Source: https://patents.google.com/patent/US9404110B2/en
Timestamp: 2018-03-25 05:35:48
Document Index: 489453767

Matched Legal Cases: ['application No. 102010007562', 'application No. 2012552313', 'application No. 2012552313', 'Application No. 11', 'application No. 10', 'application No. 11', 'application No. 10', 'application No. 2012137904', 'application No. 2012137904']

US9404110B2 - Dermatological, pharmaceutical composition suitable for oligonucleotides - Google Patents
Dermatological, pharmaceutical composition suitable for oligonucleotides Download PDF
US9404110B2
US9404110B2 US14965371 US201514965371A US9404110B2 US 9404110 B2 US9404110 B2 US 9404110B2 US 14965371 US14965371 US 14965371 US 201514965371 A US201514965371 A US 201514965371A US 9404110 B2 US9404110 B2 US 9404110B2
US14965371
US20160115478A1 (en )
Sterna Biologicals GmbH and Co KG
C12N2310/127—DNAzymes
The invention relates to a cosmetic and/or dermatological and/or pharmaceutical composition for the topical use and application of oligonucleotides, in particular antisense-oligonucleotides such as DNAzyme, siRNSs, asDNAs or ribozymes for use as an agent against inflammatory diseases by means of emulsions having a dispersed, internal, discontinous aqueous phase.
This is a division of U.S. patent application Ser. No. 13/578,013 filed Aug. 9, 2012, which is the U.S. national phase of international application No. PCT/EP2011/000648, filed Feb. 11, 2011, which claims priority to German patent application No. 102010007562.0, filed Feb. 10, 2010, the disclosure of each of which is incorporated herein by reference.
The content of the electronically submitted sequence listing in ASCII text file (Name: 46951a_SeqListing.TXT; Size: 35,775 bytes; and Date of Creation: Nov. 24, 2015) filed with the application is incorporated herein by reference in its entirety.
Oligonucleotides are substances comprised of a few nucleotides (DNA or RNA building blocks) whose nucleotide sequence consists in general of approx. 10-100 nucleotide units. Oligonucleotides are known as primers, for example, which are used in the polymerase chain reaction (PCR). Antisense oligonucleotides are oligonucleotides which have a base sequence that is complimentary to a cellular, viral or synthetic RNA or DNA and which can bind them by Watson-Crick base pairing. Such molecules are often directed against functional mRNAs. Due to the specific binding to the mRNA, ultimately the formation of the respective protein which is encoded by this mRNA is prevented by blockade of translation.
Antisense molecules may belong to various molecular classes to which antisense DNA (asDNA), “small inhibitory” RNA (siRNA), ribozymes and DNAzymes also belong. The last two groups of molecules are characterized by an inherent catalytic activity, which may lead to direct cleavage of the bound target RNA among other things.
As an example, but not exclusively, the DNAzymes of DE 103 46 487.5 are disclosed as examples of DNAzymes for producing an agent against inflammatory diseases.
It is known in general that oligonucleotides are very sensitive to naturally occurring or recombinant nucleases. Nucleases are a group of ubiquitous enzymes which usually act as hydrolases at ester linkages and catalyze the degradation of oligonucleotides. Those skilled in the art are familiar with nucleases as DNAses or RNAses.
The sensitivity of oligonucleotides to nucleases is problematical in medical use of oligonucleotides in particular. In comparison with traditional drugs, oligonucleotides can be degraded rapidly by DNAses and RNAses, which leads to a short half-life and thus to a lower bioavailability in the target cells.
In order for oligonucleotides to also be usable medicinally (in particular therapeutically), they must be protected effectively from the nucleases. To do so, great efforts are being made throughout the world with various technical approaches to modify the structure of oligonucleotides by chemical modifications, for example, in order to increase their stability or to develop drug carrier systems, i.e., so-called drug delivery systems. These drug delivery systems include, for example, liposomes, nanoparticles, viral shell capsids and protamine oligonucleotide particles.
Many of these drug delivery systems have disadvantages; for example, they do not provide adequate protection from degradation by nucleases, they have poor uptake into the target cells and tissue or they retain toxic or problematical constituents, which have a negative effect on the efficacy of the oligonucleotides. For topical application in particular, there is not currently a suitable preparation with which oligonucleotides such as DNAzymes can be formulated and administered as agents against inflammatory diseases because the formulations are not adequately protected against bacteria, fungi and nucleases. Successful use of oligonucleotides in dermatology has so far been prevented by this lack of options for protection.
The present invention relates to a cosmetic and/or dermatological and/or pharmaceutical preparation for topical use and administration of oligonucleotides as agents against diseases. The invention relates in particular to emulsions having a dispersed, discontinuous, internal aqueous phase with which oligonucleotides are formulated and administered as agents for topical application against inflammatory diseases.
A stable effective formulation in the form of an emulsion with a dispersed, discontinuous, internal aqueous phase holding oligonucleotides in a stable emulsion, effectively protecting the oligonucleotides from enzymatic degradation by nuclease while permitting good uptake into the target cells and tissue has surprisingly been found.
FIGS. 1a and 1b show the viscosity (Pa·s) of the DNAzyme-containing formulations. FIG. 1a shows WOW 146 (formulation with MgSO4). FIG. 1b shows WOW 167 (formulation with NaCl) in comparison over a period of 150 days. The stability of the WOW 146 (formulation with MgSO4) is higher than the stability of the WOW 167 (formulation with NaCl).
FIGS. 2a and 2b show the degradation of DNAzyme by DNAse from the skin, measured with HPLC. FIG. 2a shows the HPLC measurement of DNAzyme on the skin after 2 minutes. FIG. 2b shows the HPLC measurement of DNAzyme on the skin after 152 minutes. FIGS. 2a and 2b show that unprotected degradation of DNAzymes on the skin begins already after 2 minutes due to the DNAses present there and after 152 minutes the DNAzyme is almost completely degraded. This reaction takes place equally all oligonucleotides and is not limited to DNAzymes.
FIG. 3 shows the protection of the formulations containing DNAzyme with respect to degradation by DNAse. This shows the WOW 146 formulation containing DNAzyme, which exhibit 90% protection of the DNAzymes, whereas the WOW 167 formulation containing DNAzyme exhibits only 60% protection. A standard solution (0.4% DNAzyme in PBS buffer) containing DNAzyme is used as the control.
FIG. 4 shows the experimental setup of an animal model in which an inflammatory skin reaction is induced experimentally and the efficacy of agents for treatment of these diseases is tested.
1. Characterization of the Formulation
The formulation according to the invention in the form of an emulsion with a dispersed, discontinuous, internal aqueous phase is formed by using the galenical form of the water-in-oil-in-water emulsion, the so-called WOW emulsion, and/or the water-in-oil emulsion, the so-called WO emulsion, with the addition of magnesium or sodium ions. It has surprisingly been found that adding magnesium or sodium ions to the formulation is very advantageous for the effect of the oligonucleotides, improving the stability of the oligonucleotides by protecting them from enzymatic degradation and thus permitting good absorption into the target cells, e.g., skin cells, lung and intestinal epithelial cells, mucosa, nasal epithelial cells and cells of the pharyngeal space.
SUBSTANCES OF THE FORMULATION ACCORDING TO THE INVENTION
The formulation according to the invention comprises at least one lipophilic emulsifier, at least one consistency agent, at least one occlusive component, at least one organic and/or inorganic additive, water and at least one oligonucleotide.
Alternatively, the formulation according to the invention additionally comprises at least one hydrophilic emulsifier and/or at least one amphiphilic component.
Alternatively, the formulation according to the invention additionally comprises at least one humectant factor.
Alternatively, the formulation according to the invention comprises at least one preservative.
Alternatively, the formulation according to the invention comprises at least one natural or synthetic oil or wax.
The at least one lipophilic emulsifier is selected from the group comprised of sorbitan fatty acid esters, glycerol derivatives (e.g., Spans, also glycerol stearate, glycerol dioleate, glycerol monooleate).
The at least one consistency agent is selected from the group comprised of fatty acid esters (e.g., cetyl palmitate, myristyl myristate), polyethylene glycols, cera alba, microcrystalline wax, lanolin and alcohols thereof, hydrogenated castor oil, carbomers (e.g., crosslinked acrylic acid polymers) or cellulose and its derivative.
The at least one occlusive component is selected from the group comprised of saturated hydrocarbons, e.g., paraffin or polysiloxanes such as silicone oils.
The at least one organic and/or inorganic additive is a salt or an ionic liquid whose cation component comprises Na, Mg, K, Li, Ca, Fe, Cu, Ag or a combination of these elements (e.g., a mixture of NaCl, MgSO4) or a combination of these elements with organic cations (e.g., a mixture of Mg(N(SO2CF3)2)2, Mg(OSO2CF3)2 in an ionic liquid, e.g., 1-ethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium sulfate, 1-ethyl-3-methylimdazolium trifluoromethane sulfonate, 1-ethyl-3-methylimidazolium dicyanamide or 1-ethyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)amide).
The at least one hydrophilic emulsifier is selected from the group comprised of polysorbates or ethoxylated polyethylene glycols (e.g., Tweens, steareths, laureths, ceteareths) or ethoxylated ether and/or esters.
The at least one amphiphilic component is selected from the group of phospholipids, preferably lecithin or one of its derivatives.
The at least one humectant factor is selected from the group of glycerol, polyols, osmolytes.
The at least one oil is selected from the group of esterified fatty acids (e.g., decyl oleate), waxes (e.g., jojoba wax) or partially esterified glycerides (e.g., medium-chain length triglycerides) or natural oils such as soy oil, peanut oil, avocado oil, olive oil, castor oil and nut oils or seed oils.
The at least one preservative is paraben, for example.
The at least one oligonucleotide is selected from the group comprised of primers, aptamers, antisense oligonucleotides, e.g., DNAzymes, siRNAs, asDNAs or ribozyme.
As an example but not exclusively, the DNAzymes of DE 103 46 487.5 are cited as the DNAzymes; they are directed against the mRNA of the proteins GATA-3 and T-bet and are disclosed for producing an agent against inflammatory diseases. For example, the following DNAzymes are used (each individually or in combination with the others):
Name of the DNAzyme against GATA-3 mRNA sequence:
hgd1 (SEQ ID NO: 1) 5′-TCGGTCAGAggctagctacaacgaTGCGTTGCT-3′ hgd2 (SEQ ID NO: 2) 5′-GGCGTACGAggctagctacaacgaCTGCTCGGT-3′ hgd3 (SEQ ID NO: 3) 5′-GGCGGCGTAggctagctacaacgaGACCTGCTC-3′ hgd4 (SEQ ID NO: 4) 5′-CTCGGGTCAggctagctacaacgaCTGGGTAGC-3′ hgd5 (SEQ ID NO: 5) 5′-TCCTCTGCAggctagctacaacgaCGGGGTCCT-3′ hgd6 (SEQ ID NO: 6) 5′-ACTCTGCAAggctagctacaacgaTCTGCGAGC-3′ hgd7 (SEQ ID NO: 7) 5′-GGGCGACGAggctagctacaacgaTCTGCAATT-3′ hgd8 (SEQ ID NO: 8) 5′-AAGGGGCGAggctagctacaacgaGACTCTGCA-3′ hgd9 (SEQ ID NO: 9) 5′-AAAACGGGAggctagctacaacgaCAGGTTGTA-3′ hgd10 (SEQ ID NO: 10) 5′-AGAATAAAAggctagctacaacgaGGGACCAGG-3′ hgd11 (SEQ ID NO: 11) 5′-ATGGCAGAAggctagctacaacgaAAAACGGGA-3′ hgd12 (SEQ ID NO: 12) 5′-AACTGGGTAggctagctacaacgaGGCAGAATA-3′ hgd13 (SEQ ID NO: 13) 5′-ATCCAAAAAggctagctacaacgaTGGGTATGG-3′ hgd14 (SEQ ID NO: 14) 5′-AGGGGAAGAggctagctacaacgaAAAAATCCA-3′ hgd15 (SEQ ID NO: 15) 5′-TTTTAAAAAggctagctacaacgaTATCTTGGA-3′ hgd16 (SEQ ID NO: 16) 5′-GTGGGGGGAggctagctacaacgaGGGAAGGCT-3′ hgd17 (SEQ ID NO: 17) 5′-GTTGAATGAggctagctacaacgaTTGCTTTCG-3′ hgd18 (SEQ ID NO: 18) 5′-GTCGTTGAAggctagctacaacgaGATTTGCTT-3′ hgd19 (SEQ ID NO: 19) 5′-GGCCCGGAAggctagctacaacgaCCGCGCGCG-3′ hgd20 (SEQ ID NO: 20) 5′-TCACCTCCAggctagctacaacgaGGCCTCGGC-3′ hgd21 (SEQ ID NO: 21) 5′-CCGCCGTCAggctagctacaacgaCTCCATGGC-3′ hgd22 (SEQ ID NO: 22) 5′-GGTGGCTCAggctagctacaacgaCCAGCGCGG-3′ hgd23 (SEQ ID NO: 23) 5′-CGTTGAGCAggctagctacaacgaGGCGGGGTG-3′ hgd24 (SEQ ID NO: 24) 5′-CCGCGTCCAggctagctacaacgaGTAGGAGTG-3′ hgd25 (SEQ ID NO: 25) 5′-CAGCGGGTAggctagctacaacgaTGCGCCGCG-3′ hgd26 (SEQ ID NO: 26) 5′-GCACATCCAggctagctacaacgaCTCCTCCGG-3′ hgd27 (SEQ ID NO: 27) 5′-AAAAGCACAggctagctacaacgaCCACCTCCT-3′ hgd28 (SEQ ID NO: 28) 5′-TAAAAAGCAggctagctacaacgaATCCACCTC-3′ hgd29 (SEQ ID NO: 29) 5′-GACCGTCGAggctagctacaacgaGTTAAAAAG-3′ hgd30 (SEQ ID NO: 30) 5′-TTGCCTTGAggctagctacaacgaCGTCGATGT-3′ hgd31 (SEQ ID NO: 31) 5′-AGGGCGGGAggctagctacaacgaGTGGTTGCC-3′ hgd32 (SEQ ID NO:32) 5′-TGGCCCTGAggctagctacaacgaCGAGTTTCC-3′ hgd33 (SEQ ID NO: 33) 5′-ACCTCTGCAggctagctacaacgaCGTGGCCCT-3′ hgd34 (SEQ ID NO: 34) 5′-CGGAGGGTAggctagctacaacgaCTCTGCACC-3′ hgd35 (SEQ ID NO: 35) 5′-GGCGGCACAggctagctacaacgaCTGGCTCCC-3′ hgd36 (SEQ ID NO: 36) 5′-CGGGCGGCAggctagctacaacgaACCTGGCTC-3′ hgd37 (SEQ ID NO: 37) 5′-AGGGATCCAggctagctacaacgaGAAGCAGAG-3′ hgd38 (SEQ ID NO: 38) 5′-GGGTAGGGAggctagctacaacgaCCATGAAGC-3′ hgd39 (SEQ ID NO: 39) 5′-GGGCTGAGAggctagctacaacgaTCCAGGGGG-3′ hgd40 (SEQ ID NO: 40) 5′-GTGGATGGAggctagctacaacgaGTCTTGGAG-3′ hgd41 (SEQ ID NO: 41) 5′-CGTGGTGGAggctagctacaacgaGGACGTCTT-3′ hgd42 (SEQ ID NO: 42) 5′-GGGGGTAGAggctagctacaacgaGGAGAGGGG-3′ hgd43 (SEQ ID NO: 43) 5′-GGAGGAGGAggctagctacaacgaGAGGCCGGG-3′ hgd44 (SEQ ID NO: 44) 5′-GCCCCCCGAggctagctacaacgaAAGGAGGAG-3′ hgd45 (SEQ ID NO: 45) 5′-CCGGGGAGAggctagctacaacgaGTCCTTCGG-3′ hgd46 (SEQ ID NO: 46) 5′-GGACAGCGAggctagctacaacgaGGGTCCGGG-3′ hgd47 (SEQ ID NO: 47) 5′-TGGGGTGGAggctagctacaacgaAGCGATGGG-3′ hgd48 (SEQ ID NO: 48) 5′-CTTGAGGCAggctagctacaacgaTCTTTCTCG-3′ hgd49 (SEQ ID NO: 49) 5′-CACCTGGTAggctagctacaacgaTTGAGGCAC-3′ hgd50 (SEQ ID NO: 50) 5′-GCAGGGGCAggctagctacaacgaCTGGTACTT-3′ hgd51 (SEQ ID NO: 51) 5′-CCAGCTTCAggctagctacaacgaGCTGTCGGG-3′ hgd52 (SEQ ID NO: 52) 5′-GTGGGACGAggctagctacaacgaTCCAGCTTC-3′ hgd53 (SEQ ID NO: 53) 5′-GGAGTGGGAggctagctacaacgaGACTCCAGC-3′ hgd54 (SEQ ID NO: 54) 5′-ATGCTGCCAggctagctacaacgaGGGAGTGGG-3′ hgd55 (SEQ ID NO: 55) 5′-GGGCGGTCAggctagctacaacgaGCTGCCACG-3′ hgd56 (SEQ ID NO: 56) 5′-GAGGCTCCAggctagctacaacgaCCAGGGCGG-3′ hgd57 (SEQ ID NO: 57) 5′-GTGGGTCGAggctagctacaacgaGAGGAGGCT-3′ hgd58 (SEQ ID NO: 58) 5′-AGGTGGTGAggctagctacaacgaGGGGTGGTG-3′ hgd59 (SEQ ID NO: 59) 5′-ACTCGGGCAggctagctacaacgaGTAGGGCGG-3′ hgd60 (SEQ ID NO: 60) 5′-GGAGCTGTAggctagctacaacgaTCGGGCACG-3′ hgd61 (SEQ ID NO: 61) 5′-GGACTTGCAggctagctacaacgaCCGAAGCCG-3′ hgd62 (SEQ ID NO: 62) 5′-GGGCCTGGAggctagctacaacgaTTGCATCCG-3′ hgd63 (SEQ ID NO: 63) 5′-TGTGCTGGAggctagctacaacgaCGGGCCTTG-3′ hgd64 (SEQ ID NO: 64) 5′-GTTCACACAggctagctacaacgaTCCCTGCCT-3′ hgd65 (SEQ ID NO: 65) 5′-CAGTTCACAggctagctacaacgaACTCCCTGC-3′ hgd66 (SEQ ID NO: 66) 5′-CACAGTTCAggctagctacaacgaACACTCCCT-3′ hgd67 (SEQ ID NO: 67) 5′-GTTGCCCCAggctagctacaacgaAGTTCACAC-3′ hgd68 (SEQ ID NO: 68) 5′-TCGCCGCCAggctagctacaacgaAGTGGGGTC-3′ hgd69 (SEQ ID NO: 69) 5′-CCCGTGCCAggctagctacaacgaCTCGCCGCC-3′ hgd70 (SEQ ID NO: 70) 5′-GGCGTTGCAGgctagctacaacgaAGGTAGTGT-3′
Name of the DNAzymes against T-bet mRNA sequence:
td1 (SEQ ID NO: 71) 5′-TGGCTTCTAggctagctacaacgaGCCCTCGTC-3′ td2 (SEQ ID NO: 72) 5′-GGGCTCTGAggctagctacaacgaGCCTGGCTT-3′ td3 (SEQ ID NO: 73) 5′-GGGACCCCAggctagctacaacgaCGGAGCCCG-3′ td4 (SEQ ID NO: 74) 5′-GGTGGGGGAggctagctacaacgaCCCACCGGA-3′ td5 (SEQ ID NO: 75) 5′-GGCGGGGGAggctagctacaacgaCCGAGGGCC-3′ td6 (SEQ ID NO: 76) 5′-GGGCTGGGAggctagctacaacgaGGGCAGGGA-3′ td7 (SEQ ID NO: 77) 5′-CGTCGAGGAggctagctacaacgaCCGCCCCTC-3′ td8 (SEQ ID NO: 78) 5′-GGGCTGGCAggctagctacaacgaCTTCCCGTA-3′ td9 (SEQ ID NO: 79) 5′-CGATGCCCAggctagctacaacgaCCGGGGCGG-3′ td10 (SEQ ID NO: 80) 5′-GCTCCACGAggctagctacaacgaGCCCATCCG-3′ td11 (SEQ ID NO: 81) 5′-CCGGCTCCAggctagctacaacgaGATGCCCAT-3′ td12 (SEQ ID NO: 82) 5′-TCTCCGCAAggctagctacaacgaCCGGCTCCA-3′ td13 (SEQ ID NO: 83) 5′-CCGTCAGCAggctagctacaacgaGTCTCCGCA-3′ td14 (SEQ ID NO: 84) 5′-TCCCCGGCAggctagctacaacgaCGGCTCGGT-3′ td15 (SEQ ID NO: 85) 5′-CCCCCGCGAggctagctacaacgaGCTCGTCCG-3′ td16 (SEQ ID NO: 86) 5′-GTAGGGAGAggctagctacaacgaCCCAGGCTG-3′ td17 (SEQ ID NO: 87) 5′-GGGCGGGCAggctagctacaacgaCAAGGCGCC-3′ td18 (SEQ ID NO: 88) 5′-CGGGAAGGAggctagctacaacgaTCGCCCGCG-3′ td19 (SEQ ID NO: 89) 5′-TAGTCCTCAggctagctacaacgaGCGGCCCCG-3′ td20 (SEQ ID NO: 90) 5′-TCCCCGACAggctagctacaacgaCTCCAGTCC-3′ td21 (SEQ ID NO: 91) 5′-TTTCCCCGAggctagctacaacgaACCTCCAGT-3′ td22 (SEQ ID NO: 92) 5′-TGAGCGCGAggctagctacaacgaCCTCAGTTT-3′ td23 (SEQ ID NO: 93) 5′-GGACCACAAggctagctacaacgaAGGTGGTTG-3′ td24 (SEQ ID NO: 94) 5′-CTTGGACCAggctagctacaacgaAACAGGTGG-3′ td25 (SEQ ID NO: 95) 5′-AAACTTGGAggctagctacaacgaCACAACAGG-3′ td26 (SEQ ID NO: 96) 5′-CTGATTAAAggctagctacaacgaTTGGACCAC-3′ td27 (SEQ ID NO: 97) 5′-TGGTGCTGAggctagctacaacgaTAAACTTGG-3′ td28 (SEQ ID NO: 98) 5′-TGATGATCAggctagctacaacgaCTCTGTCTG-3′ td29 (SEQ ID NO: 99) 5′-TGGTGATGAggctagctacaacgaCATCTCTGT-3′ td30 (SEQ ID NO: 100) 5′-GCTTGGTGAggctagctacaacgaGATCATCTC-3′ td31 (SEQ ID NO: 101) 5′-ATGGGAACAggctagctacaacgaCCGCCGTCC-3′ td32 (SEQ ID NO: 102) 5′-GAATGGGAAggctagctacaacgaATCCGCCGT-3′ td33 (SEQ ID NO: 103) 5′-TGACAGGAAggctagctacaacgaGGGAACATC-3′ td34 (SEQ ID NO: 104) 5′-AGTAAATGAggctagctacaacgaAGGAATGGG-3′ td35 (SEQ ID NO: 105) 5′-CACAGTAAAggctagctacaacgaGACAGGAAT-3′ td36 (SEQ ID NO: 106) 5′-GCCCGGCCAggctagctacaacgaAGTAAATGA-3′ td37 (SEQ ID NO: 107) 5′-CCACAAACAggctagctacaacgaCCTGTAGTG-3′ td38 (SEQ ID NO: 108) 5′-GTCCACAAAggctagctacaacgaATCCTGTAG-3′ td39 (SEQ ID NO: 109) 5′-CCACGTCCAggctagctacaacgaAAACATCCT-3′ td40 (SEQ ID NO: 110) 5′-CCAAGACCAggctagctacaacgaGTCCACAAA-3′ td41 (SEQ ID NO: 111) 5′-CCACCAAGAggctagctacaacgaCACGTCCAC-3′ td42 (SEQ ID NO: 112) 5′-GCTGGTCCAggctagctacaacgaCAAGACCAC-3′ td43 (SEQ ID NO: 113) 5′-GCTCTGGTAggctagctacaacgaCGCCAGTGG-3′ td44 (SEQ ID NO: 114) 5′-CTGCACCCAggctagctacaacgaTTGCCGCTC-3′ td45 (SEQ ID NO: 115) 5′-CACACTGCAggctagctacaacgaCCACTTGCC-3′ td46 (SEQ ID NO: 116) 5′-CTTTCCACAggctagctacaacgaTGCACCCAC-3′ td47 (SEQ ID NO: 117) 5′-GCCTTTCCAggctagctacaacgaACTGCACCC-3′ td48 (SEQ ID NO: 118) 5′-TTCCTGGCAggctagctacaacgaGCTGCCCTC-3′ td49 (SEQ ID NO: 119) 5′-GTGGACGTAggctagctacaacgaAGGCGGTTT-3′ td50 (SEQ ID NO: 120) 5′-CCGGGTGGAggctagctacaacgaGTACAGGCG-3′ td51 (SEQ ID NO: 121) 5′-CCTGGCGCAggctagctacaacgaCCAGTGCGC-3′ td52 (SEQ ID NO: 122) 5′-CAAATGAAAggctagctacaacgaTTCCTGGCG-3′ td53 (SEQ ID NO: 123) 5′-TTTCCCAAAggctagctacaacgaGAAACTTCC-3′ td54 (SEQ ID NO: 124) 5′-ATTGTTGGAggctagctacaacgaGCCCCCTTG-3′ td55 (SEQ ID NO: 125) 5′-TGGGTCACAggctagctacaacgaTGTTGGACG-3′ td56 (SEQ ID NO: 126) 5′-TCTGGGTCAggctagctacaacgaATTGTTGGA-3′ td57 (SEQ ID NO: 127) 5′-GCACAATCAggctagctacaacgaCTGGGTCAC-3′ td58 (SEQ ID NO: 128) 5′-GGAGCACAAggctagctacaacgaCATCTGGGT-3′ td59 (SEQ ID NO: 129) 5′-ACTGGAGCAggctagctacaacgaAATCATCTG-3′ td60 (SEQ ID NO: 130) 5′-ATGGAGGGAggctagctacaacgaTGGAGCACA-3′ td61 (SEQ ID NO: 131) 5′-TGGTACTTAggctagctacaacgaGGAGGGACT-3′ td62 (SEQ ID NO: 132) 5′-GGGCTGGTAggctagctacaacgaTTATGGAGG-3′ td63 (SEQ ID NO: 133) 5′-TCAACGATAggctagctacaacgaGCAGCCGGG-3′ td64 (SEQ ID NO: 134) 5′-CCTCAACGAggctagctacaacgaATGCAGCCG-3′ td65 (SEQ ID NO: 135) 5′-TCACCTCAAggctagctacaacgaGATATGCAG-3′ td66 (SEQ ID NO: 136) 5′-CGTCGTTCAggctagctacaacgaCTCAACGAT-3′ td67 (SEQ ID NO: 137) 5′-GTAAAGATAggctagctacaacgaGCGTGTTGG-3′ td68 (SEQ ID NO: 138) 5′-AAGTAAAGAggctagctacaacgaATGCGTGTT-3′ td69 (SEQ ID NO: 139) 5′-GGCAATGAAggctagctacaacgaTGGGTTTCT-3′ td70 (SEQ ID NO: 140) 5′-TCACGGCAAggctagctacaacgaGAACTGGGT-3′ td71 (SEQ ID NO: 141) 5′-AGGCAGTCAggctagctacaacgaGGCAATGAA-3′ td72 (SEQ ID NO: 142) 5′-ATCTCGGCAggctagctacaacgaTCTGGTAGG-3′ td73 (SEQ ID NO: 143) 5′-GCTGAGTAAggctagctacaacgaCTCGGCATT-3′ td74 (SEQ ID NO: 144) 5′-TATTATCAAggctagctacaacgaTTTCAGCTG-3′ td75 (SEQ ID NO: 145) 5′-GGGTTATTAggctagctacaacgaCAATTTTCA-3′ td76 (SEQ ID NO: 146) 5′-AAGGGGTTAggctagctacaacgaTATCAATTT-3′ td77 (SEQ ID NO: 147) 5′-CTCCCGGAAggctagctacaacgaCCTTTGGCA-3′ td78 (SEQ ID NO: 148) 5′-GTACATGGAggctagctacaacgaTCAAAGTTC-3′
Components of the WOW emulsion:
The WOW emulsion comprises at least one lipophilic emulsifier, at least one amphiphilic component, at least one consistency agent, at least one occlusive component, at least one organic or inorganic additive, at least one hydrophilic emulsifier.
In particular the WOW emulsion comprises at least one lipophilic emulsifier, where the lipophilic emulsifier is selected from the group comprised of sorbitan, fatty acid esters, glycerol derivatives (e.g., Spans).
At least one amphiphilic component, where this amphiphilic component is selected from the group comprised of phospholipids, preferably lecithin or one of its derivatives.
At least one consistency agent, where the consistency agent is selected from the group comprised of fatty acid esters (e.g., cetyl palmitate, myristyl myristate), polyethylene glycols, cera alba, microcrystalline wax, lanolin, hydrogenated castor oil, protegin W, protegin WX, carbomers (e.g., crosslinked acrylic acid polymers) or cellulose and its derivatives.
At least one occlusive component, where the occlusive component is a saturated hydrocarbon selected from the group comprised of paraffin or polysiloxanes such as silicone oils.
Alternative an oil selected from the group comprised of esterified fatty acids (e.g., decyl oleate), waxes (e.g., jojoba wax) or partially esterified glycerides (e.g., medium chain length triglycerides) or natural oils such as soy oil, peanut oil, avocado oil, olive oil, castor oil and nut or seed oils.
At least one organic and/or inorganic additive preferably a salt or an ionic liquid whose cation component comprises Na, Mg, K, Li, Ca, Fe, Cu, Ag or a combination of these elements.
At least one hydrophilic emulsifier where the hydrophilic emulsifier is selected from the group comprised of polysorbates, ethoxylated polyethylene glycols (e.g., Tweens, steareths, laureths, ceteareths), ethoxylated ether, ethoxylated esters.
At least one oligonucleotide selected from the group of antisense oligonucleotides, e.g., DNAzymes, siRNAs, asDNAs or ribozymes or primers or aptamers.
Alternatively, the WOW emulsion additionally comprises at least one humectant factor where the humectant factor is selected from the group comprised of glycerol, polyols, osmolytes.
Alternatively, the WOW emulsion additionally comprises at least one preservative, e.g., paraben.
Ingredients of the WO emulsion:
The WO emulsion comprises at least one lipophilic emulsifier, at least one occlusive component, at least one consistency agent, at least one organic and/or inorganic additive, at least one hydrophilic emulsifier.
In particular the WO emulsion comprises at least one lipophilic emulsifier, where the lipophilic emulsifier is selected from the group comprised of sorbitan, fatty acid esters, glycerol derivatives (e.g., glycerol stearate, glycerol dioleates, glycerol monooleates).
At least one occlusive component wherein the occlusive component is a saturated hydrocarbon selected from the group comprised of paraffin or polysiloxanes such as silicone oils
Alternatively, an oil selected from the group comprised of esterified fatty acids (e.g., decyl oleate), waxes (e.g., jojoba wax) or partially esterified glycerides (e.g., medium chain-length triglycerides) or natural oils such as soy oil, peanut oil, avocado oil, olive oil, castor oil as well as nut or seed oils.
At least one consistency agent, where the consistency agent is selected from the group comprised of fatty acid esters (e.g., cetyl palmitate, myristyl myristate), polyethylene glycols, cera alba, microcrystalline wax, lanolin or alcohols thereof, hydrogenated castor oil, protegin W, protegin WX, carbomers (e.g., crosslinked acrylic acid polymers) or cellulose and derivatives thereof.
At least one organic and/or inorganic additive, preferably a salt or an ionic liquid whose cationic component comprises Na, Mg, K, Li, Ca, Fe, Cu, Ag or a combination of these elements.
Alternatively, the WO emulsion additionally contains at least one humectant factor wherein the humectant factor is selected from the group comprised of glycerol, polyols, osmolytes.
At least one oligonucleotide selected from the group of comprised of antisense oligonucleotides, e.g., DNAzymes, siRNAs, asDNAs or ribozymes or primers or aptamers.
Alternatively, the WO emulsion additionally comprises at least one preservative, e.g., paraben.
The pharmaceutical composition according to the invention for topical application, comprising at least one lipophilic emulsifier, at least one consistency agent, at least one occlusive component, at least one organic and/or inorganic additive and at least oligonucleotide is added by methods with which those skilled in the art are familiar.
The pharmaceutical composition according to the invention for topical application, comprising at least one lipophilic emulsifier, at least one consistency agent, at least one occlusive component, at least one organic and/or inorganic additive and at least one oligonucleotide is more or less fluid, is a shampoo, a solution, a lotion, a cream, ointment, milk, paste or foam. Alternatively, it is an aerosol and is administered via the lungs.
The pharmaceutical composition according to the invention for topical application, comprising at least one lipophilic emulsifier, at least one consistency agent, at least one occlusive component, at least one organic and/or inorganic additive and at least one oligonucleotide is more or less fluid, is used in particular in infants especially in human infants.
2. Use of an Agent for Treatment and Prevention of Diseases
The formulation according to the invention in the form of an emulsion having a dispersed internal continuous aqueous phase is suitable in particular for protecting the oligonucleotides through the addition of magnesium or sodium ions because it stabilizes the oligonucleotides and protects them from enzymatic degradation enabling good absorption into the target cells. Therefore the formulation according to the invention is suitable as a cosmetic and/or dermatologic and/or pharmaceutical preparation for topical application and administration. Due to the use of oligonucleotides, which are known to be effective against inflammatory diseases, for example, the DNAzymes of DE 103 46 487.5, the formulation according to the invention is therefore suitable for production of an agent for treatment and prevention of inflammatory diseases which is used for topical application.
The formulation according to the invention has good absorption of the oligonucleotides, e.g., the DNAzyme, in particular the DNAzyme of DE 103 46 487.5 into target cells in topical application, e.g., skin cells, lung cells and intestinal epithelial cells, mucosa, nasal epithelial cells and cells of the pharyngeal area.
EXAMPLES Example 1 Exemplary Embodiment of WOW Emulsion
The DNAzyme-containing water-in-oil-in-water emulsions (WOW emulsions) according to the invention preferably include the following ingredients:
WOW (%) (range) Lipophilic emulsifier 0.5-20 preferably sorbitan fatty acid ester, glycerol derivatives (e.g., Spans) Amphiphilic component 0.05-5 phospholipids preferably lecithin or one of its derivatives, Consistency agents, such as 0.1-5 fatty acid esters (e.g., cetyl palmitate, myristyl myristate), polyethylene glycols, cera alba, microcrystalline wax, lanolin, hydrogenated castor oil, carbomers (e.g., crosslinked acrylic acid polymers), protegin W, protegin WX or cellulose and its derivatives Occlusive component based on saturated hydrocarbons such as 1-25 paraffin or polysiloxanes such as silicone oils Alternatively, an oil selected from the group of esterified fatty acids 1-25 (e.g., decyl oleate), waxes (e.g., jojoba wax) or partially esterified glycerides (e.g., medium chain length triglycerides) or natural oils such as soy oil, peanut oil, avocado oil, olive oil, castor oil and nut or seed oils Organic and/or inorganic additives, e.g., a salt or ionic liquid whose 0.01-2 cationic component comprises Na, Mg, K, Li, Ca, Fe, Cu, Ag or a combination of these elements Oligonucleotide, e.g., one of the DNAzymes of DE 103 46 487.5 0.01-5 Water as needed Hydrophilic emulsifier such as polysorbates or ethoxylated 1-8 polyethylene glycols (e.g., Tweens, steareths, laureths, ceteareths) or ethoxylated ethers and/or esters Humectant factors (e.g., glycerol, polyols, osmolytes) 0.1-10 Preservatives such as parabens as needed
3.1.1. Selected Exemplary Embodiments (WOW 167)
WOW 167 (% w/w) Sorbitan monooleate 4 Lecithin 0.2 Paraffin 15.8 NaCl 0.074 DNAzyme hdg 40 0.4 Water to a total of 100 Steareth 20 1 Water + paraben 59
3.1.2. Selected Exemplary Embodiments (WOW 146)
WOW 146 (% w/w) Sorbitan monooleate 4 Lecithin 0.2 Paraffin 15.8 MgSO4 × 7H2O 0.308 DNAzyme hdg 40 0.4 Water to a total of 100 Water + paraben, preserved 59 Water (external phase)
Example 2 Exemplary Embodiment of a WO Emulsion
The water-in-oils emulsions according to the invention containing DNAzyme (WO emulsions) comprise the following ingredients:
WOW (%) Range A lipophilic emulsifier or a mixture of same such as sorbitan fatty acid 1-15 esters or glycerol derivatives (glycerol stearates, glycerol dioleates, glycerol monooleates) Occlusive component based on saturated hydrocarbons such as 10-70 paraffin or polysiloxanes such as silicone oils Alternatively, an oil selected from the group of esterified fatty acids 10-70 (e.g., decyl oleate), waxes (e.g., jojoba wax) or partially esterified glycerides (e.g., medium chain length triglycerides) or natural oils such as soy oil, peanut oil, avocado oil, olive oil, castor oil as well as nut or seed oils Consistency agents such as fatty acid esters (e.g., cetyl palmitate, 0.5-10 myristyl myristate), polyethylene glycols, cera alba, microcrystalline wax, lanolin, hydrogenated castor oil, protegin W, protegin WX, carbomers (e.g., crosslinked acrylic acid polymers), or cellulose and its derivatives Lanolin or its alcohols 1-8 Preservatives (e.g., parabens) as needed Organic and/or inorganic additives, e.g., a salt or ionic liquid whose 0.01-2 cation component comprises Na, Mg, K, Li, Ca, Fe, Cu, Ag or a combination of these elements Oligonucleotide, e.g., one of the DNAzymes of DE 103 46 487.5 0.01-5 Humectant factors (e.g., glycerol, polyols, osmolytes) 0.1-10
WO 126 (% w/w) Glycerol stearate 1 Glycerol monooleate (Imwitor 946) 2 Glycerol dioleate (Crossential GDO) 2 Paraffin 38 Cetyl palmitate 2 Lanolin 3 Hydrogenated castor oil 2 Preserved water + parabens to a total of 100 NaCl 0.5 DNAzyme hdg 40 0.4 Glycerol 3
WO 162 (% w/w) Glycerol stearate 1 Glycerol monooleate (Imwitor 946) 2 Glycerol dioleate (Crossential GDO) 2 Paraffin 38 Cetyl palmitate 2 Lanolin 3 Water + paraben, preserved to a total of 100 Water MgSO4 × 7H2O 1 DNAzyme hdg 40 0.4 Glycerol 3
The oligonucleotide used is preferably any of the DNAzymes hgd 1-70 and td 1-78 disclosed in DE 103 46 487.5, preferably hgd 40 as a DNAzyme directed against GATA-3 at td 69 and/or td 70 as DNAzymes directed against T-bet.
Example 3 Stability Measurements on the Formulations
The formulations WOW 167 and WOW 146 containing DNAzyme according to the invention differ in the added electrolytes in the internal aqueous phase, which is represented graphically in FIG. 1. Solutions of 0.13M are preferably used here to prepare WOW 167 and WOW 146, because formulations with higher concentrations of electrolytes reduce the stability of the formulations. Stability is measured by comparison and over a period of 150 days.
Immediately after preparing the formulation, the viscosity of the WOW 167 (formulation with NaCl) is somewhat higher at 1.4 Pa·s than the viscosity of WOW 146 (formulation with MgSO4) at 1 Pa·s. The viscosity of the WOW 146 formulation (formulation with MgSO4) drops slightly to 0.75 Pa·s within the first 5 days, then remains largely unchanged at 0.75 Pa·s. The viscosity of the WOW 167 (formulation with NaCl) drops to 0.6 Pa·s by day 50 and then drops further to 0.4 Pa·s.
WOW 146 (formulation with MgSO4) is thus much more stable than the WOW 167 (formulation with NaCl). It should be pointed out that the slight decline in viscosities does not have a negative effect on the stability or the protective action, and the emulsions do not exhibit any phase inversion or separation and are stable.
The measured droplet size is in the range of 10-20 μm.
The DNAzyme-containing formulations WO 162 and WO 126 according to the invention also differ in the electrolytes added to the internal water phase. The stability is measured by comparison and over a period of 150 days, where the viscosity remains unchanged for 150 days at approx. 4 Pa·s in the case of WO 126 and 4.2 for WO 162.
The droplet size here is in the range of 0.5-2.5 μm.
Example 4 Stability with Respect to Degradation by Nucleases Such as DNAses
The DNAzyme-containing WOW 167 and WOW 146 formulations according to the invention contain the DNAzymes of DE 103 46 487.5, namely DNAzymes hgd 1 to hgd 70 against GATA-3 and DNAzymes td 1 to td 78 against T-bet and protect them from degradation due to nucleases, in particular DNAses. It is important to protect the DNAzymes from degradation due to the DNAse, so that they can manifest their therapeutic efficacy.
A commercially available DNAse I with an activity of 105 U is used experimentally to measure the stability of the DNAzyme-containing formulations according to the invention with respect to DNAse and this was added to the WOW 167 and WOW 146 formulations. In addition, the degradation by a dermal lysate is used as a positive control. The degradation of the DNAzymes is determined by HPLC.
The measurement is performed using the following method:
Degradation of DNAzymes by DNAse from the skin
Skin: approx. 50 mg
Solution: 1 mL DNAzyme standard (0.1625 mg/mL)+3 mL RO water (water from reverse osmosis)
Method: Skin pulverized using a scalpel and mixed with solution and shaken lightly, then filtered and degraded by HPLC after 2 min, 52 min, 102 min and 152 min.
FIGS. 2a and 2b show that the DNAses naturally occurring in skin begin to degrade the DNAzyme after only 2 minutes; after 152 minutes almost the entire amount of DNAzyme is degraded.
Example 5 Protecting the DNAzymes from Degradation by DNAse Using the Formulations WOW 146, WOW 167, WO 126, WO 162 According to the Invention
Galenical formulation 20 mg
Solution: DNAse I with an activity of 105 U in Tris buffer+10 mM MgSO4
Method: 20 mg of the respective formulation is mixed with 1 mL of a DNAse I solution. After an incubation time of 1 min, the mixture is agitated lightly at a temperature of 99° C. for 10 minutes in a thermomixer to stop the activity of the DNAse. To break the emulsion, the batch is incubated in an ultrasonic bath at 50° C. for 10 minutes. Then the batch is filtered through a 0.45 μm syringe filter and HPLC analysis is performed.
FIG. 3 shows that the WOW 146 formulation offers approx. 61% protection of the DNAzymes from DNAse degradation, whereas 51% protection is achieved with the WOW 167 formulation. A standard solution containing DNAzyme (0.4% DNAzyme hgd 40 in PBS buffer (PBS=phosphate buffered saline solution ˜137 mM NaCl, 2.7 mM KCl, 12 mM Na2HPO4 and KH2PO4, pH=7.4)) was used as the control, but no active ingredient could be recovered here. The WOW 167 formulation thus provides better protection on the whole. A comparable OW galenical formulation (microemulsion: preserved water, oil components, glycerol, hydrophilic emulsifier, magnesium sulfate) with the same amount of DNAzyme also failed to show any protection and 100% of the DNAzymes was degraded.
Example 6 Stability in Comparison with Oil-in-Water Formulations (OW Formulations)
For detecting the stability of the active ingredient in the galenical formulations, the recovery of 0.4% of the DNAzyme hgd 40 active ingredient in WO 162, WOW 146 was analyzed by HPLC after one month, using as the reference a comparable OW galenical formulation (microemulsion: preserved water, oil component, glycerol, hydrophilic emulsifier, magnesium sulfate).
The recovery of DNAzyme hgd 40 in comparison with the starting value of 100% after one month was 35.98±0.16% in the OW comparative galenical formulation. The recovery after one month in the WO 162 formulation was 95.66±2.77%. The recovery after one month in the WOW formulation was 103.15±2.29%.
Example 7 Detecting the Efficacy of GATA-3 DNAzyme Formulations in the Animal Model
The efficacy of the pharmaceutical composition according to the invention for topical application comprising at least one lipophilic emulsifier, at least consistency agent, at least one occlusive component, at least one organic and/or inorganic additive and at least one oligonucleotide as the agent for treatment of inflammatory skin diseases is illustrated on the example of GATA-3 DNAzyme-containing formulations in an animal model.
To do so, a mouse model is used (see FIG. 4) in which a specific inflammation reaction in the skin of the mouse is induced by allergic sensitization using a model allergen such as ovalbumin UOVA) and subsequent repeated cutaneous administration by skin patches.
For sensitization, 10 μg OVA and 10 μg AL(OH)3 in 100 μl PBS was administered intraperitoneally. For the patch test method, 100 μg OVA and 10 μg AL(OH)3 was applied epicutaneously. For DNAzyme treatment, three formulations of 200 μg DNAzyme in 50 μl PBS were administered intradermally and/or epicutaneously.
In this model, the inflammatory reaction typical of inflammatory skin diseases is induced in the skin of mice (strain Balb) by allergic sensitization using a model allergen, e.g., ovalbumin (OVA) and subsequent repeated epicutaneous application over a skin patch.
The sensitization is accomplished by intraperitoneal administration (i.p.), while the treatment with DNAzyme is achieved by intradermal (i.d.) administration or epicutaneous (e.c.) administration of various formulations.
In addition to the typical histological changes in the skin, the expected increased expression of the target gene, e.g., GATA-3 and the Th2 cytokines IL-4, IL-5 and IL-13 in the inflamed area of skin is triggered and detected. By using the DNAzyme-containing formulations according to the invention as the agents for treatment of inflamed skin diseases, a therapeutic efficacy becomes apparent. Thus a significantly reduced inflammation score in mice was achieved after treating them with DNAzyme formulations. Furthermore, it was demonstrated histologically that after this treatment, pure inflammation cells in particular CD4+ T-lymphocytes could be found in the inflamed areas of skin.
For example, the following were used:
Sensitization: 10 μg OVA+10 μg Al(OH)3 in 100 μL PBS, administered intraperitoneally
Patches: 100 μg OVA+10 μg Al(OH)3 administered cutaneously by the patch test method
Treatment: 200 μg DNAzyme in 50 μL PBS applied topically intradermally and/or epicutaneously in the formulations WOW 146, WO 162 and WOW 167
The analyses have shown that the formulations containing DNAzyme are effective against experimentally-induced inflammatory skin diseases.
What is claimed: 1. A pharmaceutical composition for topical application in the form of either (i) a water-in-oil-in-water emulsion or (ii) a water-in-oil emulsion, each of said emulsions comprising a dispersed, discontinuous, internal aqueous phase, the composition comprising: (a) at least one lipophilic emulsifier selected from the group consisting of a sorbitan fatty acid ester or a glycerol derivative; (b) at least one consistency agent; (c) at least one synthetic oil, natural oil, wax, or saturated hydrocarbon selected from the group consisting of paraffin or a polysiloxane; (d) at least one additive comprising a salt or an ionic liquid, wherein the cation component of the salt or of the ionic liquid comprises an element selected from the group consisting of Na, Mg, K, Li, Ca, Fe, Cu, Ag and a combination of these elements; and, (e) at least one antisense oligonucleotide held by the internal aqueous phase, wherein the at least one antisense oligonucleotide is a DNAzyme directed against the mRNA coding for T-bet protein, the DNAzyme selected from a group consisting of DNAzymes according to SEQ ID NO 71 to SEQ ID NO 148. 2. The pharmaceutical composition according to claim 1, further comprising at least one hydrophilic emulsifier selected from the group consisting of polysorbates, ethoxylated polyethylene glycols, ethoxylated ethers, and ethoxylated esters. 3. The pharmaceutical composition according to claim 1, further comprising at least one amphiphilic component. 4. The pharmaceutical composition according to claim 3, wherein the amphiphilic component is a phospholipid. 5. The pharmaceutical composition according to claim 1, further comprising at least one humectant factor. 6. The pharmaceutical composition according to claim 5, wherein the humectant factor is selected from the group consisting of glycerol, polyols, and osmolytes. 7. The pharmaceutical composition according to claim 1, further comprising at least one preservative. 8. The pharmaceutical composition according to claim 1, wherein the lipophilic emulsifier is a sorbitan monooleate. 9. The pharmaceutical composition according to claim 1, wherein the lipophilic emulsifier is a glycerol derivative selected from the group consisting of Spans, glycerol stearate, glycerol dioleate, and glycerol monooleate. 10. The pharmaceutical composition according to claim 1, wherein the consistency agent is selected from the group consisting of a fatty acid ester, a polyethylene glycol, cera alba, a microcrystalline wax, lanolin, hydrogenated castor oil, protegin W, protegin WX, carbomers, and cellulose. 11. The pharmaceutical composition according to claim 1, wherein the synthetic oil is an esterified fatty acid or a partially esterified glyceride. 12. The pharmaceutical composition according to claim 1, wherein the natural oil is selected from the group consisting of soy oil, peanut oil, avocado oil, olive oil, castor oil, a seed oil, and a nut oil. 13. The pharmaceutical composition according to claim 1, wherein the wax is jojoba wax. 14. The pharmaceutical composition according to claim 7, wherein the preservative is paraben. 15. The pharmaceutical composition according to claim 1, wherein the cation is selected from the group consisting of Na, Mg, and a combination thereof. 16. The pharmaceutical composition according to claim 1, wherein the salt is selected from the group consisting of NaCl, MgSO4, Mg(N(SO2CF3)2)2, and Mg(OSO2CF3)2. 17. The pharmaceutical composition according to claim 1, wherein the ionic liquid is selected from the group consisting of 1-ethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium sulfate, 1-ethyl-3-methylimdazolium trifluoromethane sulfonate, 1-ethyl-3-methylimidazolium dicyanamide, and 1-ethyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)amide).
US14965371 2010-02-10 2015-12-10 Dermatological, pharmaceutical composition suitable for oligonucleotides Active US9404110B2 (en)
DE102010007562 2010-02-10
DE102010007562.0 2010-02-10
DE201010007562 DE102010007562A1 (en) 2010-02-10 2010-02-10 Dermatological pharmaceutical composition suitable for oligonucleotides
PCT/EP2011/000648 WO2011098285A3 (en) 2010-02-10 2011-02-11 Dermatological, pharmaceutical composition suitable for oligonucleotides
US201213578013 true 2012-10-16 2012-10-16
US14965371 US9404110B2 (en) 2010-02-10 2015-12-10 Dermatological, pharmaceutical composition suitable for oligonucleotides
US13578013 Division US9249413B2 (en) 2010-02-10 2011-02-11 Dermatological, pharmaceutical composition suitable for oligonucleotides
PCT/EP2011/000648 Division WO2011098285A3 (en) 2010-02-10 2011-02-11 Dermatological, pharmaceutical composition suitable for oligonucleotides
US201213578013 Division 2012-10-16 2012-10-16
US20160115478A1 true US20160115478A1 (en) 2016-04-28
US9404110B2 true US9404110B2 (en) 2016-08-02
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US13578013 Active 2031-06-17 US9249413B2 (en) 2010-02-10 2011-02-11 Dermatological, pharmaceutical composition suitable for oligonucleotides
US14965371 Active US9404110B2 (en) 2010-02-10 2015-12-10 Dermatological, pharmaceutical composition suitable for oligonucleotides
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WO2017205506A1 (en) * 2016-05-24 2017-11-30 Emory University Particles with rna cleaving nucleobase polymers and uses for managing inflammatory disorders
WO2000009673A1 (en) 1998-08-13 2000-02-24 Johnson & Johnson Research Pty. Limited Dnazymes and methods for treating hpv-related disorders
JP2002515514A (en) 1998-05-21 2002-05-28 アイシス・ファーマシューティカルス・インコーポレーテッド Compositions and methods for topical Oku逹 oligonucleotide
DE10238298A1 (en) 2002-08-21 2004-03-04 Beiersdorf Ag Use of antisense oligonucleotides for the treatment of degenerative skin
DE10254214A1 (en) 2002-11-20 2004-06-09 Beiersdorf Ag Oligoribonucleotides for the treatment of degenerative skin by RNA interference
DE10346487A1 (en) 2003-10-02 2005-05-12 Transmit Technologietransfer A method for producing a cell and / or tissue and / or disease phase specific medicament
DE102004010547A1 (en) 2004-03-03 2005-11-17 Beiersdorf Ag Oligoribonucleotides for the treatment of irritative and / or inflammatory skin conditions by RNA interference
WO2007013411A1 (en) 2005-07-25 2007-02-01 Kaneka Corporation Anti-sense nucleic acid derived from organism
US7291721B2 (en) 2001-11-14 2007-11-06 Centocor, Inc. Anti-IL-6 antibodies, compositions, methods and uses
JP2009506763A (en) 2005-09-01 2009-02-19 エル、ベ、エム、アッシュ、ルシェルシュＬ Ｖ Ｍ Ｈ Ｒｅｃｈｅｒｃｈｅ New tyrosinase-specific anti-gene oligonucleotides as depigmenting agents
US20130030038A1 (en) 2010-02-10 2013-01-31 Thomas Schmidts Dermatological, Pharmaceutical Composition Suitable for Oligonucleotides
US20110213014A1 (en) 1998-05-21 2011-09-01 Isis Pharmaceuticals, Inc. Compositions and methods for topical delivery of oligonucleotides
US20050096287A1 (en) 1998-05-21 2005-05-05 Mehta Rahul C. Compositions and methods for topical delivery of oligonucleotides
RU2249458C2 (en) 1999-07-28 2005-04-10 Авентис Фарма Дойчланд Гмбх Oligonucleotide for inhibiting human eg5 expression
US7955597B2 (en) 2001-11-14 2011-06-07 Centocor, Inc. Anti-IL-6 antibodies, compositions, methods and uses
US7833786B2 (en) 2001-11-14 2010-11-16 Centocor, Inc. Anti-IL-6 antibodies, compositions, methods and uses
US7612182B2 (en) 2001-11-14 2009-11-03 Centocor, Inc. Anti-IL-6 antibodies, compositions, methods and uses
RU2318829C2 (en) 2001-11-14 2008-03-10 Сентокор, Инк. Anti-il-6 antibodies, methods and using
US20140141013A1 (en) 2001-11-14 2014-05-22 Janssen Biotech, Inc. Anti-il-6 antibodies, compositions, methods and uses
US20060025363A1 (en) 2002-08-21 2006-02-02 Ute Breitenbach Use of antisense oligonucleotides for the treatment of degenerative skin conditions
US20060058256A1 (en) 2002-11-20 2006-03-16 Beiersdorf Ag Oligoribonucleotides for the treatment of degenerative skin conditions by RNA interference
US20080182808A1 (en) 2002-11-20 2008-07-31 Beiersdorf, Inc. Oligoribonucleotides for the treatment of degenerative skin conditions by rna interference
US8247544B2 (en) 2003-10-02 2012-08-21 Sterna Biologicals Gmbh & Co. Kg Method for producing a cell and/or tissue and/or disease phase specific medicament
US8119789B2 (en) 2003-10-02 2012-02-21 Sterna Biologicals Gmbh & Co. Kg Method for the production of a cell and/or tissue and/or disease phase specific medicament
JP2007507438A (en) 2003-10-02 2007-03-29 フィリップス−ウニベルジテート・マールブルク The method of producing cells and / or tissue and / or disease phase specific agents
US20050261222A1 (en) 2004-03-03 2005-11-24 Beiersdorf Ag Oligoribonucleotides for the treatment of irritative or inflammatory skin symptoms through RNA interference
US20090209623A1 (en) 2005-07-25 2009-08-20 Jon Tomono Anti-sense nucleic acid derived from organism
US20100047192A1 (en) 2005-09-01 2010-02-25 L.V.M.H. Recherche Novel tyrosinase-specific anti-gene oligonucleotides as depigmenting agents
English-language translation of Official Action issued Jul. 7, 2015, by the Japanese Patent Office in Japanese counterpart priority application No. 2012552313.
English-language translation of Official Action issued Jul. 7, 2015, by the Russian Patent Office in Russian counterpart priority application No. 2012552313.
German-language Communication pursuant to Article 94(3) EPC issued Mar. 16, 2015 by the European Patent Office for European Application No. 11 705 444.5-1455.
German-language Official Action issued Jan. 13, 2011, by the German Patent Office in connection with the German counterpart priority application No. 10 2010 007 562.0.
German-language Official Action issued Jun. 25, 2014, by the European Patent Office in connection with the German counterpart priority application No. 11 705 444.5.
International Search Report and Written Opinion for corresponding international application No. PCT/EP2011/000648, mailing date Feb. 6, 2012 (includes English translation of written opinion).
Japanese-language Official Action issued Dec. 24, 2014, by the Japanese Patent Office in connection with the Japanese counterpart priority application No. 10-2012-552313.
Russian-language Decision on Grant issued Jun. 26, 2015, by the Russian Patent Office in Russian counterpart priority application No. 2012137904.
Russian-language Official Action issued Feb. 2, 2015, by the Russian Patent Office in connection with the Russian counterpart priority application No. 2012137904/15(061502).
Uzagare et al., Application of ionic liquid 1-methoxyethyl-3-methyl imidazolium methanesulfonate in nucleoside chemistry, Green Chem., 5:370-2 (2003).
JP5896921B2 (en) 2016-03-30 grant
RU2570378C9 (en) 2016-03-10 grant
US20130030038A1 (en) 2013-01-31 application
WO2011098285A3 (en) 2012-04-19 application
JP2013527134A (en) 2013-06-27 application
RU2570378C2 (en) 2015-12-10 grant
EP2533763A2 (en) 2012-12-19 application
RU2012137904A (en) 2014-03-20 application
WO2011098285A8 (en) 2011-12-29 application
WO2011098285A2 (en) 2011-08-18 application
CA2827049A1 (en) 2011-08-18 application
US9249413B2 (en) 2016-02-02 grant
DE102010007562A1 (en) 2011-08-11 application
US20160115478A1 (en) 2016-04-28 application
Dombrowski et al. 2011 Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions
US20070020213A1 (en) 2007-01-25 Foamable composition combining a polar solvent and a hydrophobic carrier
Fang et al. 2006 Effect of liposome encapsulation of tea catechins on their accumulation in basal cell carcinomas
WO2014028763A1 (en) 2014-02-20 Exosome-based therapeutics against neurodegenerative disorders
Elias et al. 2005 Skin barrier
Liu et al. 2011 Terpene microemulsions for transdermal curcumin delivery: effects of terpenes and cosurfactants
Lee et al. 2014 Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage
Giannantoni et al. 2006 New frontiers in intravesical therapies and drug delivery
Som et al. 2012 Status of surfactants as penetration enhancers in transdermal drug delivery
Hon et al. 2013 Barrier repair therapy in atopic dermatitis: an overview
Schumacher 2010 Transient receptor potential channels in pain and inflammation: therapeutic opportunities
Bhardwaj et al. 2010 Physicochemical properties of extruded and non-extruded liposomes containing the hydrophobic drug dexamethasone
Puglia et al. 2001 Evaluation of in vitro percutaneous absorption of lorazepam and clonazepam from hydro-alcoholic gel formulations
US20100184670A1 (en) 2010-07-22 Antisense compositions and methods for modulating contact hypersensitivity or contact dermatitis
WO2009123764A2 (en) 2009-10-08 Compositions and use of epas1 inhibitors
Salerno et al. 2010 Study of in vitro drug release and percutaneous absorption of fluconazole from topical dosage forms
US20090022779A1 (en) 2009-01-22 Phenytoin Formulations, and Uses Thereof in Wound Healing
Chen et al. 2014 Topical delivery of siRNA into skin using SPACE-peptide carriers
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