottom ply and aligned between the bottom ply and the top ply, a channel formed in the top ply and the top ply adheres to the bottom ply to seal the cosmetic sample within the sampler without leakage, and the well, walls, and channel when located adjacent resist compression, wherein the improvement comprises: an applicator located within the channel and attached to the top ply; whereby the bottom ply and the top ply are formed by an embossing/debossing process; whereby a user positions said applicator upon the sample, said applicator collects some of the sample, and then a user places said applicator upon her face to test the sample. 11. The sampler of claim 10 wherein said applicator has a length, width, and height to fit within the channel and the channel fits within the well. 12. The sampler of claim 10 wherein said applicator is absorbent fabric. 13. The sampler of claim 12 further comprising: said applicator being cellulose, organic, or inorganic non-woven material, and said applicator joining to the top ply by one of welding, fusing, pressing, or gluing. 14. The sampler of claim 1 wherein one of said top ply and bottom ply having a pressure sensitive adhesive provided thereon to allow for connection of said sampler to any printed matter.

A fragrance sampler is provided which is made from a bottom ply and a top ply of material and an applicator. A cosmetic sample, such as a wet fragrance, is deposited on the bottom ply. An absorbent applicator attaches to the top ply. The applicator collects a portion from the sample and then applies the portion for testing. A well is formed in at least one of the plies and is sized and shaped to surround the sample. An applicator joins to the other ply and fits within the well. The two plies are then adhered together to form a liquid tight seal between the plies and around the well.1. A sampler for inserting into printed matter such as a magazine or a mass mailing, the sampler including: a bottom ply having a top surface and a bottom surface; a well formed in said bottom ply to receive a cosmetic sample; a top ply having a top surface and a bottom surface; one or more walls formed in at least the bottom ply; a channel formed in the top ply; and, an applicator located within said channel and attached to said top ply; whereby said channel, said walls, and said well when placed adjacent resist compression; whereby said top ply adheres to said bottom ply to form a liquid tight seal to prevent leakage of the cosmetic sample. 2. The sampler of claim 1 wherein said well is a depression; said channel has dimensions at least as large as said well, such that said walls align and said channel fits over said well; and, said top surface of said top ply adheres to said top surface of said bottom ply. 3. The sampler of claim 2 further comprising: a frame upon said top surface of said bottom ply outside of said well; and, a frame upon said top surface of said top ply outside of said channel, whereby said frames adhere together by heat sealing, sonic sealing, or waterproof adhesive, with said channel atop said well. 4. The sampler of claim 2 further comprising: said channel, said well, and said walls having a generally rectangular cross section and radiused corners; and, said well having a texture to retain the cosmetic sample. 5. The sampler of claim 2 wherein said well, said channel, and said applicator have a mutually similar shape in plan view of one of round, oval, ovoid, rectangular, or polygonal. 6. The sampler of claim 2 wherein said applicator is absorbent fabric and said top ply and said bottom ply are a laminate of one of metal foil, polyester, or polyethylene. 7. The sampler of claim 6 further comprising: said applicator having cellulose, organic, or inorganic non-woven material, and said applicator joining to said top ply by one of welding, fusing, pressing, or gluing. 8. The sampler of claim 1 wherein said top ply is symmetric to said bottom ply and said applicator is symmetric to said well, said bottom ply has a pressure sensitive adhesive to connect said sampler to the printed matter, and said top ply and said bottom ply are formed by an embossing/debossing process. 9. The sampler of claim 4 wherein said walls are one of single or double. 10. A sampler for inserting into printed matter such as a magazine or a mass mailing has a bottom ply with a top surface and a bottom surface, the bottom ply having a pressure sensitive adhesive to connect the sampler into printed matter, a well formed in the bottom ply with a texture to store a cosmetic sample, a top ply with a top surface and a bottom surface and position opposite the bottom ply, one or more walls formed in at least the bottom ply and aligned between the bottom ply and the top ply, a channel formed in the top ply and the top ply adheres to the bottom ply to seal the cosmetic sample within the sampler without leakage, and the well, walls, and channel when located adjacent resist compression, wherein the improvement comprises: an applicator located within the channel and attached to the top ply; whereby the bottom ply and the top ply are formed by an embossing/debossing process; whereby a user positions said applicator upon the sample, said applicator collects some of the sample, and then a user places said applicator upon her face to test the sample. 11. The sampler of claim 10 wherein said applicator has a length, width, and height to fit within the channel and the channel fits within the well. 12. The sampler of claim 10 wherein said applicator is absorbent fabric. 13. The sampler of claim 12 further comprising: said applicator being cellulose, organic, or inorganic non-woven material, and said applicator joining to the top ply by one of welding, fusing, pressing, or gluing. 14. The sampler of claim 1 wherein one of said top ply and bottom ply having a pressure sensitive adhesive provided thereon to allow for connection of said sampler to any printed matter.

An improved excipient comprising substantially homogeneous particles of a compressible, high functionality granular microcrystalline cellulose based excipient is provided. The improved excipient comprises microcrystalline cellulose and a binder, and optionally a disintegrant, and is formed by spraying a homogeneous slurry of the components. The excipient provides enhanced flowability/good flow properties, excellent/high compactibility, and increased API loading and blendability as compared to the individual components, and as compared to conventional excipients formed from the same materials. The improved excipient has strong intraparticle bonding bridges between the components, resulting in a unique structural morphology including significant open structures or hollow pores. The presence of these pores provides a surface roughness that is the ideal environment for improved blending with an API.

1. A composition comprising: about 90% to about 99% microcrystalline cellulose; and about 1% to about 10% at least one binder; wherein the microcrystalline cellulose and binder are indistinguishable when viewed with a SEM, thereby forming substantially homogeneous-particles. 2. The composition of claim 1 wherein the composition includes: about 95% to about 99% microcrystalline cellulose; and about 1% to about 5% at least one binder. 3. The composition of claim 1 wherein the composition includes: about 97% to about 99% microcrystalline cellulose; and about 1% to about 3% at least one binder. 4. The composition of claim 1 wherein the binder includes hydroxypropyl methylcellulose. 5. The composition of claim 1 wherein the excipient is formed by homogenizing/spray dry granulating an aqueous slurry comprised of the microcrystalline cellulose and binder. 6. The composition of claim 1 wherein the aerated bulk density is 0.2-0.3 g/cc. 7. A method or making an excipient comprising: mixing a microcrystalline cellulose (MCC) slurry with a cross-linked polyvinylpyrrolidone slurry to form a MCC/cross-linked polyvinylpyrrolidone slurry; mixing a binder comprising hydroxypropyl methylcellulose in water to form a viscous hydroxypropyl methylcellulose slurry; homogenizing the hydroxypropyl methylcellulose slurry with the MCC/cross-linked polyvinylpyrrolidone slurry to form a homogenized slurry; and spray dry granulating the homogenized slurry through a rotary nozzle at an RPM of between 12,000 and 25,000 to form substantially homogeneous and substantially spherical particles of excipient wherein the microcrystalline cellulose and hydroxypropyl methylcellulose are indistinguishable when viewed with a SEM, and wherein said excipient is a complete and universal directly compressible excipient with a flowability index over 80. 8. (canceled) 9. (canceled) 10. (canceled) 11. (canceled) 12. (canceled) 13. (canceled) 14. (canceled) 15. (canceled) 16. pharmaceutical tablet comprising: at least one active pharmaceutical ingredient; a disintegrant; and an excipient of substantially homogeneous particles including: a) microcrystalline cellulose; and b) at least one binder. 17. The tablet of claim 16 wherein the excipient includes: about 90% to about 99% microcrystalline cellulose; and about 1% to about 10% at least one binder. 18. The tablet of claim 16 wherein the excipient includes: about 95% to about 99% microcrystalline cellulose; and about 1% to about 5% at least one binder. 19. The tablet of claim 16 wherein the excipient includes: about 97% to about 99% microcrystalline cellulose; and about 1% to about 3% at least one binder. 20. The tablet of claim 16 wherein the binder includes hydroxypropyl methylcellulose. 21. A method of making a pharmaceutical tablet comprising: mixing at least one active pharmaceutical ingredient with a disintegrant and an excipient of substantially homogeneous particles including: a) microcrystalline cellulose; and b) at least one binder; and compressing the mixture to form a tablet. 22. The method of claim 21 wherein the excipient includes: about 90% to about 99% microcrystalline cellulose; and about 1% to about 10% at least one binder. 23. The method of claim 21 wherein the excipient includes: about 95% to about 99% microcrystalline cellulose; and about 1% to about 5% at least one binder. 24. The method of claim 21 wherein the excipient includes: about 97% to about 99% microcrystalline cellulose; and about 1% to about 3% at least one binder. 25. The method of claim 21 wherein the binder includes hydroxypropyl methylcellulose. 26. A composition comprising: about 75% to about 98% microcrystalline cellulose; about 1% to about 10% at least one binder; and about 1% to about 20% at least one disintegrant; wherein the microcrystalline cellulose, binder and disintegrant are indistinguishable when viewed with a SEM, thereby forming substantially homogeneous, substantially spherical particles. 27. The composition of claim 26 wherein the composition includes: about 80% to about 90% microcrystalline cellulose; about 2% to about 8% at least one binder; and about 3% to about 12% at least one disintegrant. 28. The composition of claim 26 wherein the composition includes: about 85% to about 93% microcrystalline cellulose; about 2% to about 5% at least one binder; and about 10% at least one disintegrant. 29. The composition of claim 26 wherein the binder includes hydroxypropyl methylcellulose and the disintegrant includes cross-linked polyvinylpyrrolidone. 30. The composition of claim 26 wherein the excipient is formed by spraying an aqueous slurry comprised of the microcrystalline cellulose, binder and disintegrant. 31. A method of making an excipient comprising: mixing a MCC slurry with a disintegrant slurry to form a MCC/disintegrant slurry; mixing a binder in water to form a viscous binder slurry; homogenizing the binder slurry with the MCC/disintegrant slurry to form a homogenized slurry; and spray dry granulating the homogenized slurry to form substantially homogeneous, substantially spherical particles of excipient. 32. The method of claim 31 wherein: about 75% to about 98% microcrystalline cellulose; about 1% to about 10% at least one binder; and about 1% to about 20% at least one disintegrant. 33. The method of claim 31 comprising: about 80% to about 90% microcrystalline cellulose; about 2% to about 8% at least one binder; and about 3% to about 12% at least one disintegrant. 34. The method of claim 31 comprising: about 85% to about 93% microcrystalline cellulose; about 2% to about 5% at least one binder; and about 10% at least one disintegrant. 35. The method of claim 31 wherein the binder includes hydroxypropyl methylcellulose and the disintegrant includes cross-linked polyvinylpyrrolidone. 36. (canceled) 37. The method of claim 7 comprising: about 75% to about 98% microcrystalline cellulose; about 1% to about 10% hydroxypropyl methylcellulose; and about 1% to about 20% cross-linked polyvinylpyrrolidone. 38. The method of claim 7 comprising: about 80% to about 90% microcrystalline cellulose; about 2% to about 8% hydroxypropyl methylcellulose; and about 3% to about 12% cross-linked polyvinylpyrrolidone. 39. The method of claim 7 comprising: about 85% to about 93% microcrystalline cellulose; about 2% to about 5% hydroxypropyl methylcellulose; and about 10% cross-linked polyvinylpyrrolidone. 40. A pharmaceutical tablet comprising: at least one active pharmaceutical ingredient; and an excipient of substantially homogeneous, substantially spherical particles including: a) microcrystalline cellulose; b) at least one binder; and c) at least one disintegrant. 41. The tablet of claim 40 wherein the at least one active pharmaceutical ingredient comprises about 1% to about 50% of the tablet. 42. The tablet of claim 40 further comprising a glidant, wherein the at least one active pharmaceutical ingredient comprises at least about 50% of the tablet, and wherein the tablet further comprises a glidant. 43. The tablet of claim 40 wherein the excipient includes: about 75% to about 98% microcrystalline cellulose; about 1% to about 10% at least one binder; and about 1% to about 20% at least one disintegrant. 44. The tablet of claim 40 wherein the excipient includes: about 80% to about 90% microcrystalline cellulose; about 2% to about 8% at least one binder; and about 3% to about 12% at least one disintegrant. 45. The tablet of claim 40 wherein the excipient includes: about 85% to about 93% microcrystalline cellulose; about 2% to about 5% at least one binder; and about 10% at least one disintegrant. 46. The tablet of claim 40 wherein the binder includes hydroxypropyl methylcellulose and the disintegrant includes cross-linked polyvinylpyrrolidone. 47. The tablet of claim 40 wherein the active pharmaceutical ingredient includes ibuprofen. 48. The tablet of claim 40 wherein the active pharmaceutical ingredient includes atorvastatin calcium. 49. A method of making a pharmaceutical tablet comprising: mixing at least one active pharmaceutical ingredient with an excipient of substantially homogeneous, substantially spherical particles according to claim 26; and compressing the mixture to form a tablet. 50. The method of claim 49 wherein the tablet is formed by a rotary tabletting machine. 51. The method of claim 49 further including coating the tablet.

An improved excipient comprising substantially homogeneous particles of a compressible, high functionality granular microcrystalline cellulose based excipient is provided. The improved excipient comprises microcrystalline cellulose and a binder, and optionally a disintegrant, and is formed by spraying a homogeneous slurry of the components. The excipient provides enhanced flowability/good flow properties, excellent/high compactibility, and increased API loading and blendability as compared to the individual components, and as compared to conventional excipients formed from the same materials. The improved excipient has strong intraparticle bonding bridges between the components, resulting in a unique structural morphology including significant open structures or hollow pores. The presence of these pores provides a surface roughness that is the ideal environment for improved blending with an API.1. A composition comprising: about 90% to about 99% microcrystalline cellulose; and about 1% to about 10% at least one binder; wherein the microcrystalline cellulose and binder are indistinguishable when viewed with a SEM, thereby forming substantially homogeneous-particles. 2. The composition of claim 1 wherein the composition includes: about 95% to about 99% microcrystalline cellulose; and about 1% to about 5% at least one binder. 3. The composition of claim 1 wherein the composition includes: about 97% to about 99% microcrystalline cellulose; and about 1% to about 3% at least one binder. 4. The composition of claim 1 wherein the binder includes hydroxypropyl methylcellulose. 5. The composition of claim 1 wherein the excipient is formed by homogenizing/spray dry granulating an aqueous slurry comprised of the microcrystalline cellulose and binder. 6. The composition of claim 1 wherein the aerated bulk density is 0.2-0.3 g/cc. 7. A method or making an excipient comprising: mixing a microcrystalline cellulose (MCC) slurry with a cross-linked polyvinylpyrrolidone slurry to form a MCC/cross-linked polyvinylpyrrolidone slurry; mixing a binder comprising hydroxypropyl methylcellulose in water to form a viscous hydroxypropyl methylcellulose slurry; homogenizing the hydroxypropyl methylcellulose slurry with the MCC/cross-linked polyvinylpyrrolidone slurry to form a homogenized slurry; and spray dry granulating the homogenized slurry through a rotary nozzle at an RPM of between 12,000 and 25,000 to form substantially homogeneous and substantially spherical particles of excipient wherein the microcrystalline cellulose and hydroxypropyl methylcellulose are indistinguishable when viewed with a SEM, and wherein said excipient is a complete and universal directly compressible excipient with a flowability index over 80. 8. (canceled) 9. (canceled) 10. (canceled) 11. (canceled) 12. (canceled) 13. (canceled) 14. (canceled) 15. (canceled) 16. pharmaceutical tablet comprising: at least one active pharmaceutical ingredient; a disintegrant; and an excipient of substantially homogeneous particles including: a) microcrystalline cellulose; and b) at least one binder. 17. The tablet of claim 16 wherein the excipient includes: about 90% to about 99% microcrystalline cellulose; and about 1% to about 10% at least one binder. 18. The tablet of claim 16 wherein the excipient includes: about 95% to about 99% microcrystalline cellulose; and about 1% to about 5% at least one binder. 19. The tablet of claim 16 wherein the excipient includes: about 97% to about 99% microcrystalline cellulose; and about 1% to about 3% at least one binder. 20. The tablet of claim 16 wherein the binder includes hydroxypropyl methylcellulose. 21. A method of making a pharmaceutical tablet comprising: mixing at least one active pharmaceutical ingredient with a disintegrant and an excipient of substantially homogeneous particles including: a) microcrystalline cellulose; and b) at least one binder; and compressing the mixture to form a tablet. 22. The method of claim 21 wherein the excipient includes: about 90% to about 99% microcrystalline cellulose; and about 1% to about 10% at least one binder. 23. The method of claim 21 wherein the excipient includes: about 95% to about 99% microcrystalline cellulose; and about 1% to about 5% at least one binder. 24. The method of claim 21 wherein the excipient includes: about 97% to about 99% microcrystalline cellulose; and about 1% to about 3% at least one binder. 25. The method of claim 21 wherein the binder includes hydroxypropyl methylcellulose. 26. A composition comprising: about 75% to about 98% microcrystalline cellulose; about 1% to about 10% at least one binder; and about 1% to about 20% at least one disintegrant; wherein the microcrystalline cellulose, binder and disintegrant are indistinguishable when viewed with a SEM, thereby forming substantially homogeneous, substantially spherical particles. 27. The composition of claim 26 wherein the composition includes: about 80% to about 90% microcrystalline cellulose; about 2% to about 8% at least one binder; and about 3% to about 12% at least one disintegrant. 28. The composition of claim 26 wherein the composition includes: about 85% to about 93% microcrystalline cellulose; about 2% to about 5% at least one binder; and about 10% at least one disintegrant. 29. The composition of claim 26 wherein the binder includes hydroxypropyl methylcellulose and the disintegrant includes cross-linked polyvinylpyrrolidone. 30. The composition of claim 26 wherein the excipient is formed by spraying an aqueous slurry comprised of the microcrystalline cellulose, binder and disintegrant. 31. A method of making an excipient comprising: mixing a MCC slurry with a disintegrant slurry to form a MCC/disintegrant slurry; mixing a binder in water to form a viscous binder slurry; homogenizing the binder slurry with the MCC/disintegrant slurry to form a homogenized slurry; and spray dry granulating the homogenized slurry to form substantially homogeneous, substantially spherical particles of excipient. 32. The method of claim 31 wherein: about 75% to about 98% microcrystalline cellulose; about 1% to about 10% at least one binder; and about 1% to about 20% at least one disintegrant. 33. The method of claim 31 comprising: about 80% to about 90% microcrystalline cellulose; about 2% to about 8% at least one binder; and about 3% to about 12% at least one disintegrant. 34. The method of claim 31 comprising: about 85% to about 93% microcrystalline cellulose; about 2% to about 5% at least one binder; and about 10% at least one disintegrant. 35. The method of claim 31 wherein the binder includes hydroxypropyl methylcellulose and the disintegrant includes cross-linked polyvinylpyrrolidone. 36. (canceled) 37. The method of claim 7 comprising: about 75% to about 98% microcrystalline cellulose; about 1% to about 10% hydroxypropyl methylcellulose; and about 1% to about 20% cross-linked polyvinylpyrrolidone. 38. The method of claim 7 comprising: about 80% to about 90% microcrystalline cellulose; about 2% to about 8% hydroxypropyl methylcellulose; and about 3% to about 12% cross-linked polyvinylpyrrolidone. 39. The method of claim 7 comprising: about 85% to about 93% microcrystalline cellulose; about 2% to about 5% hydroxypropyl methylcellulose; and about 10% cross-linked polyvinylpyrrolidone. 40. A pharmaceutical tablet comprising: at least one active pharmaceutical ingredient; and an excipient of substantially homogeneous, substantially spherical particles including: a) microcrystalline cellulose; b) at least one binder; and c) at least one disintegrant. 41. The tablet of claim 40 wherein the at least one active pharmaceutical ingredient comprises about 1% to about 50% of the tablet. 42. The tablet of claim 40 further comprising a glidant, wherein the at least one active pharmaceutical ingredient comprises at least about 50% of the tablet, and wherein the tablet further comprises a glidant. 43. The tablet of claim 40 wherein the excipient includes: about 75% to about 98% microcrystalline cellulose; about 1% to about 10% at least one binder; and about 1% to about 20% at least one disintegrant. 44. The tablet of claim 40 wherein the excipient includes: about 80% to about 90% microcrystalline cellulose; about 2% to about 8% at least one binder; and about 3% to about 12% at least one disintegrant. 45. The tablet of claim 40 wherein the excipient includes: about 85% to about 93% microcrystalline cellulose; about 2% to about 5% at least one binder; and about 10% at least one disintegrant. 46. The tablet of claim 40 wherein the binder includes hydroxypropyl methylcellulose and the disintegrant includes cross-linked polyvinylpyrrolidone. 47. The tablet of claim 40 wherein the active pharmaceutical ingredient includes ibuprofen. 48. The tablet of claim 40 wherein the active pharmaceutical ingredient includes atorvastatin calcium. 49. A method of making a pharmaceutical tablet comprising: mixing at least one active pharmaceutical ingredient with an excipient of substantially homogeneous, substantially spherical particles according to claim 26; and compressing the mixture to form a tablet. 50. The method of claim 49 wherein the tablet is formed by a rotary tabletting machine. 51. The method of claim 49 further including coating the tablet.

A biologic-adsorbent, e.g., protein-adsorbent, material is prepared by forming a polymeric substrate into structures having high surface area topography whose biologic adsorbing properties can be controlled. Biologic adsorption by these structures of optimized high surface area topography is increased by mild treating of the surfaces, e.g., by oxygen plasma, without substantially altering topography. Structures can have tailored geometric features including microstructures, e.g., pillars, with a diameter from 100 nm-50 μm and height greater than 1 μm.

1. A biologic adsorbent structure comprising a polymeric substrate having a substantially fixed topography, said substantially fixed topography comprising substructures having dimensions ranging from about 100 nanometers to about 50 microns, said substructures formed by contact with a shaped surface for imparting increased surface area to at least one surface thereof, said polymeric substrate formed from a polymeric material comprising a cyclic poly(olefin), a non-cyclic polyolefin or blends thereof, wherein said at least one surface is plasma-treated. 2. The structure of claim 1, wherein said polymeric material comprises a cyclic poly(olefin). 3. The structure of claim 2, wherein said cyclic poly(olefin) comprises an amorphous polyolefin having a cyclic structure polymerized by ring opening metathesis polymerization of norbornene or norbornene derivatives, followed by hydrogenation of double bonds. 4. The structure of claim 1, wherein each surface of said polymeric substrate has a substantially fixed topography comprising substructures having dimensions ranging from about 100 nanometers to about 50 microns, formed by contact with a shaped surface for imparting increased surface area, and is plasma-treated. 5. The structure of claim 1 wherein said topography is formed by imprinting or hot embossing. 6. The structure of claim 5 wherein said imprinted topography is obtained using a mold prepared using at least one of photolithography and polycarbonate membrane. 7. The structure of claim 1, wherein said at least one surface has a water contact angle no greater than 60 degrees. 8. The structure of claim 1, wherein said polymeric material provides a water contact angle of 60 degrees or greater when tested in the form of a flat untreated film or untreated film with substructures. 9. The structure of claim 1 wherein said at least one plasma-treated surface is an oxygen plasma-treated surface. 10. The structure of claim 9 wherein said at least one plasma-treated surface is a surface treated at 50 to 150 watts for 15 to 45 seconds. 11. The structure of claim 9 wherein said at least one plasma-treated surface is a surface treated at 75 to 125 watts for 25 to 35 seconds. 12. The structure of claim 9 wherein said at least one surface is plasma-treated under conditions sufficient to increase wettability to an extent sufficient to provide a water contact angle of no greater than 60 degrees without substantially altering the topography. 13. The structure of claim 1 wherein said topography comprises pillar-like substructures having an average cross-section width ranging from 100 nanometers to 50 microns, an average height ranging from 1 to 50 microns, and an aspect ratio ranging from 0.1 to 50. 14. The structure of claim 13 wherein the topography comprises pillar-like substructures having an average cross-section width ranging from 1 to 10 microns, an average height ranging from 3 to 20 microns, and an aspect ratio ranging from 1 to 20. 15. The structure of claim 1 wherein the ratio of increased surface area compared to a flat surface is at least 1.01. 16. The structure of claim 15 wherein the ratio of increased surface area compared to a flat surface is at least 1.1. 17. The structure of claim 16 wherein the pillar-like substructures are spaced apart at an average inter-structural spacing of from 100 nanometers to 100 microns. 18. The structure of claim 17 wherein the pillar-like substructures are spaced apart at an average inter-structural spacing of from 1 to 50 microns. 19. The structure of claim 1 wherein the pillar-like substructures have a protrusion density of from about 1×105 to about 6×108 protrusions/cm2. 20. The structure of claim 19 wherein the pillar-like substructures have a protrusion density of from about 1×107 to about 5×107 protrusions per cm2. 21. A diagnostic test device comprising the structure of claim 1 whose surface is capable of adsorbing a biologic analyte. 22. A method for preparing a biologic adsorbent structure which comprises: a) contacting a polymeric mass comprising a cyclic poly(olefin), a non-cyclic polyolefin, or blends thereof, with a shaped surface to form a polymeric substrate having an increased surface area on at least one surface of the polymeric substrate to provide a surface of substantially fixed topography; and b) plasma-treating the at least one surface of substantially fixed topography to increase wettability as measured by water contact angle, without substantially altering the topography. 23. The method of claim 22, wherein said polymeric material comprises a cyclic poly(olefin). 24. The method of claim 23 wherein the polymeric substrate provides a water contact angle of 60 degrees or greater when tested in the form of a flat untreated film or untreated film with substructures. 25. The method of claim 22 wherein the cyclic poly(olefin) comprises an amorphous polyolefin having a cyclic structure polymerized by ring opening metathesis polymerization of norbornene or norbornene derivatives, followed by hydrogenation of double bonds. 26. The method of claim 22 wherein said contacting is formed by imprinting or hot embossing. 27. The method of claim 26 wherein said imprinting uses a mold prepared using at least one of photolithography and polycarbonate membrane. 28. The method of claim 22 wherein the plasma-treated surface is treated with oxygen plasma. 29. The method of claim 28 wherein the plasma-treated surface is treated at 50 to 150 watts for 15 to 45 seconds. 30. The method of claim 28 wherein the plasma-treated surface is treated at 75 to 125 watts for 25 to 35 seconds. 31. The method of claim 28 wherein the plasma-treated surface is treated under conditions sufficient to increase wettability to an extent sufficient to provide a water contact angle of no greater than 60 degrees without substantially altering the topography. 32. The method of claim 22 wherein the topography comprises pillar-like substructures having an average cross-section width ranging from 100 nanometers to 50 microns, an average height ranging from 1 to 50 microns, and an aspect ratio ranging from 0.1 to 50. 33. The method of claim 32 wherein the topography comprises pillar-like substructures having an average cross-section width ranging from 1 to 10 microns, an average height ranging from 3 to 20 microns, and an aspect ratio ranging from 1 to 20. 34. The method of claim 22 wherein the ratio of increased surface area compared to a flat surface is at least 1.01. 35. The method of claim 34 wherein the ratio of increased surface area compared to a flat surface is at least 1.1. 36. The method of claim 32 wherein the pillar-like substructures are spaced apart at an average inter-structural spacing of from 100 nanometers to 100 microns. 37. The method of claim 36 wherein the pillar-like substructures are spaced apart at an average inter-structural spacing of from 1 to 50 microns. 38. The method of claim 32 wherein the pillar-like substructures have a protrusion density of from about 1×105 to about 6×108 protrusions/cm2. 39. The method of claim 38 wherein the pillar-like substructures have a protrusion density of from about 1×107 to about 5×107 protrusions per cm2. 40. A method for modulating the amount of biologic uptake of a polymeric structure mass comprising a cyclic poly(olefin), a non-cyclic polyolefin, or blends thereof, the polymeric structure having a substantially fixed topography and high surface area whose biologic uptake is otherwise not a function of surface area which comprises surface treating the polymeric structure by plasma treatment to increase wettability without substantially altering the topography. 41. The method of claim 40 wherein the increase in wettability is determined by measuring a reduction in water contact angle for the treated surface compared to the untreated surface. 42. The method of claim 40 wherein the biologic is selected from at least one of sugar, lipid, protein, nucleic acid, and polynucleotide. 43. The method of claim 40 wherein the plasma treatment is oxygen plasma treatment. 44. The method of claim 43 wherein the plasma-treated surface is treated under conditions sufficient to increase wettability to an extent sufficient to provide a water contact angle of no greater than 60 degrees without substantially altering the topography. 45. The method of claim 40 wherein the plasma-treated surface is treated under conditions sufficient to increase wettability to an extent sufficient to provide a water contact angle of no greater than 60 degrees without substantially altering the topography.

A biologic-adsorbent, e.g., protein-adsorbent, material is prepared by forming a polymeric substrate into structures having high surface area topography whose biologic adsorbing properties can be controlled. Biologic adsorption by these structures of optimized high surface area topography is increased by mild treating of the surfaces, e.g., by oxygen plasma, without substantially altering topography. Structures can have tailored geometric features including microstructures, e.g., pillars, with a diameter from 100 nm-50 μm and height greater than 1 μm.1. A biologic adsorbent structure comprising a polymeric substrate having a substantially fixed topography, said substantially fixed topography comprising substructures having dimensions ranging from about 100 nanometers to about 50 microns, said substructures formed by contact with a shaped surface for imparting increased surface area to at least one surface thereof, said polymeric substrate formed from a polymeric material comprising a cyclic poly(olefin), a non-cyclic polyolefin or blends thereof, wherein said at least one surface is plasma-treated. 2. The structure of claim 1, wherein said polymeric material comprises a cyclic poly(olefin). 3. The structure of claim 2, wherein said cyclic poly(olefin) comprises an amorphous polyolefin having a cyclic structure polymerized by ring opening metathesis polymerization of norbornene or norbornene derivatives, followed by hydrogenation of double bonds. 4. The structure of claim 1, wherein each surface of said polymeric substrate has a substantially fixed topography comprising substructures having dimensions ranging from about 100 nanometers to about 50 microns, formed by contact with a shaped surface for imparting increased surface area, and is plasma-treated. 5. The structure of claim 1 wherein said topography is formed by imprinting or hot embossing. 6. The structure of claim 5 wherein said imprinted topography is obtained using a mold prepared using at least one of photolithography and polycarbonate membrane. 7. The structure of claim 1, wherein said at least one surface has a water contact angle no greater than 60 degrees. 8. The structure of claim 1, wherein said polymeric material provides a water contact angle of 60 degrees or greater when tested in the form of a flat untreated film or untreated film with substructures. 9. The structure of claim 1 wherein said at least one plasma-treated surface is an oxygen plasma-treated surface. 10. The structure of claim 9 wherein said at least one plasma-treated surface is a surface treated at 50 to 150 watts for 15 to 45 seconds. 11. The structure of claim 9 wherein said at least one plasma-treated surface is a surface treated at 75 to 125 watts for 25 to 35 seconds. 12. The structure of claim 9 wherein said at least one surface is plasma-treated under conditions sufficient to increase wettability to an extent sufficient to provide a water contact angle of no greater than 60 degrees without substantially altering the topography. 13. The structure of claim 1 wherein said topography comprises pillar-like substructures having an average cross-section width ranging from 100 nanometers to 50 microns, an average height ranging from 1 to 50 microns, and an aspect ratio ranging from 0.1 to 50. 14. The structure of claim 13 wherein the topography comprises pillar-like substructures having an average cross-section width ranging from 1 to 10 microns, an average height ranging from 3 to 20 microns, and an aspect ratio ranging from 1 to 20. 15. The structure of claim 1 wherein the ratio of increased surface area compared to a flat surface is at least 1.01. 16. The structure of claim 15 wherein the ratio of increased surface area compared to a flat surface is at least 1.1. 17. The structure of claim 16 wherein the pillar-like substructures are spaced apart at an average inter-structural spacing of from 100 nanometers to 100 microns. 18. The structure of claim 17 wherein the pillar-like substructures are spaced apart at an average inter-structural spacing of from 1 to 50 microns. 19. The structure of claim 1 wherein the pillar-like substructures have a protrusion density of from about 1×105 to about 6×108 protrusions/cm2. 20. The structure of claim 19 wherein the pillar-like substructures have a protrusion density of from about 1×107 to about 5×107 protrusions per cm2. 21. A diagnostic test device comprising the structure of claim 1 whose surface is capable of adsorbing a biologic analyte. 22. A method for preparing a biologic adsorbent structure which comprises: a) contacting a polymeric mass comprising a cyclic poly(olefin), a non-cyclic polyolefin, or blends thereof, with a shaped surface to form a polymeric substrate having an increased surface area on at least one surface of the polymeric substrate to provide a surface of substantially fixed topography; and b) plasma-treating the at least one surface of substantially fixed topography to increase wettability as measured by water contact angle, without substantially altering the topography. 23. The method of claim 22, wherein said polymeric material comprises a cyclic poly(olefin). 24. The method of claim 23 wherein the polymeric substrate provides a water contact angle of 60 degrees or greater when tested in the form of a flat untreated film or untreated film with substructures. 25. The method of claim 22 wherein the cyclic poly(olefin) comprises an amorphous polyolefin having a cyclic structure polymerized by ring opening metathesis polymerization of norbornene or norbornene derivatives, followed by hydrogenation of double bonds. 26. The method of claim 22 wherein said contacting is formed by imprinting or hot embossing. 27. The method of claim 26 wherein said imprinting uses a mold prepared using at least one of photolithography and polycarbonate membrane. 28. The method of claim 22 wherein the plasma-treated surface is treated with oxygen plasma. 29. The method of claim 28 wherein the plasma-treated surface is treated at 50 to 150 watts for 15 to 45 seconds. 30. The method of claim 28 wherein the plasma-treated surface is treated at 75 to 125 watts for 25 to 35 seconds. 31. The method of claim 28 wherein the plasma-treated surface is treated under conditions sufficient to increase wettability to an extent sufficient to provide a water contact angle of no greater than 60 degrees without substantially altering the topography. 32. The method of claim 22 wherein the topography comprises pillar-like substructures having an average cross-section width ranging from 100 nanometers to 50 microns, an average height ranging from 1 to 50 microns, and an aspect ratio ranging from 0.1 to 50. 33. The method of claim 32 wherein the topography comprises pillar-like substructures having an average cross-section width ranging from 1 to 10 microns, an average height ranging from 3 to 20 microns, and an aspect ratio ranging from 1 to 20. 34. The method of claim 22 wherein the ratio of increased surface area compared to a flat surface is at least 1.01. 35. The method of claim 34 wherein the ratio of increased surface area compared to a flat surface is at least 1.1. 36. The method of claim 32 wherein the pillar-like substructures are spaced apart at an average inter-structural spacing of from 100 nanometers to 100 microns. 37. The method of claim 36 wherein the pillar-like substructures are spaced apart at an average inter-structural spacing of from 1 to 50 microns. 38. The method of claim 32 wherein the pillar-like substructures have a protrusion density of from about 1×105 to about 6×108 protrusions/cm2. 39. The method of claim 38 wherein the pillar-like substructures have a protrusion density of from about 1×107 to about 5×107 protrusions per cm2. 40. A method for modulating the amount of biologic uptake of a polymeric structure mass comprising a cyclic poly(olefin), a non-cyclic polyolefin, or blends thereof, the polymeric structure having a substantially fixed topography and high surface area whose biologic uptake is otherwise not a function of surface area which comprises surface treating the polymeric structure by plasma treatment to increase wettability without substantially altering the topography. 41. The method of claim 40 wherein the increase in wettability is determined by measuring a reduction in water contact angle for the treated surface compared to the untreated surface. 42. The method of claim 40 wherein the biologic is selected from at least one of sugar, lipid, protein, nucleic acid, and polynucleotide. 43. The method of claim 40 wherein the plasma treatment is oxygen plasma treatment. 44. The method of claim 43 wherein the plasma-treated surface is treated under conditions sufficient to increase wettability to an extent sufficient to provide a water contact angle of no greater than 60 degrees without substantially altering the topography. 45. The method of claim 40 wherein the plasma-treated surface is treated under conditions sufficient to increase wettability to an extent sufficient to provide a water contact angle of no greater than 60 degrees without substantially altering the topography.

The present invention relates to compositions and methods for disease control in plants. The compositions for use in the methods of the invention include glyphosate as the active compound. In addition, methods and compositions are disclosed to prevent and treat pest infection in glyphosate tolerant plants.

1. A method of controlling a fungal or fungal-like pathogen induced disease in a glyphosate tolerant onion plant comprising, identifying an onion plant in need of disease control; and contacting said onion plant with an effective amount of a composition having glyphosate, whereby said disease of said onion plant is controlled. 2. The method of claim 1, wherein said disease is caused by an Oomycete 3. The method of claim 2, wherein said Oomycete is Peronospora destructor. 4. The method of claim 1, wherein said effective amount of said composition having glyphosate yields an application of a concentration of glyphosate between about 8 ounces/acre to about 128 ounces/acre. 5. The method of claim 1, wherein said onion is contacted with said effective amount of glyphosate before infection by said pathogen. 6. The method of claim 1, wherein said onion is contacted with said effective amount of glyphosate after infection by said pathogen. 7. A method of treating a mildew disease in a glyphosate tolerant onion plant comprising, identifying an onion plant in need of mildew disease treatment; and contacting said onion plant with an effective amount of a composition having glyphosate, whereby said mildew disease of said onion plant is treated. 8. The method of claim 7, wherein said mildew disease is caused by a Peronospora species. 9. The method of claim 8, wherein said Peronospora species is Peronospora destructor. 10. The method of claim 7, wherein said effective amount of said composition having glyphosate yields an application of a concentration of glyphosate between about 8 ounces/acre to about 128 ounces/acre. 11. A method of controlling a fungal or fungal-like pathogen induced disease in a glyphosate tolerant pea plant comprising, identifying a pea plant in need of disease control; and contacting said pea plant with an effective amount of a composition having glyphosate, whereby said disease of said pea plant is controlled. 12. The method of claim 11, wherein said disease is caused by a pathogen of the family Erysiphaceae 13. The method of claim 12, wherein said pathogen is Erysiphe pisi. 14. The method of claim 11, wherein said effective amount of said composition having glyphosate yields an application of a concentration of glyphosate between about 8 ounces/acre to about 128 ounces/acre. 15. The method of claim 11, wherein said pea plant is contacted with said effective amount of glyphosate before infection by said plant. 16. A method of treating powdery mildew disease in a glyphosate tolerant pea plant comprising, identifying a pea plant in need of powdery mildew disease treatment; and contacting said pea plant with an effective amount of a composition having glyphosate, whereby said powdery mildew disease of said pea plant is treated. 17. The method of claim 16, wherein said powdery mildew disease is caused by Erysiphe pisi. 18. The method of claim 16, wherein said effective amount of said composition having glyphosate yields an application of a concentration of glyphosate between about 8 ounces/acre to about 128 ounces/acre.

The present invention relates to compositions and methods for disease control in plants. The compositions for use in the methods of the invention include glyphosate as the active compound. In addition, methods and compositions are disclosed to prevent and treat pest infection in glyphosate tolerant plants.1. A method of controlling a fungal or fungal-like pathogen induced disease in a glyphosate tolerant onion plant comprising, identifying an onion plant in need of disease control; and contacting said onion plant with an effective amount of a composition having glyphosate, whereby said disease of said onion plant is controlled. 2. The method of claim 1, wherein said disease is caused by an Oomycete 3. The method of claim 2, wherein said Oomycete is Peronospora destructor. 4. The method of claim 1, wherein said effective amount of said composition having glyphosate yields an application of a concentration of glyphosate between about 8 ounces/acre to about 128 ounces/acre. 5. The method of claim 1, wherein said onion is contacted with said effective amount of glyphosate before infection by said pathogen. 6. The method of claim 1, wherein said onion is contacted with said effective amount of glyphosate after infection by said pathogen. 7. A method of treating a mildew disease in a glyphosate tolerant onion plant comprising, identifying an onion plant in need of mildew disease treatment; and contacting said onion plant with an effective amount of a composition having glyphosate, whereby said mildew disease of said onion plant is treated. 8. The method of claim 7, wherein said mildew disease is caused by a Peronospora species. 9. The method of claim 8, wherein said Peronospora species is Peronospora destructor. 10. The method of claim 7, wherein said effective amount of said composition having glyphosate yields an application of a concentration of glyphosate between about 8 ounces/acre to about 128 ounces/acre. 11. A method of controlling a fungal or fungal-like pathogen induced disease in a glyphosate tolerant pea plant comprising, identifying a pea plant in need of disease control; and contacting said pea plant with an effective amount of a composition having glyphosate, whereby said disease of said pea plant is controlled. 12. The method of claim 11, wherein said disease is caused by a pathogen of the family Erysiphaceae 13. The method of claim 12, wherein said pathogen is Erysiphe pisi. 14. The method of claim 11, wherein said effective amount of said composition having glyphosate yields an application of a concentration of glyphosate between about 8 ounces/acre to about 128 ounces/acre. 15. The method of claim 11, wherein said pea plant is contacted with said effective amount of glyphosate before infection by said plant. 16. A method of treating powdery mildew disease in a glyphosate tolerant pea plant comprising, identifying a pea plant in need of powdery mildew disease treatment; and contacting said pea plant with an effective amount of a composition having glyphosate, whereby said powdery mildew disease of said pea plant is treated. 17. The method of claim 16, wherein said powdery mildew disease is caused by Erysiphe pisi. 18. The method of claim 16, wherein said effective amount of said composition having glyphosate yields an application of a concentration of glyphosate between about 8 ounces/acre to about 128 ounces/acre.

The present invention relates to new types of metal complexes. Such compounds can be used as active components (=functional materials) in a series of different types of applications which can be classed within the electronics industry in the widest sense. The inventive compounds are described by the structure 1 and the formulae (1) to (60).

1.-30. (canceled) 31. A compound of the following formula [V-L3]M wherein M is a metal selected from the group consisting of Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu; V is a bridging unit, which contains from 1 to 80 atoms and which connects the three partial ligands L; L is the same or different on each occurrence and is a bidentate ligand comprising two cyclic groups Cy1 and Cy2, which are the same or different on each occurrence substituted or unsubstituted, saturated, unsaturated or aromatic homo- or heterocycles, which are each bonded ionically, covalently or coordinatively to the metal via a ring atom or via an atom bonded exocyclically to the homo- or heterocycle. 32. A compound according to claim 31 which comprises the structure 1, containing at least one metal Met, which has the same meaning as M in claim 31, coordinated to a polypodal ligand Lig of the structure 2, where V is a bridging unit, wherein V contains from 1 to 80 atoms and the three part-ligands L1, L2 and L3 which may be the same or different at each instance are covalently bonded to one another, and where the three part-ligands L1, L2 and L3 satisfy the structure 3 wherein Cy1 and Cy2 are the same or different at each instance and correspond to substituted or unsubstituted, saturated, unsaturated or aromatic homo- or heterocycles or part-homo- or part-heterocycles of a fused system, which are each bonded ionically, covalently or coordinatively to the metal via a ring atom or via an atom bonded exocyclically to the homo- or heterocycle, and that the compounds of the structure 1 are uncharged. 33. The compound as claimed in claim 31, wherein L1=L2=L3. 34. The compound as claimed in claim 31, wherein L1≠L2. 35. The compound as claimed in claim 31, wherein the linking unit V contains, as the linking atom, an element of main group 3, 4 or 5, or a 3- to 6-membered homo- or heterocycle. 36. The compound as claimed in claim 31, wherein the polypodal ligand Lig of the structure 4 generates facial coordination geometry on the metal Met 37. The compound as claimed in claim 31, wherein the polypodal ligand Lig of the structure 5 generates meridional coordination geometry on the metal Met 38. The compound as claimed in claim 31, wherein the compound is selected from the compounds (1) to (8) where the symbols and indices are each defined as follows: M is Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu; L is the same or different at each instance and is C, N or P; Q is the same or different at each instance and is O, S, Se, Te or N; T is the same or different at each instance and is N, P or C; X is the same or different at each instance and is CR, N or P; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1. 39. The compound as claimed in claim 31, wherein the compound is selected from the compounds (9) to (12) wherein M is Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu; L is the same or different at each instance and is C, N or P; Q is the same or different at each instance and is O, S, Se, Te or N; T is the same or different at each instance and is N, P or C; X is the same or different at each instance and is CR, N or P; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1 and n is 1 or 2. 40. The compound as claimed in claim 31, wherein the compound is selected from the compounds (13) to (30) wherein M is Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu; L is the same or different at each instance and is C, N or P; Q is the same or different at each instance and is O, S, Se, Te or N; T is the same or different at each instance and is N, P or C; X is the same or different at each instance and is CR, N or P; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1 and n is 1 or 2. 41. The compound as claimed in claim 31, selected from the compounds (31) to (41) wherein M is Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu; L is the same or different at each instance and is C, N or P; Q is the same or different at each instance and is O, S, Se, Te or N; T is the same or different at each instance and is N, P or C; X is the same or different at each instance and is CR, N or P; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1 and n is 1 or 2. 42. A compound which is selected from the compounds of compounds (42) to (82) wherein T is the same or different at each instance and is N, P or C; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1 X is the same or different at each instance and is CR or P; wherein the symbols and indices T, X, Y, Z, R, R1, and c are defined above, wherein Q is the same or different at each instance and is O, S, Se, Te or N; wherein the symbols and indices T, X, Y, Z, R, R1, and c are defined above, L is the same or different at each instance and is C or P; where the symbols and indices L, Q, T, X, Y, Z, R, R1, and c are defined above and n is 1 or 2. 43. The compound as claimed in claims 38, wherein the symbol M is Al, Ga, In, Sc, Y, La, Ru, Os, Rh, Ir or Au. 44. The compound as claimed in claims 38, wherein the symbol L is C. 45. The compound as claimed in claims 38, wherein the symbol Q=O or S. 46. The compound as claimed in claims 38, wherein the symbol T is N. 47. The compound as claimed in claims 38, wherein the symbol X is CR. 48. The compound as claimed in claims 38, wherein the symbol Z is B, CH, CR1, COR1, CF, CCl, CBr, SiR, N, P, PO, RC(CR2)3, RC(CR2CR2)3, cis,cis-1,3,5-cyclohexyl, RSi(CR2)3, RSi(CR2CR2)3, N(CR2)3, N(C=0)3, or N(CR2CR2)3. 49. The compound as claimed in claims 38, wherein the symbol Y is O or S. 50. The compound as claimed in claims 38, wherein the symbol R is H, F, CI, Br, I, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 6 carbon atoms or an aryl or heteroaryl group which has from 3 to 8 carbon atoms and may be substituted by one or more nonaromatic R radicals, in which a plurality of substituents R, either on the same ring or on the two different rings, together may in turn form a further mono- or polycyclic, aliphatic or aromatic ring system. 51. The compound as claimed in claims 38, wherein the polycyclic ring system optionally formed by the R radical(s) corresponds to benzene, 1- or 2-naphthalene, 1-, 2- or 9-anthracene, 2-, 3- or 4-pyridine, 2-, 4- or 5-pyrimidine, 2-pyrazine, 3- or 4-25 pyridazine, 2-, 3-, 4-, 5-, 6-, 7- or 8-quinoline, 2- or 3-pyrrole, 3-, 4- or 5-pyrazole, 2-, 4- or 5-imidazole, 2- or 3-thiophene, 2- or 3-selenophene, 2- or 3-furan, 2-(1,3,4-oxadiazole), indole or carbazole. 52. A process for preparing the compound as claimed in claim 42, which comprises reacting the compounds (42) to (82) with metal alkoxides of the formula (83), with metal ketoketonates of the formula (84) or metal halides of the formula (85), where the symbol R1 and Hal=F, CI, Br, I. 53. The compound as claimed in claim 31, wherein the compound has a purity (determined by means of 1H NMR and/or HPLC) is more than 99%. 54. A conjugated, semiconjugated or nonconjugated polymer or dendrimer containing one or more compounds of the structure (1) as claimed in claim 32 or of the compounds (1) to (41) wherein M is Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu; L is the same or different at each instance and is C, N or P; Q is the same or different at each instance and is O, S, Se, Te or N; T is the same or different at each instance and is N, P or C; X is the same or different at each instance and is CR, N or P; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1 and n is 1 or 2. 55. A conjugated, semiconjugated or nonconjugated polymer or dendrimer as claimed in claim 54, in which one or more of the R radicals is a bond to the polymer or dendrimer. 56. The polymer as claimed in claim 54, wherein the polymer is selected from the group of polyfluorenes, poly-spiro-bifluorenes, poly-para-phenylenes, polycarbazoles, polyvinylcarbazoles, polythiophenes, or else from copolymers which have a plurality of these units. 57. The polymer as claimed in claim 54, wherein the polymer is soluble in organic solvents. 58. An electronic component comprising at least one polymer or dendrimer as claimed in claim 54. 59. The electronic component as claimed in claim 58, wherein the electronic component is an organic light-emitting diode (OLED), organic integrated circuit (O-IC), organic field-effect transistor (OFET), organic thin-film transistor (OTFT), organic solar cell (O-SC) or organic laser diode (O-laser).

The present invention relates to new types of metal complexes. Such compounds can be used as active components (=functional materials) in a series of different types of applications which can be classed within the electronics industry in the widest sense. The inventive compounds are described by the structure 1 and the formulae (1) to (60).1.-30. (canceled) 31. A compound of the following formula [V-L3]M wherein M is a metal selected from the group consisting of Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu; V is a bridging unit, which contains from 1 to 80 atoms and which connects the three partial ligands L; L is the same or different on each occurrence and is a bidentate ligand comprising two cyclic groups Cy1 and Cy2, which are the same or different on each occurrence substituted or unsubstituted, saturated, unsaturated or aromatic homo- or heterocycles, which are each bonded ionically, covalently or coordinatively to the metal via a ring atom or via an atom bonded exocyclically to the homo- or heterocycle. 32. A compound according to claim 31 which comprises the structure 1, containing at least one metal Met, which has the same meaning as M in claim 31, coordinated to a polypodal ligand Lig of the structure 2, where V is a bridging unit, wherein V contains from 1 to 80 atoms and the three part-ligands L1, L2 and L3 which may be the same or different at each instance are covalently bonded to one another, and where the three part-ligands L1, L2 and L3 satisfy the structure 3 wherein Cy1 and Cy2 are the same or different at each instance and correspond to substituted or unsubstituted, saturated, unsaturated or aromatic homo- or heterocycles or part-homo- or part-heterocycles of a fused system, which are each bonded ionically, covalently or coordinatively to the metal via a ring atom or via an atom bonded exocyclically to the homo- or heterocycle, and that the compounds of the structure 1 are uncharged. 33. The compound as claimed in claim 31, wherein L1=L2=L3. 34. The compound as claimed in claim 31, wherein L1≠L2. 35. The compound as claimed in claim 31, wherein the linking unit V contains, as the linking atom, an element of main group 3, 4 or 5, or a 3- to 6-membered homo- or heterocycle. 36. The compound as claimed in claim 31, wherein the polypodal ligand Lig of the structure 4 generates facial coordination geometry on the metal Met 37. The compound as claimed in claim 31, wherein the polypodal ligand Lig of the structure 5 generates meridional coordination geometry on the metal Met 38. The compound as claimed in claim 31, wherein the compound is selected from the compounds (1) to (8) where the symbols and indices are each defined as follows: M is Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu; L is the same or different at each instance and is C, N or P; Q is the same or different at each instance and is O, S, Se, Te or N; T is the same or different at each instance and is N, P or C; X is the same or different at each instance and is CR, N or P; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1. 39. The compound as claimed in claim 31, wherein the compound is selected from the compounds (9) to (12) wherein M is Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu; L is the same or different at each instance and is C, N or P; Q is the same or different at each instance and is O, S, Se, Te or N; T is the same or different at each instance and is N, P or C; X is the same or different at each instance and is CR, N or P; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1 and n is 1 or 2. 40. The compound as claimed in claim 31, wherein the compound is selected from the compounds (13) to (30) wherein M is Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu; L is the same or different at each instance and is C, N or P; Q is the same or different at each instance and is O, S, Se, Te or N; T is the same or different at each instance and is N, P or C; X is the same or different at each instance and is CR, N or P; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1 and n is 1 or 2. 41. The compound as claimed in claim 31, selected from the compounds (31) to (41) wherein M is Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu; L is the same or different at each instance and is C, N or P; Q is the same or different at each instance and is O, S, Se, Te or N; T is the same or different at each instance and is N, P or C; X is the same or different at each instance and is CR, N or P; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1 and n is 1 or 2. 42. A compound which is selected from the compounds of compounds (42) to (82) wherein T is the same or different at each instance and is N, P or C; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1 X is the same or different at each instance and is CR or P; wherein the symbols and indices T, X, Y, Z, R, R1, and c are defined above, wherein Q is the same or different at each instance and is O, S, Se, Te or N; wherein the symbols and indices T, X, Y, Z, R, R1, and c are defined above, L is the same or different at each instance and is C or P; where the symbols and indices L, Q, T, X, Y, Z, R, R1, and c are defined above and n is 1 or 2. 43. The compound as claimed in claims 38, wherein the symbol M is Al, Ga, In, Sc, Y, La, Ru, Os, Rh, Ir or Au. 44. The compound as claimed in claims 38, wherein the symbol L is C. 45. The compound as claimed in claims 38, wherein the symbol Q=O or S. 46. The compound as claimed in claims 38, wherein the symbol T is N. 47. The compound as claimed in claims 38, wherein the symbol X is CR. 48. The compound as claimed in claims 38, wherein the symbol Z is B, CH, CR1, COR1, CF, CCl, CBr, SiR, N, P, PO, RC(CR2)3, RC(CR2CR2)3, cis,cis-1,3,5-cyclohexyl, RSi(CR2)3, RSi(CR2CR2)3, N(CR2)3, N(C=0)3, or N(CR2CR2)3. 49. The compound as claimed in claims 38, wherein the symbol Y is O or S. 50. The compound as claimed in claims 38, wherein the symbol R is H, F, CI, Br, I, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 6 carbon atoms or an aryl or heteroaryl group which has from 3 to 8 carbon atoms and may be substituted by one or more nonaromatic R radicals, in which a plurality of substituents R, either on the same ring or on the two different rings, together may in turn form a further mono- or polycyclic, aliphatic or aromatic ring system. 51. The compound as claimed in claims 38, wherein the polycyclic ring system optionally formed by the R radical(s) corresponds to benzene, 1- or 2-naphthalene, 1-, 2- or 9-anthracene, 2-, 3- or 4-pyridine, 2-, 4- or 5-pyrimidine, 2-pyrazine, 3- or 4-25 pyridazine, 2-, 3-, 4-, 5-, 6-, 7- or 8-quinoline, 2- or 3-pyrrole, 3-, 4- or 5-pyrazole, 2-, 4- or 5-imidazole, 2- or 3-thiophene, 2- or 3-selenophene, 2- or 3-furan, 2-(1,3,4-oxadiazole), indole or carbazole. 52. A process for preparing the compound as claimed in claim 42, which comprises reacting the compounds (42) to (82) with metal alkoxides of the formula (83), with metal ketoketonates of the formula (84) or metal halides of the formula (85), where the symbol R1 and Hal=F, CI, Br, I. 53. The compound as claimed in claim 31, wherein the compound has a purity (determined by means of 1H NMR and/or HPLC) is more than 99%. 54. A conjugated, semiconjugated or nonconjugated polymer or dendrimer containing one or more compounds of the structure (1) as claimed in claim 32 or of the compounds (1) to (41) wherein M is Al, Ga, In, Tl, P, As, Sb, Bi, Sc, Y, La, V, Nb, Ta, Cr, Mo, W, Fe, Ru, Os, Co, Rh, Ir, Cu, Au, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or Lu; L is the same or different at each instance and is C, N or P; Q is the same or different at each instance and is O, S, Se, Te or N; T is the same or different at each instance and is N, P or C; X is the same or different at each instance and is CR, N or P; Y is the same or different at each instance and is NR1, O, S, Se, Te, SO, SeO, TeO, SO2, SeO2 or TeO2; Z is B, BR, B(CR2)3, B(CR2CR2)3, CR, COH, COR1, CF, CCl, CBr, C—I, CNR1 2, RC(CR2)3, RC(CR2CR2)3, RC(SiR2)3, RC(SiR2CR2)3, RC(CR2SiR2)3, RC(SiR2SiR2)3, cis,cis-1,3,5-cyclohexyl, 1,3,5-(CR2)3C6H3, SiR, SiOH, SiOR1, RSi(CR2)3, RSi(CR2CR2)3, RSi(SiR2)3, RSi(SiR2CR2)3, RSi(CR2SiR2)3, RSi(SiR2SiR2)3, N, N(CR2)3, N(C═O)3, N(CR2CR2)3, NO, P, As, Sb, Bi, PO, AsO, SbO, BiO, PSe, AsSE, SbSe, BiSe, PTe, AsTe, SbTe or BiTe; R is the same or different at each instance and is H, F, Cl, Br, I, NO2, CN, a straight-chain or branched or cyclic alkyl or alkoxy group having from 1 to 20 carbon atoms, in which one or more nonadjacent CH2 groups is optionally replaced by —R1C═CR1—, —C≡C—, Si(R1)2, Ge(R1)2, Sn(R1)2, C═O, C═S, C═Se, C═NR1, —O—, —S—, —NR1— or —CONR1—, and in which one or more hydrogen atoms is optionally replaced by F, or an aryl or heteroaryl group which has from 1 to 14 carbon atoms and is optionally substituted by one or more nonaromatic R radicals, where a plurality of substituents R, both on the same ring and on the two different rings, together optionally forms a further mono- or polycyclic, aliphatic or aromatic ring system; R1 is the same or different at each instance and is an aliphatic or aromatic hydrocarbon radical having from 1 to 20 carbon atoms; c is the same or different at each instance and is 0 or 1 and n is 1 or 2. 55. A conjugated, semiconjugated or nonconjugated polymer or dendrimer as claimed in claim 54, in which one or more of the R radicals is a bond to the polymer or dendrimer. 56. The polymer as claimed in claim 54, wherein the polymer is selected from the group of polyfluorenes, poly-spiro-bifluorenes, poly-para-phenylenes, polycarbazoles, polyvinylcarbazoles, polythiophenes, or else from copolymers which have a plurality of these units. 57. The polymer as claimed in claim 54, wherein the polymer is soluble in organic solvents. 58. An electronic component comprising at least one polymer or dendrimer as claimed in claim 54. 59. The electronic component as claimed in claim 58, wherein the electronic component is an organic light-emitting diode (OLED), organic integrated circuit (O-IC), organic field-effect transistor (OFET), organic thin-film transistor (OTFT), organic solar cell (O-SC) or organic laser diode (O-laser).

A person-support structure includes a control system, an input configured to communicate a first input signal to the control system, and a sensor configured to sense a physiological characteristic of a person supported on the person-support structure. The sensor communicates a second input signal corresponding to the physiological characteristic to the control system. The control system calculates a condition score as a function of the first input signal and the physiological characteristic of the person and compares the condition score to a predetermined threshold. The control system scales at least one of the physiological characteristic of the person and the predetermined threshold as a function of the first input signal.

1. A method of predicting the onset of an adverse condition, the method comprising receiving a first input signal from an input, receiving a second input signal corresponding to a physiological characteristic of a person supported on a person-support structure, calculating a condition score as a function of the first input signal on the physiological characteristic, and alerting a caregiver if the condition score is greater than a predetermined threshold, wherein at least one of the physiological characteristic and the predetermined threshold is scaled as a function of the first input signal. 2. The method of claim 1, wherein the first input signal corresponds to at least one of a user input, person-support surface status information, a care facility characteristic, a history of a person, and the status of a bed frame. 3. The method of claim 1, wherein the first input signal comprises at least one of a fall risk assessment score and a pressure ulcer risk assessment score. 4. The method of claim 1, further comprising configuring a person-support system as a function of the condition score. 5. The method of claim 4, wherein the person-support system includes at least one of a bed frame and a person-support surface. 6. The method of claim 1, further comprising controlling a fluid supply to a person-support surface as a function of the condition score. 7. The method of claim 1, wherein the first input signal includes information corresponding to a characteristic of a care facility. 8. The method of claim 7, wherein the characteristic of the care facility includes at least one of a staffing level of the care facility, at least one of a date and a time of day, an amount of people in the care facility, a size of the care facility, a location of a caregiver within the care facility, a progress of a caregiver on their rounds, and an acuity of people in the care facility. 9. The method of claim 1, wherein the first input signal includes information corresponding to the history of a person. 10. The method of claim 9, wherein the history of the person includes at least one of a preference of a person, a tendency of a person, an electronic medical record (EMR) of a person, a pressure ulcer risk assessment of a person, a medical history of a person, an allergy of a person, a medical condition of a person, a diagnosis of a person, a medication chart for a person, a caregiver assignment history for a person, and a habit of a person.

A person-support structure includes a control system, an input configured to communicate a first input signal to the control system, and a sensor configured to sense a physiological characteristic of a person supported on the person-support structure. The sensor communicates a second input signal corresponding to the physiological characteristic to the control system. The control system calculates a condition score as a function of the first input signal and the physiological characteristic of the person and compares the condition score to a predetermined threshold. The control system scales at least one of the physiological characteristic of the person and the predetermined threshold as a function of the first input signal.1. A method of predicting the onset of an adverse condition, the method comprising receiving a first input signal from an input, receiving a second input signal corresponding to a physiological characteristic of a person supported on a person-support structure, calculating a condition score as a function of the first input signal on the physiological characteristic, and alerting a caregiver if the condition score is greater than a predetermined threshold, wherein at least one of the physiological characteristic and the predetermined threshold is scaled as a function of the first input signal. 2. The method of claim 1, wherein the first input signal corresponds to at least one of a user input, person-support surface status information, a care facility characteristic, a history of a person, and the status of a bed frame. 3. The method of claim 1, wherein the first input signal comprises at least one of a fall risk assessment score and a pressure ulcer risk assessment score. 4. The method of claim 1, further comprising configuring a person-support system as a function of the condition score. 5. The method of claim 4, wherein the person-support system includes at least one of a bed frame and a person-support surface. 6. The method of claim 1, further comprising controlling a fluid supply to a person-support surface as a function of the condition score. 7. The method of claim 1, wherein the first input signal includes information corresponding to a characteristic of a care facility. 8. The method of claim 7, wherein the characteristic of the care facility includes at least one of a staffing level of the care facility, at least one of a date and a time of day, an amount of people in the care facility, a size of the care facility, a location of a caregiver within the care facility, a progress of a caregiver on their rounds, and an acuity of people in the care facility. 9. The method of claim 1, wherein the first input signal includes information corresponding to the history of a person. 10. The method of claim 9, wherein the history of the person includes at least one of a preference of a person, a tendency of a person, an electronic medical record (EMR) of a person, a pressure ulcer risk assessment of a person, a medical history of a person, an allergy of a person, a medical condition of a person, a diagnosis of a person, a medication chart for a person, a caregiver assignment history for a person, and a habit of a person.

Provided herein is a method comprising: (a) obtaining a mixture of multiple sets of oligonucleotides, wherein the oligonucleotides within each set each comprise a terminal indexer sequence and can be assembled to produce a synthon; and (b) hybridizing the oligonucleotide mixture to an array, thereby spatially-separating the different sets of oligonucleotides from one another. In some embodiments the method may comprise (c) contacting the array with a solution, thereby producing, for each feature bound by the oligonucleotides, a discrete droplet comprising the feature and, optionally, placing an immiscible liquid over the droplets, thereby producing, for each feature bound by the oligonucleotides, a discrete reaction chamber defined by a droplet. The method may further comprise incubating the array under conditions by which a synthon is assembled in each of the reaction chambers. Other embodiments are also provided.

1. A method comprising: (a) obtaining a mixture of multiple sets of oligonucleotides, wherein the oligonucleotides within each set each comprise a terminal indexer sequence and can be assembled to produce a synthon; and (b) hybridizing the oligonucleotide mixture to an array, thereby spatially-separating the different sets of oligonucleotides from one another. 2. The method of claim 1, wherein the oligonucleotides are single-stranded oligonucleotides. 3. The method of claim 1, wherein the mixture comprises double-stranded oligonucleotides. 4. The method of claim 1, wherein the oligonucleotides are single stranded and comprise a 3′ hairpin. 5. The method of claim 4, comprising: contacting the array with a solution comprising a polymerase and nucleotides, thereby extending the hairpin and producing, for each feature bound by the oligonucleotides, a set of double-stranded extension products. 6. The method of claim 1, wherein the oligonucleotides hybridize directly to oligonucleotides that are immobilized on the array. 7. The method of claim 1, wherein the oligonucleotides hybridize via an adaptor to oligonucleotides that are immobilized on the array. 8. The method of claim 7, wherein the method comprises: contacting the array with a solution comprising a polymerase and nucleotides, thereby extending the adaptor and producing, for each feature bound by the oligonucleotides, a set of double-stranded extension product. 9. The method of claim 1, further comprising (c) contacting the array with a solution, thereby producing, for each feature bound by the oligonucleotides, a discrete droplet comprising the feature. 10. The method of claim 9, further comprising placing an immiscible liquid over the droplets, thereby producing, for each feature bound by the oligonucleotides, a discrete reaction chamber defined by a droplet. 11. The method of claim 10, further comprising incubating the array under conditions by which a synthon is assembled in each of the reaction chambers. 12. The method of claim 11, wherein the droplets comprise double-stranded oligonucleotides or double-stranded extension products, and the solution comprises a Type IIs restriction endonuclease, a DNA ligase and ATP, wherein the products of digestion of the double-stranded oligonucleotides or double-stranded extension products by the Type IIs restriction endonuclease are ligated to one another in a defined order by the DNA ligase in the discrete reaction chambers, thereby producing a synthon. 13. The method of claim 11, wherein the oligonucleotides are single-stranded oligonucleotides and the method comprises: cleaving the terminal indexer sequence from the oligonucleotides to release single-stranded primers; and assembling the synthon by polymerase chain assembly. 14. The method of claim 9, further comprising separating the synthons from the array. 15. A composition comprising multiple sets of oligonucleotides, wherein the oligonucleotides within each set comprise a terminal indexer sequence and can be assembled to produce a synthon. 16. The composition of claim 15, wherein the oligonucleotides, in their double-stranded form, are digestible by a Type IIs restriction enzyme to produce fragments that can be assembled by polymerase chain assembly or ordered ligation. 17. An apparatus comprising: a planar support, a plurality of spatially distinct droplets on a surface of the planar support, and an immiscible liquid covering the droplets, wherein the apparatus comprises a plurality of reaction chambers defined by the droplets, and each reaction chamber comprises a different synthon.

Provided herein is a method comprising: (a) obtaining a mixture of multiple sets of oligonucleotides, wherein the oligonucleotides within each set each comprise a terminal indexer sequence and can be assembled to produce a synthon; and (b) hybridizing the oligonucleotide mixture to an array, thereby spatially-separating the different sets of oligonucleotides from one another. In some embodiments the method may comprise (c) contacting the array with a solution, thereby producing, for each feature bound by the oligonucleotides, a discrete droplet comprising the feature and, optionally, placing an immiscible liquid over the droplets, thereby producing, for each feature bound by the oligonucleotides, a discrete reaction chamber defined by a droplet. The method may further comprise incubating the array under conditions by which a synthon is assembled in each of the reaction chambers. Other embodiments are also provided.1. A method comprising: (a) obtaining a mixture of multiple sets of oligonucleotides, wherein the oligonucleotides within each set each comprise a terminal indexer sequence and can be assembled to produce a synthon; and (b) hybridizing the oligonucleotide mixture to an array, thereby spatially-separating the different sets of oligonucleotides from one another. 2. The method of claim 1, wherein the oligonucleotides are single-stranded oligonucleotides. 3. The method of claim 1, wherein the mixture comprises double-stranded oligonucleotides. 4. The method of claim 1, wherein the oligonucleotides are single stranded and comprise a 3′ hairpin. 5. The method of claim 4, comprising: contacting the array with a solution comprising a polymerase and nucleotides, thereby extending the hairpin and producing, for each feature bound by the oligonucleotides, a set of double-stranded extension products. 6. The method of claim 1, wherein the oligonucleotides hybridize directly to oligonucleotides that are immobilized on the array. 7. The method of claim 1, wherein the oligonucleotides hybridize via an adaptor to oligonucleotides that are immobilized on the array. 8. The method of claim 7, wherein the method comprises: contacting the array with a solution comprising a polymerase and nucleotides, thereby extending the adaptor and producing, for each feature bound by the oligonucleotides, a set of double-stranded extension product. 9. The method of claim 1, further comprising (c) contacting the array with a solution, thereby producing, for each feature bound by the oligonucleotides, a discrete droplet comprising the feature. 10. The method of claim 9, further comprising placing an immiscible liquid over the droplets, thereby producing, for each feature bound by the oligonucleotides, a discrete reaction chamber defined by a droplet. 11. The method of claim 10, further comprising incubating the array under conditions by which a synthon is assembled in each of the reaction chambers. 12. The method of claim 11, wherein the droplets comprise double-stranded oligonucleotides or double-stranded extension products, and the solution comprises a Type IIs restriction endonuclease, a DNA ligase and ATP, wherein the products of digestion of the double-stranded oligonucleotides or double-stranded extension products by the Type IIs restriction endonuclease are ligated to one another in a defined order by the DNA ligase in the discrete reaction chambers, thereby producing a synthon. 13. The method of claim 11, wherein the oligonucleotides are single-stranded oligonucleotides and the method comprises: cleaving the terminal indexer sequence from the oligonucleotides to release single-stranded primers; and assembling the synthon by polymerase chain assembly. 14. The method of claim 9, further comprising separating the synthons from the array. 15. A composition comprising multiple sets of oligonucleotides, wherein the oligonucleotides within each set comprise a terminal indexer sequence and can be assembled to produce a synthon. 16. The composition of claim 15, wherein the oligonucleotides, in their double-stranded form, are digestible by a Type IIs restriction enzyme to produce fragments that can be assembled by polymerase chain assembly or ordered ligation. 17. An apparatus comprising: a planar support, a plurality of spatially distinct droplets on a surface of the planar support, and an immiscible liquid covering the droplets, wherein the apparatus comprises a plurality of reaction chambers defined by the droplets, and each reaction chamber comprises a different synthon.

A transdermal patch suitable for releasing a stimulant such as caffeine for an extended time period utilizes a pressure sensitive acrylic adhesive matrix constituted by an acrylic adhesive having hydroxy groups and an acrylic adhesive without hydroxy, groups, preferably in a respective weight ratio of about 2 to about 3. A suitable caffeine source is caffeine-fortified guarana seed extract, caffeine, a caffeine salt, and the like.

1. A transdermal patch comprising a backing layer; a pressure-sensitive acrylic adhesive matrix on the backing layer; and a xanthine composition comprising guarana seed extract and caffeine in said matrix; and a protective release sheet over the pressure-sensitive acrylic adhesive matrix; the pressure-sensitive acrylic adhesive matrix comprising a mixture of an acrylic adhesive having hydroxy groups and an acrylic adhesive without hydroxy groups. 2. The transdermal patch in accordance with claim 1 wherein the xanthine composition is present in the matrix in an amount in the range of about 5 to about 50 percent by weight, based on the total weight of the adhesive matrix. 3. The transdermal patch in accordance with claim 1 wherein the acrylic adhesive having hydroxy groups and the acrylic adhesive without hydroxy groups are present in the matrix in a respective weight ratio in the range of about 0.5 to about 5. 4. The transdermal patch in accordance with claim 1 wherein the acrylic adhesive having hydroxy groups and the acrylic adhesive without hydroxy groups are present in the matrix in a respective weight ratio of about 2 to about 3. 5. The transdermal patch in accordance with claim 1 wherein the pressure-sensitive acrylic adhesive matrix further contains polyvinylpyrrolidone and propylene glycol. 6. The transdermal patch in accordance with claim 1 wherein the polyvinyl pyrrolidone is present in an amount in the range of about 2 to about 5 percent by weight of the total weight of the adhesive matrix and has a weight average molecular weight in the range of about 2,000 to about 11,000 Daltons, and propylene glycol is present in an amount in the range of about 2 to about 10 percent by weight of the total weight of the adhesive matrix. 7. The transdermal patch in accordance with claim 1 wherein the thickness of pressure-sensitive acrylic adhesive matrix is in the range of about 3 mils to about 5 mils. 8. The transdermal patch in accordance with claim 1 wherein the xanthine composition comprises a caffeine fortified guarana seed extract and caffeine. 9. The transdermal patch in accordance with claim 8 further containing cocoa. 10. The transdermal patch in accordance with claim 9 and containing about 0.5 to about 5 percent by weight cocoa, based on total weight of the adhesive matrix. 11. The transdermal patch in accordance with claim 8 wherein the caffeine is present in an amount in the range of about 6 to about 40 percent by weight of the total weight of the adhesive matrix, and cocoa is present in an amount in the range of about 1 to about 3 percent by weight, based on total weight of the adhesive matrix. 12. The transdermal patch in accordance with claim 1 wherein the acrylic adhesive having hydroxy groups is a self-crosslinking acrylic copolymer of 2-hydroxyethyl acrylate, 2-ethylhexyl acrylate, acrylic acid, and methyl methacrylate, containing aluminum acetonate and t-amyl peroxypivalate as cross-linking agents, and wherein the acrylic adhesive without hydroxy groups is a copolymer of 2-ethylhexyl acrylate, acrylic acid, butyl acrylate and vinyl acetate. 13. The transdermal patch in accordance with claim 1 wherein the release sheet is a silicone treated polyurethane film. 14. The transdermal patch in accordance with claim 1 wherein the foam backing layer is an ethylene-vinyl acetate foam. 15. The transdermal patch in accordance with claim 1 wherein the adhesive matrix is at least 3 mils thick. 16. A transdermal patch comprising an occlusive foam backing layer; a pressure-sensitive acrylic adhesive matrix on the backing layer; guarana seed extract in said adhesive matrix; caffeine in said adhesive matrix; cocoa in said adhesive matrix; and the adhesive matrix comprising a mixture of an acrylic adhesive having hydroxy groups and an acrylic adhesive without hydroxy groups. 17. The transdermal patch in accordance with claim 16 further including arnica in said adhesive matrix. 18. The transdermal patch in accordance with claim 16 containing about 2.5 percent by weight caffeine fortified guarana seed extract, about 6 percent by weight caffeine, and about 1 percent by weight cocoa, based on total weight of the adhesive matrix, and wherein the weight ratio of acrylic adhesive having hydroxy groups to acrylic adhesive without hydroxy groups is about 2.9. 19. The transdermal patch in accordance with claim 16 further containing up to about 10 percent by weight arnica extract, based on total weight of the adhesive matrix. 20. The transdermal patch in accordance with claim 1 further containing an arnica extract in an amount of up to about 10 percent by weight, based on total weight of the adhesive matrix.

A transdermal patch suitable for releasing a stimulant such as caffeine for an extended time period utilizes a pressure sensitive acrylic adhesive matrix constituted by an acrylic adhesive having hydroxy groups and an acrylic adhesive without hydroxy, groups, preferably in a respective weight ratio of about 2 to about 3. A suitable caffeine source is caffeine-fortified guarana seed extract, caffeine, a caffeine salt, and the like.1. A transdermal patch comprising a backing layer; a pressure-sensitive acrylic adhesive matrix on the backing layer; and a xanthine composition comprising guarana seed extract and caffeine in said matrix; and a protective release sheet over the pressure-sensitive acrylic adhesive matrix; the pressure-sensitive acrylic adhesive matrix comprising a mixture of an acrylic adhesive having hydroxy groups and an acrylic adhesive without hydroxy groups. 2. The transdermal patch in accordance with claim 1 wherein the xanthine composition is present in the matrix in an amount in the range of about 5 to about 50 percent by weight, based on the total weight of the adhesive matrix. 3. The transdermal patch in accordance with claim 1 wherein the acrylic adhesive having hydroxy groups and the acrylic adhesive without hydroxy groups are present in the matrix in a respective weight ratio in the range of about 0.5 to about 5. 4. The transdermal patch in accordance with claim 1 wherein the acrylic adhesive having hydroxy groups and the acrylic adhesive without hydroxy groups are present in the matrix in a respective weight ratio of about 2 to about 3. 5. The transdermal patch in accordance with claim 1 wherein the pressure-sensitive acrylic adhesive matrix further contains polyvinylpyrrolidone and propylene glycol. 6. The transdermal patch in accordance with claim 1 wherein the polyvinyl pyrrolidone is present in an amount in the range of about 2 to about 5 percent by weight of the total weight of the adhesive matrix and has a weight average molecular weight in the range of about 2,000 to about 11,000 Daltons, and propylene glycol is present in an amount in the range of about 2 to about 10 percent by weight of the total weight of the adhesive matrix. 7. The transdermal patch in accordance with claim 1 wherein the thickness of pressure-sensitive acrylic adhesive matrix is in the range of about 3 mils to about 5 mils. 8. The transdermal patch in accordance with claim 1 wherein the xanthine composition comprises a caffeine fortified guarana seed extract and caffeine. 9. The transdermal patch in accordance with claim 8 further containing cocoa. 10. The transdermal patch in accordance with claim 9 and containing about 0.5 to about 5 percent by weight cocoa, based on total weight of the adhesive matrix. 11. The transdermal patch in accordance with claim 8 wherein the caffeine is present in an amount in the range of about 6 to about 40 percent by weight of the total weight of the adhesive matrix, and cocoa is present in an amount in the range of about 1 to about 3 percent by weight, based on total weight of the adhesive matrix. 12. The transdermal patch in accordance with claim 1 wherein the acrylic adhesive having hydroxy groups is a self-crosslinking acrylic copolymer of 2-hydroxyethyl acrylate, 2-ethylhexyl acrylate, acrylic acid, and methyl methacrylate, containing aluminum acetonate and t-amyl peroxypivalate as cross-linking agents, and wherein the acrylic adhesive without hydroxy groups is a copolymer of 2-ethylhexyl acrylate, acrylic acid, butyl acrylate and vinyl acetate. 13. The transdermal patch in accordance with claim 1 wherein the release sheet is a silicone treated polyurethane film. 14. The transdermal patch in accordance with claim 1 wherein the foam backing layer is an ethylene-vinyl acetate foam. 15. The transdermal patch in accordance with claim 1 wherein the adhesive matrix is at least 3 mils thick. 16. A transdermal patch comprising an occlusive foam backing layer; a pressure-sensitive acrylic adhesive matrix on the backing layer; guarana seed extract in said adhesive matrix; caffeine in said adhesive matrix; cocoa in said adhesive matrix; and the adhesive matrix comprising a mixture of an acrylic adhesive having hydroxy groups and an acrylic adhesive without hydroxy groups. 17. The transdermal patch in accordance with claim 16 further including arnica in said adhesive matrix. 18. The transdermal patch in accordance with claim 16 containing about 2.5 percent by weight caffeine fortified guarana seed extract, about 6 percent by weight caffeine, and about 1 percent by weight cocoa, based on total weight of the adhesive matrix, and wherein the weight ratio of acrylic adhesive having hydroxy groups to acrylic adhesive without hydroxy groups is about 2.9. 19. The transdermal patch in accordance with claim 16 further containing up to about 10 percent by weight arnica extract, based on total weight of the adhesive matrix. 20. The transdermal patch in accordance with claim 1 further containing an arnica extract in an amount of up to about 10 percent by weight, based on total weight of the adhesive matrix.

Disclosed are active ingredient combinations of alkylamidothiazoles and one or more cosmetically or dermatologically acceptable UV-filter substances.

1.-15. (canceled) 16. An active ingredient combination of one or more alkylamidothiazoles and one or more cosmetically or dermatologically acceptable UV-filter substances. 17. The active ingredient combination of claim 16, wherein the one or more UV-filter substances comprise one or more of butylmethoxydibenzoylmethane, ethylhexyl methoxycinnamate, octocrylene, ethylhexyl salicylate, phenylbenzimidazolesulfonic acid, disodium phenyldibenzimidazoletetrasulfonate, benzophenone-3, drometrizole trisiloxane, benzophenone-4, homosalate, benzene-1,4-di(2-oxo-3-bornylidenemethyl-10-sulfonic acid, polysilicone-15, ethylhexyl triazone, diethylhexyl-butamidotriazone, isoamyl p-methoxycinnamate, diethylamino hydroxybenzoyl hexyl benzoate, methylenebisbenzotriazolyltetramethylbutylphenol, bisethylhexyloxyphenolmethoxy-phenyltriazine, 4-methylbenzylidenecamphor, 2,4-bis-[5-] (dimethylpropyl)-benz-oxazol-2-yl-(4-phenyl)imino]-6-(2-ethylhexyl)imino-1,3,5-triazine, (2-{4-[2-(4-diethylamino-2-hydroxybenzoyl)benzoyl]piperazine-1-carbonyl}-phenyl)-(4-diethyl-amino-2-hydroxy-phenyl)methanone. 18. The active ingredient combination of claim 16, wherein the one or more UV-filter substances comprise at least one water-soluble UV-filter substance. 19. The active ingredient combination of claim 16, wherein the one or more UV-filter substances comprise at least one oil-soluble UV-filter substance. 20. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which R1, R2, X and Y are different, partly identical or completely identical and, independently of one another, represent: R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24-alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylaryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24 alkyl-morpholino, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alkyl-piperazino-N-alkyl; R2=H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-hydroxyalkyl (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched); X=—H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-aryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), —C1-C24-heteroaryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), -aryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), -phenyl, -2,4-dihydroxyphenyl, -2,3-dihydroxyphenyl, -2,4-dimethoxyphenyl, -2,3-dimethoxyphenyl; Y=H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-aryl, —C1-C24-heteroaryl, —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), -aryl, -phenyl, -2,4-dihydroxyphenyl, -2,3-dihydroxyphenyl, -2,4-dimethoxyphenyl, -2,3-dimethoxyphenyl, —COO-alkyl, —COO-alkenyl, —COO-cycloalkyl, —COO-aryl, —COO-heteroaryl; and X, Y can also form a fused aromatic ring system and can form with one another aromatic or aliphatic homo- or heterocyclic ring systems with up to n ring-forming atoms, where n can assume values from 5 to 8, and the respective ring systems can in turn be substituted with up to n−1 alkyl groups, hydroxyl groups, carboxyl groups, amino groups, nitrile functions, sulfur-containing substituents, ester groups and/or ether groups, and wherein the one or more alkylamidothiazoles are be present as a free base and/or as a cosmetically and dermatologically acceptable salt thereof. 21. The active ingredient combination of claim 20, wherein X represents a substituted phenyl group. 22. The active ingredient combination of claim 20, wherein the one or more alkylamidothiazoles comprise one or more alkylamidothiazoles of formula in which the substituents Z independently represent —H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN, acetyl. 23. The active ingredient combination of claim 22, wherein the one or more alkylamidothiazoles comprise one or more alkylamidothiazoles of formula in which the substituent Z represents —H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN. 24. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24 alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylaryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24-alkyl-morpholine, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alky-piperazino-N-alkyl; R2=H, —C1-C24-alkyl (linear and branched); Z=—H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN, acetyl. 25. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24-alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkyl-aryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24 alkyl-morpholino, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alkyl-piperazino-N-alkyl; R2=H. 26. The active ingredient combination of claim 16, wherein the combination comprises one or more of the following alkylamidothiazoles: and 27. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles in the form of one or more of a halide, a carbonate, an ascorbate, a sulfate, an acetate, a phosphate. 28. A cosmetic or dermatological preparation, wherein the preparation comprises the active ingredient combination of claim 16. 29. The preparation of claim 28, wherein the preparation comprises from 0.000001% to 10% by weight of the active ingredient combination, based on a total weight of the preparation. 30. The preparation of claim 29, wherein the preparation comprises from 0.0001% to 3% by weight of the active ingredient combination. 31. The preparation of claim 29, wherein the preparation comprises from 0.001% to 1% by weight of the active ingredient combination. 32. The preparation of claim 28, wherein the preparation comprises a total of from 0.00001% by weight to 10% by weight of the one or more UV-filter substances, based on a total weight of the preparation. 33. The preparation of claim 32, wherein the preparation comprises a total of from 0.001% by weight to 5% by weight of the one or more UV-filter substances. 34. The preparation of claim 32, wherein the preparation comprises a total of from 0.005% by weight to 3% by weight of the one or more UV-filter substances. 35. A method of lightening human skin, wherein the method comprises applying to human skin to be lightened the preparation of claim 28.

Disclosed are active ingredient combinations of alkylamidothiazoles and one or more cosmetically or dermatologically acceptable UV-filter substances.1.-15. (canceled) 16. An active ingredient combination of one or more alkylamidothiazoles and one or more cosmetically or dermatologically acceptable UV-filter substances. 17. The active ingredient combination of claim 16, wherein the one or more UV-filter substances comprise one or more of butylmethoxydibenzoylmethane, ethylhexyl methoxycinnamate, octocrylene, ethylhexyl salicylate, phenylbenzimidazolesulfonic acid, disodium phenyldibenzimidazoletetrasulfonate, benzophenone-3, drometrizole trisiloxane, benzophenone-4, homosalate, benzene-1,4-di(2-oxo-3-bornylidenemethyl-10-sulfonic acid, polysilicone-15, ethylhexyl triazone, diethylhexyl-butamidotriazone, isoamyl p-methoxycinnamate, diethylamino hydroxybenzoyl hexyl benzoate, methylenebisbenzotriazolyltetramethylbutylphenol, bisethylhexyloxyphenolmethoxy-phenyltriazine, 4-methylbenzylidenecamphor, 2,4-bis-[5-] (dimethylpropyl)-benz-oxazol-2-yl-(4-phenyl)imino]-6-(2-ethylhexyl)imino-1,3,5-triazine, (2-{4-[2-(4-diethylamino-2-hydroxybenzoyl)benzoyl]piperazine-1-carbonyl}-phenyl)-(4-diethyl-amino-2-hydroxy-phenyl)methanone. 18. The active ingredient combination of claim 16, wherein the one or more UV-filter substances comprise at least one water-soluble UV-filter substance. 19. The active ingredient combination of claim 16, wherein the one or more UV-filter substances comprise at least one oil-soluble UV-filter substance. 20. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which R1, R2, X and Y are different, partly identical or completely identical and, independently of one another, represent: R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24-alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylaryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24 alkyl-morpholino, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alkyl-piperazino-N-alkyl; R2=H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-hydroxyalkyl (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched); X=—H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-aryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), —C1-C24-heteroaryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), -aryl (optionally mono- or polysubstituted with —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN), -phenyl, -2,4-dihydroxyphenyl, -2,3-dihydroxyphenyl, -2,4-dimethoxyphenyl, -2,3-dimethoxyphenyl; Y=H, —C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C24-aryl, —C1-C24-heteroaryl, —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), -aryl, -phenyl, -2,4-dihydroxyphenyl, -2,3-dihydroxyphenyl, -2,4-dimethoxyphenyl, -2,3-dimethoxyphenyl, —COO-alkyl, —COO-alkenyl, —COO-cycloalkyl, —COO-aryl, —COO-heteroaryl; and X, Y can also form a fused aromatic ring system and can form with one another aromatic or aliphatic homo- or heterocyclic ring systems with up to n ring-forming atoms, where n can assume values from 5 to 8, and the respective ring systems can in turn be substituted with up to n−1 alkyl groups, hydroxyl groups, carboxyl groups, amino groups, nitrile functions, sulfur-containing substituents, ester groups and/or ether groups, and wherein the one or more alkylamidothiazoles are be present as a free base and/or as a cosmetically and dermatologically acceptable salt thereof. 21. The active ingredient combination of claim 20, wherein X represents a substituted phenyl group. 22. The active ingredient combination of claim 20, wherein the one or more alkylamidothiazoles comprise one or more alkylamidothiazoles of formula in which the substituents Z independently represent —H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN, acetyl. 23. The active ingredient combination of claim 22, wherein the one or more alkylamidothiazoles comprise one or more alkylamidothiazoles of formula in which the substituent Z represents —H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN. 24. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24 alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkylaryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24-alkyl-morpholine, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alky-piperazino-N-alkyl; R2=H, —C1-C24-alkyl (linear and branched); Z=—H, —OH, —F, —Cl, —Br, —I, —OMe, —NH2, —CN, acetyl. 25. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles of formula in which R1=—C1-C24-alkyl (linear and branched), —C1-C24-alkenyl (linear and branched), —C1-C8-cycloalkyl, —C1-C8-cycloalkyl-alkylhydroxy, —C1-C24-alkylhydroxy (linear and branched), —C1-C24 alkylamine (linear and branched), —C1-C24-alkylaryl (linear and branched), —C1-C24-alkyl-aryl-alkyl-hydroxy (linear and branched), —C1-C24-alkylheteroaryl (linear and branched), —C1-C24-alkyl-O—C1-C24-alkyl (linear and branched), —C1-C24 alkyl-morpholino, —C1-C24 alkyl-piperidino, —C1-C24 alkyl-piperazino, —C1-C24 alkyl-piperazino-N-alkyl; R2=H. 26. The active ingredient combination of claim 16, wherein the combination comprises one or more of the following alkylamidothiazoles: and 27. The active ingredient combination of claim 16, wherein the combination comprises one or more alkylamidothiazoles in the form of one or more of a halide, a carbonate, an ascorbate, a sulfate, an acetate, a phosphate. 28. A cosmetic or dermatological preparation, wherein the preparation comprises the active ingredient combination of claim 16. 29. The preparation of claim 28, wherein the preparation comprises from 0.000001% to 10% by weight of the active ingredient combination, based on a total weight of the preparation. 30. The preparation of claim 29, wherein the preparation comprises from 0.0001% to 3% by weight of the active ingredient combination. 31. The preparation of claim 29, wherein the preparation comprises from 0.001% to 1% by weight of the active ingredient combination. 32. The preparation of claim 28, wherein the preparation comprises a total of from 0.00001% by weight to 10% by weight of the one or more UV-filter substances, based on a total weight of the preparation. 33. The preparation of claim 32, wherein the preparation comprises a total of from 0.001% by weight to 5% by weight of the one or more UV-filter substances. 34. The preparation of claim 32, wherein the preparation comprises a total of from 0.005% by weight to 3% by weight of the one or more UV-filter substances. 35. A method of lightening human skin, wherein the method comprises applying to human skin to be lightened the preparation of claim 28.

The present invention relates to the application of modafinil in cocaine addiction. The modafinil used is its dextro-rotatory enantiomer (S modafinil), having a release time of less than 1 hour and wakening effect of less than 4 hours. It is absorbed orally as a pharmaceutical composition, each unit dose including from 25 to 200 mg of S modafinil. The pharmaceutical composition can be used as a substitute treatment for cocaine addicts.

1. An application of modafinil in the substitution treatment of cocaine addicts, consisting of using a pharmaceutical composition in which said modafinil is in the form of its dextro-rotary enantiomer, S modafinil, wherein the dose of S modafinil absorbed by the patient is from 50 to 100 mg per unit dose. 2. The application according to claim 1, wherein said S modafinil has a release of less than 1 hour from its absorption by the patient. 3. The application according to claim 2, wherein said release is located between 15 and 30 minutes. 4. The application according to claim 1, wherein the effect of said S modafinil is less than 4 hours from its absorption by the patient. 5. The application according to claim 4, wherein said effect is less than 2 hours. 6. The application according to claim 1, wherein said pharmaceutical composition is absorbed via an oral route. 7. The application according to claim 6, wherein said S modafinil is obtained by the technology of the supercritical fluid, said S modafinil being absorbed at the surface of granules made in an inert support. 8. The application according to claim 1, wherein said pharmaceutical composition appears as tablets. 9. The application according to claim 1, wherein said pharmaceutical composition appears as semi-solid gelatin capsules, each gelatin capsule comprising: modafinil, in the form of its enantiomer S, an oleic complex comprising at least one compound selected from polyethylene glycols having a molecular weight comprised between 300 and 500 daltons and from glycerides, and an emulsifier. 10. The application according to claim 9, wherein the S modafinil/oleic complex ratio is located between 35 and 65%. 11. The application according to claim 10, wherein said ratio is located between 40 and 60%.

The present invention relates to the application of modafinil in cocaine addiction. The modafinil used is its dextro-rotatory enantiomer (S modafinil), having a release time of less than 1 hour and wakening effect of less than 4 hours. It is absorbed orally as a pharmaceutical composition, each unit dose including from 25 to 200 mg of S modafinil. The pharmaceutical composition can be used as a substitute treatment for cocaine addicts.1. An application of modafinil in the substitution treatment of cocaine addicts, consisting of using a pharmaceutical composition in which said modafinil is in the form of its dextro-rotary enantiomer, S modafinil, wherein the dose of S modafinil absorbed by the patient is from 50 to 100 mg per unit dose. 2. The application according to claim 1, wherein said S modafinil has a release of less than 1 hour from its absorption by the patient. 3. The application according to claim 2, wherein said release is located between 15 and 30 minutes. 4. The application according to claim 1, wherein the effect of said S modafinil is less than 4 hours from its absorption by the patient. 5. The application according to claim 4, wherein said effect is less than 2 hours. 6. The application according to claim 1, wherein said pharmaceutical composition is absorbed via an oral route. 7. The application according to claim 6, wherein said S modafinil is obtained by the technology of the supercritical fluid, said S modafinil being absorbed at the surface of granules made in an inert support. 8. The application according to claim 1, wherein said pharmaceutical composition appears as tablets. 9. The application according to claim 1, wherein said pharmaceutical composition appears as semi-solid gelatin capsules, each gelatin capsule comprising: modafinil, in the form of its enantiomer S, an oleic complex comprising at least one compound selected from polyethylene glycols having a molecular weight comprised between 300 and 500 daltons and from glycerides, and an emulsifier. 10. The application according to claim 9, wherein the S modafinil/oleic complex ratio is located between 35 and 65%. 11. The application according to claim 10, wherein said ratio is located between 40 and 60%.