Source: http://www.google.com/patents/US8138144?dq=inventor:%22Arthur+R.+Hair%22&ei=VAy0Tsa4NYTl0QGQiqWiBA
Timestamp: 2016-09-28 00:43:15
Document Index: 776191405

Matched Legal Cases: ['Application No. 60', 'Application No. 2', 'Application No. 02759416', 'Application No. 02759416', 'Application No. 2003', 'Application No. 2003', 'Application No. 02759416', 'art 1']

Patent US8138144 - Antimicrobial cationic peptides and formulations thereof - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsCompositions and methods for making and using therapeutic formulations of antimicrobial cationic peptides are provided. The antimicrobial cationic peptide formulations may be used, for example, in the treatment of microorganism-caused infections, which infections may be systemic, such as a septicemia,...http://www.google.com/patents/US8138144?utm_source=gb-gplus-sharePatent US8138144 - Antimicrobial cationic peptides and formulations thereofAdvanced Patent SearchTry the new Google Patents, with machine-classified Google Scholar results, and Japanese and South Korean patents.Publication numberUS8138144 B2Publication typeGrantApplication numberUS 10/865,687Publication dateMar 20, 2012Filing dateJun 10, 2004Priority dateAug 21, 2001Fee statusPaidAlso published asCA2456477A1, CA2456477C, DE60239707D1, EP1469876A2, EP1469876B1, US6835536, US8466102, US8927487, US9227999, US20030171281, US20050049182, US20120202735, US20130296227, US20150087580, WO2003015809A2, WO2003015809A3, WO2003015809A9Publication number10865687, 865687, US 8138144 B2, US 8138144B2, US-B2-8138144, US8138144 B2, US8138144B2InventorsTimothy J. Krieger, Patricia J. McNicolOriginal AssigneeCarrus Capital CorporationExport CitationBiBTeX, EndNote, RefManPatent Citations (54), Non-Patent Citations (126), Referenced by (6), Classifications (19), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetAntimicrobial cationic peptides and formulations thereof
US 8138144 B2Abstract
1. A composition, comprising an antimicrobial cationic peptide at a concentration ranging from about 0.01% to about 10% (w/w), a viscosity-increasing agent at a concentration of about 0.5% to about 5% (w/w), a solvent, and a monocarboxylate buffering agent at a concentration ranging from about 1 mM to about 200 mM, wherein the cationic peptide is a peptide of up to 35 amino acids comprising 11B7CN (SEQ ID NO: 23) wherein the viscosity-increasing agent is hydroxyethyl cellulose, the solvent is water and glycerin, and the monocarboxylate buffering agent is lactate, wherein the solvent comprises glycerin at a concentration up to about 20% (w/w), and wherein the composition is a gel and has a pH ranging from about 3 to about 8.
2. The composition according to claim 1 wherein the cationic peptide consists of the amino acid sequence 11B7CN (SEQ ID NO:23).
3. The composition of claim 2, wherein the concentration of the antimicrobial cationic peptide is in a range from about 1% to about 3% (w/w), the concentration of hydroxyethyl cellulose is in a range of about 1% to about 2% (w/w), and the concentration of glycerin is in a range of about 9% to about 11% (w/w).
4. The composition according to claim 1 having a pH ranging from about 3.5 to about 7.
5. The composition according to claim 1, wherein the solvent comprising comprises glycerin at a concentration ranging from about 9% to about 11% (w/w).
6. The composition according to claim 1, further comprising a preservative, wherein the preservative comprises benzoic acid, benzyl alcohol, phenoxyethanol, methylparaben, propylparaben, or a combination thereof.
7. The composition according to claim 1 comprising hydroxyethyl cellulose at a concentration ranging from about 1% to about 2% (w/w).
8. The composition according to claim 1 comprising hydroxyethyl cellulose at a concentration ranging from about 1% to about 3% (w/w).
9. The composition according to claim 1, further comprising a second viscosity-increasing agent selected from dextran, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.
10. The composition according to claim 9 comprising, as the second viscosity-increasing agent, dextran at a concentration ranging from about 0.1% to about 5% (w/w).
11. The composition according to claim 9 comprising, as the second viscosity-increasing agent, dextran at a concentration ranging from about 0.5% to about 1% (w/w).
12. The composition according to claim 9 comprising, as the second viscosity-increasing agent, polyvinylpyrrolidone at a concentration ranging from about 0.1% to about 5% (w/w).
13. The composition according to claim 9 comprising, as the second viscosity-increasing agent, polyvinylpyrrolidone at a concentration ranging from about 0.5% to about 1% (w/w).
14. The composition of claim 1, wherein the concentration of the antimicrobial cationic peptide is in a range from about 0.5% to about 5% (w/w).
15. The composition of claim 1, wherein the concentration of the antimicrobial cationic peptide is in a range from about 1% to about 3% (w/w).
16. A method for treating or preventing inflammation at a target site, comprising applying to the target site a composition of claim 1.
17. The method of claim 16, further comprising inserting a medical device at the target site after applying the composition.
18. The method of claim 16, further comprising inserting a medical device at the target site before applying the composition.
19. The method of claim 16, wherein applying the composition to the target site comprises applying the composition to a medical device prior to inserting the device at the target site.
20. The method according to any one of claim 17, 18 or 19 wherein the device comprises a catheter.
21. The method according to claim 20, wherein the catheter comprises a central venous catheter.
22. The method according to claim 20, wherein the catheter is selected from the group consisting of a vascular dialysis catheter, a pulmonary artery catheter, a periotoneal dialysis catheter, and an umbilical catheter.
23. The method of claim 16, wherein the target site is a skin.
24. The method of claim 23, wherein the skin further comprises acne.
25. The method of claim 16, wherein the target site is a mucosa.
26. A method for treating an infection caused by a prokaryotic or cukaryotic organism at a target site, comprising applying to the target site a composition of claim 1.
27. The method of claim 26, wherein the infection is caused by a prokaryotic organism, wherein the prokaryotic organism is a bacterium.
28. The method of claim 26, wherein the infection at a target site is associated with a medical device at the target site.
29. The method of claim 28, comprising applying the composition prior to or after inserting a medical device at the target.
30. The method of claim 28, wherein applying the composition to the target site comprises applying the composition to the medical device prior to inserting the device at the target site.
31. The method according to any one of claim 29 or 30, wherein the medical device comprises a catheter.
32. A method for treating acne at a target site comprising applying to the target site a composition of claim 1.
33. The method of claim 32, wherein the acne is acne vulgaris.
34. The method of claim 33, wherein the cationic peptide comprises the amino acid sequence 11B7CN (SEQ ID NO: 23).
35. The method of claim 32, wherein the acne is caused by Propionibacterium acne.
36. The method of claim 32, wherein the cationic peptide comprises the amino acid sequence 11B7CN (SEQ ID NO: 23).
37. A composition comprising an antimicrobial cationic peptide from about 0.01% to 10% w/w, a viscosity-increasing agent, and a solvent, wherein the cationic peptide is a peptide of up to 35 amino acids comprising the amino acid sequence 11B7CN (SEQ ID NO: 23), the viscosity-increasing agent is hydroxyethyl cellulose at a concentration of about 0.5% to about 5% (w/w), and the solvent is water and glycerin, wherein the solvent comprises glycerin at a concentration up to about 20% (w/w), and wherein the composition is a gel.
38. The composition of claim 37, wherein the cationic peptide 11B7CN (SEQ ID NO: 23) is at a concentration ranging from about 2.0% to about 4.0% (w/w).
39. The composition of claim 38, wherein the cationic peptide 11B7CN (SEQ ID NO: 23) is at a concentration of about 2.5% (w/w).
40. The composition of claim 38, wherein the cationic peptide 11B7CN (SEQ ID NO: 23) is at a concentration of about 3.0% (w/w).
41. The composition of claim 37, wherein the glycerin in the solvent is at a concentration ranging from 9% to 11% (w/w).
42. The composition of claim 37, wherein the glycerin in the solvent is at a concentration of 10% (w/w).
43. The composition of claim 37, wherein the hydroxyethyl cellulose is at a concentration ranging from 1.0% to 3.0% (w/w).
44. The composition of claim 37, wherein the hydroxyethyl cellulose is at a concentration of 2.0% (w/w).
45. The composition of claim 37, further comprising benzoic acid.
46. The composition of claim 37, further comprising a monocarboxylate or dicarboxylate buffering agent in a concentration in a range of about 1 mM to about 200 mM and wherein the composition has a pH ranging from about 3.5 to about 7.
47. The composition of claim 46, wherein the buffering agent comprises a monocarboxylate.
48. The composition according to claim 46 wherein the buffering agent is selected from the group consisting of acetate, fumarate, lactate, malonate, succinate, and tartrate.
49. A pharmaceutical composition comprising an antimicrobial cationic peptide at a concentration ranging from about 0.01% to about 10% (w/w); a viscosity-increasing agent at a concentration of about 0.5% to about 5% (w/w); a solvent selected from water, glycerin, propylene glycol, isopropanol, ethanol, and methanol; and a monocarboxylate buffering agent at a concentration ranging from about 1 mM to about 200 mM; wherein the cationic peptide is a peptide of up to 35 amino acids comprising 11B7CN (SEQ ID NO: 23), the viscosity-increasing agent is hydroxyethyl cellulose, and the composition is a gel and has a pH ranging from about 3 to about 8.
50. The pharmaceutical composition of claim 49, wherein the concentration of the antimicrobial cationic peptide is in a range from about 1% to about 3% (w/w).
51. The pharmaceutical composition of claim 49, wherein the concentration of the antimicrobial cationic peptide is in a range from about 2.5% to about 3.5% (w/w).
52. The pharmaceutical composition of claim 50, wherein the solvent is water and glycerin, wherein the solvent comprises glycerin at a concentration up to about 20% (w/w).
53. The pharmaceutical composition of claim 52, wherein the concentration of glycerin in the solvent is in a range of about 9% to about 11% (w/w).
54. The pharmaceutical composition of claim 53, wherein the concentration of hydroxyethyl cellulose is in a range of about 1% to about 3% (w/w).
55. The pharmaceutical composition of claim 52, wherein the concentration of hydroxyethyl cellulose is in a range of about 1% to about 3% (w/w).
56. The pharmaceutical composition of claim 49, wherein the composition has a pH ranging from about 3.5 to about 7.
This application is a Continuation of allowed U.S. patent application Ser. No. 10/225,087, filed Aug. 20, 2002, which claims the benefit of U.S. Provisional Patent Application No. 60/314,232 filed Aug. 21, 2001, where these applications are incorporated herein by reference in their entireties.
Yet another clinical indication, although not life-threatening, is the most common skin disease of adolescence and early adulthood, acne vulgaris, or acne as it is generally called. In addition to psychological effects, such as anxiety, depression and withdrawl from society, studies have also shown that acne vulgaris can directly and significantly affects a patient's quality of life. Antibiotic agents have been extensively used for the treatment of acne for several decades; however, there is a growing concern that with the use of antibiotics to treat acne, drug-resistant microorganisms will inevitably emerge.
To address the issue of ever increasing drug-resistant microorganisms, investigations have turned to new classes of antibiotics, such as antimicrobial peptides. Antimicrobial peptides are found in evolutionarily diverse species including, for example, prokaryotes, plants, insects, and mammals. Antimicrobial peptides may be anionic, but most known antimicrobial peptides are cationic. Multiple families of antimicrobial cationic peptides are known and these peptides encompass a wide variety of structural motifs, yet all of these cationic peptides have similar physicochemical properties. For example, most known antimicrobial cationic peptides are cationic at neutral pH, are generally less than 10 kDa, and are amphipathically “sided” in solution such that hydrophobic side chains are regionalized. Many antimicrobial cationic peptides are known, including defensins, cecropins, melittins, magainins, indolicidins, and protegrins. The advantages of cationic peptides are their ability to kill target cells rapidly, their broad spectrum of activity, and their activity against some of the more serious antibiotic-resistant and clinically relevant pathogens. Most importantly, antimicrobial peptide-resistant microorganisms are relatively difficulty to select in vitro. However, some antimicrobial peptides have been found to be toxic (e.g., bee venom, wasp venom, and scorpion toxin), some have been found to have reduced activity in vivo (due to factors such as high mono- and divalent cation concentrations, polyanions, serum, apolipoprotein A-1, serpins, and proteases, although many peptides are not affected by these factors), and some have been found to be less potent than conventional antibiotics.
In one aspect, the present invention provides a composition that comprises an antimicrobial cationic peptide, a viscosity-increasing agent, and a solvent. In certain embodiments the solvent is water, glycerin, propylene glycol, isopropanol, ethanol, or methanol. In certain other embodiments, the solvent is glycerin at a concentration ranging from about 0.1% to about 20% or from about 9% to about 11%. In still other embodiments, the solvent is propylene glycol at a concentration ranging from about 0.1% to about 20% or from about 9% to about 11%. In yet other embodiments, the solvent comprises at least one of water, glycerin, propylene glycol, isopropanol, ethanol, and methanol. In further embodiments, the solvent comprises at least one of water at a concentration up to 99%, glycerin at a concentration up to 20%, propylene glycol at a concentration up to 20%, ethanol at a concentration up to 99%, and methanol at a concentration up to 99%.
In certain embodiments, the viscosity-increasing agent is dextran, polyvinylpyrrolidone, hydroxyethyl cellulose, or hydroxypropyl methylcellulose. In another embodiment, the viscosity-increasing agent is hydroxyethyl cellulose at a concentration ranging from about 0.5% to about 5% or from about 1% to about 3%. In another embodiment, the viscosity-increasing agent is hydroxypropyl methylcellulose at a concentration ranging from about 1% to about 3%. In other embodiments, the viscosity-increasing agent is dextran at a concentration ranging from about 0.1% to about 5% or from about 0.5% to about 1%. In certain other embodiments, where the viscosity-increasing agent is hydroxyethyl cellulose, the composition further comprises a second viscosity-increasing agent of dextran, polyvinylpyrrolidone, or hydroxypropyl methylcellulose. In one embodiment, the second viscosity-increasing agent is polyvinylpyrrolidone. In related embodiments, the polyvinylpyrrolidone is at a concentration ranging from about 0.1% to about 5% or from about 0.5% to about 1%. In another embodiment, the second viscosity-increasing agent is hydroxypropyl methylcellulose. In related embodiments, the hydroxypropyl methylcellulose is at a concentration ranging from about 1% to about 3%. In certain other embodiments, where the viscosity-increasing agent is hydroxypropyl methylcellulose, the composition further comprises a second viscosity-increasing agent of dextran, or dextran at concentration ranging from about 0.1% to about 5% or from about 0.5% to about 1%. In still other embodiments, where the viscosity-increasing agent is hydroxypropyl methylcellulose, the composition further comprises a second viscosity-increasing agent of polyvinylpyrrolidone, or polyvinylpyrrolidone at a concentration ranging from about 0.1% to about 5% or from about 0.5% to about 1%. In certain embodiments, the first viscosity-increasing agent comprises hydroxyethyl cellulose at a concentration up to about 3% and second viscosity-increasing agent comprises hydroxypropyl methylcellulose at a concentration up to about 3%.
In still other embodiments, any of the aforementioned compositions further comprise a humectant. In one embodiment, the humectant is sorbitol or glycerol. In further embodiments, any of the aforementioned compositions further comprise a preservative. In one embodiment, the preservative comprises benzoic acid, benzyl alcohol, phenoxyethanol, methylparaben, propylparaben, or a combination thereof. As used herein, any reference to an acid may include a free acid, a salt, and any ester thereof. In other embodiments, any of the aforementioned compositions further comprise a humectant and a preservative.
In another aspect there is provided a composition comprising an antimicrobial cationic peptide, a viscosity-increasing agent, a solvent, a humectant, and a buffering agent. In one embodiment, the humectant is sorbitol or glycerol. In certain embodiments, the buffering agent is at a concentration ranging from about 1 mM to about 200 mM. In other embodiments, the buffering agent comprises a monocarboxylate or a dicarboxylate. In further embodiments, the buffering agent is acetate, fumarate, lactate, malonate, succinate, or tartrate. In yet another embodiment, the composition has a pH ranging from about 3 to about 8. In another embodiment, the composition further comprises a preservative. In one embodiment, the preservative comprises benzoic acid, benzyl alcohol, phenoxyethanol, methylparaben, propylparaben, or a combination thereof. In certain embodiments, the solvent is water, glycerin, propylene glycol, isopropanol, ethanol, or methanol. In certain other embodiments, the solvent is glycerin at a concentration ranging from about 0.1% to about 20% or from about 9% to about 11%. In still other embodiments, the solvent is propylene glycol at a concentration ranging from about 0.1% to about 20% or from about 9% to about 11%. In yet other embodiments, the solvent comprises at least one of water, glycerin, propylene glycol, isopropanol, ethanol, and methanol. In further embodiments, the solvent comprises at least one of water at a concentration up to 99%, glycerin at a concentration up to 20%, propylene glycol at a concentration up to 20%, ethanol at a concentration up to 99%, and methanol at a concentration up to 99%.
In certain embodiments, the antimicrobial cationic peptide is an indolicidin or an analog or derivative thereof in any one of the aforementioned compositions. In other embodiments, the cationic peptide is at a concentration ranging from about 0.01% to about 10% or from about 0.5% to about 1.5% in any one of the aforementioned compositions. In yet other embodiments, the cationic peptide is a peptide of up to 35 amino acids, comprising one of the following sequences: 11B7CN, 11B32CN, 11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN, 11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN, 11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN, 11J68CN, Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN in any one of the aforementioned compositions.
In still another aspect, the present invention provides a composition comprising an antimicrobial cationic peptide, a buffering agent, and a solvent. In other embodiments, the composition further comprises a humectant. In one embodiment, the humectant is sorbitol or glycerol. In another embodiment, the composition further comprises a preservative. In one embodiment, the preservative comprises benzoic acid, benzyl alcohol, phenoxyethanol, methylparaben, propylparaben, or a combination thereof. In yet other embodiments, the composition further comprises a viscosity-increasing agent of dextran, polyvinylpyrrolidone, hydroxyethyl cellulose, or hydroxypropyl methylcellulose. In another embodiment, the viscosity-increasing agent is hydroxyethyl cellulose at a concentration ranging from about 1% to about 3%. In another embodiment, the viscosity-increasing agent is hydroxypropyl methylcellulose at a concentration ranging from about 1% to about 3%. In certain other embodiments, wherein the viscosity-increasing agent is hydroxyethyl cellulose, the composition further comprises a second viscosity-increasing agent of hydroxypropyl methylcellulose. In another embodiment, the viscosity-increasing agent is hydroxyethyl cellulose at a concentration up to about 3% and the second viscosity-increasing agent is hydroxypropyl methylcellulose at a concentration up to about 3%. In a further embodiment, the composition further comprises an acne medicament of retinoid, vitamin D3, or corticosteroid, and analogues or derivatives thereof.
In certain embodiments the solvent is water, glycerin, propylene glycol, isopropanol, ethanol, or methanol. In certain embodiments, the solvent is glycerin at a concentration ranging from about 9% to about 11%. In another embodiment, the solvent is propylene glycol at a concentration ranging from about 9% to about 11%. In yet other embodiments, the solvent comprises at least one of water, glycerin, propylene glycol, isopropanol, ethanol, and methanol. In further embodiments, the solvent comprises at least one of water at a concentration up to 99%, glycerin at a concentration up to 20%, propylene glycol at a concentration up to 20%, ethanol at a concentration up to 99%, and methanol at a concentration up to 99%.
In a further aspect, the present invention provides a composition comprising (a) an antimicrobial cationic peptide wherein the cationic peptide is a peptide of up to 35 amino acids comprising one of the following: 11B7CN, 11B32CN, 11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN, 11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN, 11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN, 11J68CN, Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN; (b) a viscosity-increasing agent wherein the viscosity-increasing agent is hydroxyethyl cellulose at a concentration of about 1.2% to about 1.8%; (c) a buffer wherein the buffer is lactate at a concentration ranging from about 4 mM to about 6 mM; (d) a solvent wherein the solvent comprises glycerin at a concentration ranging from about 9% to about 11% and water at a concentration ranging from about 85% to about 90%; and (e) a pH ranging from about 3.5 to about 7. In certain embodiments, the cationic peptide is at a concentration ranging from about 0.8% to about 1.2%. In yet another embodiment, provided are methods to reduce microflora, or to treat or prevent an infection, at a target site, the target site may be skin, and the skin may further comprise acne. In another embodiment, the composition may be applied to a target site to treat or prevent or ameliorate inflammation, such as inflammation associated with acne (or with an implanted or indwelling medical device).
In still another aspect, the present invention provides a composition comprising (a) an antimicrobial cationic peptide wherein the cationic peptide is a peptide of up to 35 amino acids comprising one of the following: 11B7CN, 11B32CN, 11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN, 11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN, 11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN, 11J68CN, Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN; (b) a viscosity-increasing agent wherein the viscosity-increasing agent is hydroxyethyl cellulose at a concentration of about 1.2% to about 1.8%; (c) a solvent wherein the solvent comprises glycerin at a concentration ranging from about 9% to about 11% and water at a concentration ranging from about 85% to about 90%; (d) a preservative wherein the preservative is benzoic acid at a concentration ranging from about 20 mM to about 30 mM; and (e) a pH ranging from about 3.5 to about 4.7. In certain embodiments, the cationic peptide is at a concentration ranging from about 0.8% to about 1.2%, or ranging from about 2.5% to about 3.5%. In yet another embodiment, provided are methods to reduce microflora, or to treat or prevent an infection, at a target site, the target site may be skin, and the skin may further comprise acne. In another embodiment, the composition may be applied to a target site to treat or prevent or ameliorate inflammation, such as inflammation associated with acne (or with an implanted or indwelling medical device).
An antimicrobial cationic peptide of the present invention may be a recombinant peptide or a synthetic peptide, and is preferably a recombinant peptide. Peptides may be synthesized by standard chemical methods, including synthesis by automated procedure. In general, peptide analogues are synthesized based on the standard solid-phase Fmoc protection strategy with HATU as the coupling agent. The peptide is cleaved from the solid-phase resin with trifluoroacetic acid containing appropriate scavengers, which also deprotects side chain functional groups. Crude peptide is further purified using preparative reversed-phase chromatography. Other purification methods, such as partition chromatography, gel filtration, gel electrophoresis, or ion-exchange chromatography may be used. Other synthesis techniques, known in the art, such as the tBoc protection strategy, or use of different coupling reagents or the like can be employed to produce equivalent peptides. Peptides may be synthesized as a linear molecule or as branched molecules. Branched peptides typically contain a core peptide that provides a number of attachment points for additional peptides. Lysine is most commonly used for the core peptide because it has one carboxyl functional group and two (alpha and epsilon) amine functional groups. Other diamino acids can also be used. Preferably, either two or three levels of geometrically branched lysines are used; these cores form a tetrameric and octameric core structure, respectively (Tam, Proc. Natl. Acad. Sci. USA 85:5409, 1988).
An antimicrobial cationic peptide is a peptide that typically exhibits a positive charge at a pH ranging from about 3 to about 10 (i.e., has an isoelectric point of at least about 9), and contains at least one basic amino acid (e.g., arginine, lysine, histidine). In addition, an antimicrobial cationic peptide generally comprises an amino acid sequence having a molecular mass of about 0.5 kDa (i.e., approximately five amino acids in length) to about 10 kDa (i.e., approximately 100 amino acids in length), or a molecular mass of any integer, or fraction thereof (including a tenth and one hundredth of an integer), ranging from about 0.5 kDa to about 10 kDa. Preferably, an antimicrobial cationic peptide has a molecular mass ranging from about 0.5 kDa to about 5 kDa (i.e., approximately from about 5 amino acids to about 45 amino acids in length), more preferably from about 1 kDa to about 4 kDa (i.e., approximately from about 10 amino acids to about 35 amino acids in length), and most preferably from about 1 kDa to about 2 kDa (i.e., approximately from about 10 amino acids to about 18 amino acids in length). In another preferred embodiment, the antimicrobial cationic peptide is part of a larger peptide or polypeptide sequence having, for example, a total of up to 100 amino acids, more preferably up to 50 amino acids, even more preferably up to 35 amino acids, and most preferably up to 15 amino acids. The present invention contemplates an antimicrobial cationic peptide having an amino acid sequence of 5 to 100 amino acids, with the number of amino acids making up the peptide sequence comprising any integer in that range. An antimicrobial cationic peptide may exhibit antibacterial activity, anti-endotoxin activity, antifungal activity, antiparasite activity, antiviral activity, anticancer activity, anti-inflammatory activity, wound healing activity, and synergistic activity with other peptides or antimicrobial compounds, or a combination thereof.
Exemplary antimicrobial peptides include, but are not limited to, cecropins, normally made by lepidoptera (Steiner et al., Nature 292:246, 1981) and diptera (Merrifield et al., Ciba Found. Symp. 186:5, 1994), by porcine intestine (Lee et al., Proc. Nat'l Acad. Sci. USA 86:9159, 1989), by blood cells of a marine protochordate (Zhao et al., FEBS Lett. 412:144, 1997); synthetic analogs of cecropin A, melittin, and cecropin-melittin chimeric peptides (Wade et al., Int. J. Pept. Protein Res. 40:429, 1992); cecropin B analogs (Jaynes et al., Plant Sci. 89:43, 1993); chimeric cecropin A/B hybrids (D�ring, Mol. Breed. 2:297, 1996); magainins (Zasloff, Proc. Nat'l Acad. Sci USA 84:5449, 1987); cathelin-associated antimicrobial peptides from leukocytes of humans, cattle, pigs, mice, rabbits, and sheep (Zanetti et al., FEBS Lett. 374:1, 1995); vertebrate defensins, such as human neutrophil defensins [HNP 1-4]; paneth cell defensins of mouse and human small intestine (Oulette and Selsted, FASEB J. 10:1280, 1996; Porter et al., Infect. Immun. 65:2396, 1997); vertebrate β-defensins, such as HBD-1 of human epithelial cells (Zhao et al., FEBS Lett. 368:331, 1995); HBD-2 of inflamed human skin (Harder et al., Nature 387:861, 1997); bovine β-defensins (Russell et al., Infect. Immun. 64:1565, 1996); plant defensins, such as Rs-AFP1 of radish seeds (Fehlbaum et al., J. Biol. Chem. 269:33159, 1994); α- and β-thionins (Stuart et al., Cereal Chem. 19:288, 1942; Bohlmann and Apel, Annu. Rev. Physiol. Plant Mol. Biol. 42:227, 1991); γ-thionins (Broekaert et al., Plant Physiol. 108:1353, 1995); the anti-fungal drosomycin (Fehlbaum et al., J. Biol. Chem. 269:33159, 1994); apidaecins, produced by honey bee, bumble bee, cicada killer, hornet, yellow jacket, and wasp (Casteels et al., J. Biol. Chem. 269:26107, 1994; Levashina et al., Eur. J. Biochem. 233:694, 1995); cathelicidins, such as indolicidin and derivatives or analogues thereof from bovine neutrophils (Falla et al., J. Biol. Chem. 277:19298, 1996); bacteriocins, such as nisin (Delves-Broughton et al., Antonie van Leeuwenhoek J. Microbiol. 69:193, 1996); and the protegrins and tachyplesins, which have antifungal, antibacterial, and antiviral activities (Tamamura et al., Biochim. Biophys. Acta 1163:209, 1993; Aumelas et al., Eur. J. Biochem. 237:575, 1996; Iwanga et al., Ciba Found. Symp. 186:160, 1994).
In certain embodiments, preferred antimicrobial cationic peptides of the present invention are indolicidins or analogs or derivatives thereof (see Table 2 of Example 1). Natural indolicidins may be isolated from a variety of organisms, and, for example, the indolicidin isolated from bovine neutrophils is a 13 amino acid peptide, which is tryptophan-rich and amidated at the C-terminus (see Selsted et al., J. Biol. Chem. 267:4292, 1992). As noted above, a preferred indolicidin or analog or derivative thereof comprises 5 to 45 amino acids, more preferably 7 to 35 amino acids, even more preferably 8 to 25 amino acids, and most preferably 10 to 14 amino acids (see, e.g., Table 2). The indolicidins or analogs or derivatives thereof of the present invention may be used at a concentration ranging from about 0.01% to about 10%, preferably from about 0.5% to about 5%, and more preferably from either about 1% to about 3% or about 4% to about 6%, depending on the intended use and formulation ingredients (where “about” is �10% of the indicated value). In certain embodiments, the antimicrobial cationic peptide is an indolicidin or an analog or derivative thereof in any one of the aforementioned compositions. In preferred embodiments, the antimicrobial cationic peptide is a peptide of up to 35 amino acids, comprising one of the following sequences: 11B7CN, 11B32CN, 11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN, 11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN, 11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN, 11J68CN, Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN, which may be used in any one of the compositions described herein.
The amino acid designations are herein set forth as either the standard one- or three-letter code. Unless otherwise indicated, a named amino acid refers to the L-enantiomer. Polar amino acids include asparagine (Asp or N) and glutamine (Gln or Q); as well as basic amino acids such as arginine (Arg or R), lysine (Lys or K), histidine (His or H), and derivatives thereof; and acidic amino acids such as aspartic acid (Asp or D) and glutamic acid (Glu or E), and derivatives thereof. Hydrophobic amino acids include tryptophan (Trp or W), phenylalanine (Phe or F), isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V), and derivatives thereof; as well as other non-polar amino acids such as glycine (Gly or G), alanine (Ala or A), proline (Pro or P), and derivatives thereof. Amino acids of intermediate polarity include serine (Ser or S), threonine (Thr or T), tyrosine (Tyr or Y), cysteine (Cys or C), and derivatives thereof. A capital letter indicates an L-enantiomer amino acid; a small letter indicates a D-enantiomer amino acid. For example, some modified amino acids may include 2,3-diamino butyric acid, 3- or 4-mercaptoproline derivatives, N5-acetyl-N5-hydroxy-L-ornitine, and α-N-hydroxyamino acids. An antimicrobial cationic peptide analog or derivative thereof may include any one or combination of the above-noted alterations to the natural peptide, or any other modification known in the art.
The indolicidins or analogs or derivatives thereof of the present invention may be used individually, or may be used in combination with one or more different indolicidins or analogs or derivatives thereof, with one or more antimicrobial cationic peptides, and one or more conventional antimicrobial agents, as described herein. Thus, synergistic combinations of an antimicrobial cationic peptide and an antimicrobial agent may permit a reduction in the dosage of one or both agents in order to achieve a similar or improved therapeutic effect. This would allow the use of smaller doses and, therefore, would decrease the potential incidence of toxicity (e.g., from aminoglycosides) and lowering costs of expensive antimicrobials (e.g., vancomycin). Concurrent or sequential administration of an antimicrobial cationic peptide formulation and an antimicrobial agent composition is expected to provide more effective treatment of infections caused by a variety of microorganisms (e.g., bacteria, viruses, fungi, and parasites). In particular, successful treatment or prevention of infectious disease can be achieved by using the antimicrobial cationic peptides and antmicrobial agents at doses below what is normally a therapeutically effective dose when these antimicrobials are used individually. Alternatively, the antibiotic agent and antimicrobial cationic peptide formulation can be administered using a normally effective therapeutic dose for each antimicrobial, but wherein the combination of the two agents provides even more potent effects.
As noted above, the preferred antimicrobial cationic peptides may be used in a synergistic combination with other known antimicrobial agents. Antibacterial agents include, but are not limited to, penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones. Examples of antibiotic agents include, but are not limited to, Penicillin G (CAS Registry No.: 61-33-6); Methicillin (CAS Registry No.: 61-32-5); Nafcillin (CAS Registry No.: 147-52-4); Oxacillin (CAS Registry No.: 66-79-5); Cloxacillin (CAS Registry No.: 61-72-3); Dicloxacillin (CAS Registry No.: 3116-76-5); Ampicillin (CAS Registry No.: 69-53-4); Amoxicillin (CAS Registry No.: 26787-78-0); Ticarcillin (CAS Registry No.: 34787-01-4); Carbenicillin (CAS Registry No.: 4697-36-3); Mezlocillin (CAS Registry No.: 51481-65-3); Azlocillin (CAS Registry No.: 37091-66-0); Piperacillin (CAS Registry No.: 61477-96-1); Imipenem (CAS Registry No.: 74431-23-5); Aztreonam (CAS Registry No.: 78110-38-0); Cephalothin (CAS Registry No.: 153-61-7); Cefazolin (CAS Registry No.: 25953-19-9); Cefaclor (CAS Registry No.: 70356-03-5); Cefamandole formate sodium (CAS Registry No.: 42540-40-9); Cefoxitin (CAS Registry No.: 35607-66-0); Cefuroxime (CAS Registry No.: 55268-75-2); Cefonicid (CAS Registry No.: 61270-58-4); Cefinetazole (CAS Registry No.: 56796-20-4); Cefotetan (CAS Registry No.: 69712-56-7); Cefprozil (CAS Registry No.: 92665-29-7); Lincomycin (CAS Registry No.: 154-21-2); Linezolid (CAS Registry No.: 165800-03-3); Loracarbef (CAS Registry No.: 121961-22-6); Cefetamet (CAS Registry No.: 65052-63-3); Cefoperazone (CAS Registry No.: 62893-19-0); Cefotaxime (CAS Registry No.: 63527-52-6); Ceftizoxime (CAS Registry No.: 68401-81-0); Ceftriaxone (CAS Registry No.: 73384-59-5); Ceftazidime (CAS Registry No.: 72558-82-8); Cefepime (CAS Registry No.: 88040-23-7); Cefixime (CAS Registry No.: 79350-37-1); Cefpodoxime (CAS Registry No.: 80210-62-4); Cefsulodin (CAS Registry No.: 62587-73-9); Fleroxacin (CAS Registry No.: 79660-72-3); Nalidixic acid (CAS Registry No.: 389-08-2); Norfloxacin (CAS Registry No.: 70458-96-7); Ciprofloxacin (CAS Registry No.: 85721-33-1); Ofloxacin (CAS Registry No.: 82419-36-1); Enoxacin (CAS Registry No.: 74011-58-8); Lomefloxacin (CAS Registry No.: 98079-51-7); Cinoxacin (CAS Registry No.: 28657-80-9); Doxycycline (CAS Registry No.: 564-25-0); Minocycline (CAS Registry No.: 10118-90-8); Tetracycline (CAS Registry No.: 60-54-8); Amikacin (CAS Registry No.: 37517-28-5); Gentamicin (CAS Registry No.: 1403-66-3); Kanamycin (CAS Registry No.: 8063-07-8); Netilmicin (CAS Registry No.: 56391-56-1); Tobramycin (CAS Registry No.: 32986-56-4); Streptomycin (CAS Registry No.: 57-92-1); Azithromycin (CAS Registry No.: 83905-01-5); Clarithromycin (CAS Registry No.: 81103-11-9); Erythromycin (CAS Registry No.: 114-07-8); Erythromycin estolate (CAS Registry No.: 3521-62-8); Erythromycin ethyl succinate (CAS Registry No.: 41342-53-4); Erythromycin glucoheptonate (CAS Registry No.: 23067-13-2); Erythromycin lactobionate (CAS Registry No.: 3847-29-8); Erythromycin stearate (CAS Registry No.: 643-22-1); Vancomycin (CAS Registry No.: 1404-90-6); Teicoplanin (CAS Registry No.: 61036-64-4); Chloramphenicol (CAS Registry No.: 56-75-7); Clindamycin (CAS Registry No.: 18323-44-9); Trimethoprim (CAS Registry No.: 738-70-5); Sulfamethoxazole (CAS Registry No.: 723-46-6); Nitrofurantoin (CAS Registry No.: 67-20-9); Rifampin (CAS Registry No.: 13292-46-1); Mupirocin (CAS Registry No.: 12650-69-0); Metronidazole (CAS Registry No.: 443-48-1); Cephalexin (CAS Registry No.: 15686-71-2); Roxithromycin (CAS Registry No.: 80214-83-1); Co-amoxiclavuanate; combinations of Piperacillin and Tazobactam; and their various salts, acids, bases, and other derivatives.
Other techniques known in the art may be utilized to construct monoclonal antibodies (see Huse et al., Science 246:1275-1281, 1989; Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-5732, 1989; Alting-Mees et al., Strategies in Molecular Biology 3:1-9, 1990; describing recombinant techniques). These techniques include cloning heavy and light chain immunoglobulin cDNA in suitable vectors, such as λImmunoZap(H) and λ ImmunoZap(L). These recombinants may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; Sastry et al., supra). Positive plaques may subsequently be converted into non-lytic plasmids to allow high-level expression of monoclonal antibody fragments in a host, such as E. coli. Similarly, portions or fragments of antibodies, such as Fab and Fv fragments, may also be constructed utilizing conventional enzymatic digestion or recombinant DNA techniques to yield isolated variable regions of an antibody. Within one embodiment, the genes that encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the variable region. In addition, techniques may be utilized to change a “murine” antibody to a “human” antibody, without altering the binding specificity of the antibody to the antimicrobial cationic peptide and analog or derivative thereof.
Although one objective in constructing a cationic peptide variant may be to improve its activity, it may also be desirable to alter the amino acid sequence of a naturally occurring cationic peptide to enhance its production in a recombinant host cell. The presence of a particular codon may have an adverse effect on expression in a particular host; therefore, a DNA sequence encoding the desired cationic peptide is optimized for a particular host system, such as prokaryotic or eukaryotic cells. For example, a nucleotide sequence encoding a radish cationic peptide may include a codon that is commonly found in radish, but is rare for E. coli. The presence of a rare codon may have an adverse effect on protein levels when the radish cationic peptide is expressed in recombinant E. coli. Methods for altering nucleotide sequences to alleviate the codon usage problem are well known to those of skill in the art (see, e.g., Kane, Curr. Opin. Biotechnol. 6:494, 1995; Makrides, Microbiol. Rev. 60:512, 1996; and Brown (Ed.), Molecular Biology LabFax, BIOS Scientific Publishers, Ltd., 1991, which provides a Codon Usage Table at page 245 through page 253).
A DNA sequence encoding a cationic peptide is introduced into an expression vector appropriate for the host. In preferred embodiments, the gene is cloned into a vector to create a fusion protein. The fusion partner is chosen to contain an anionic region, such that a bacterial host is protected from the toxic effect of the peptide. This protective region effectively neutralizes the antimicrobial effects of the peptide and also may prevent peptide degradation by host proteases. The fusion partner (carrier protein) of the invention may further function to transport the fusion peptide to inclusion bodies, the periplasm, the outer membrane, or the extracellular environment. Carrier proteins suitable in the context of this invention specifically include, but are not limited to, glutathione-S-transferase (GST), protein A from Staphylococcus aureus, two synthetic IgG-binding domains (ZZ) of protein A, outer membrane protein F, β-galactosidase (lacZ), and various products of bacteriophage λ and bacteriophage T7. From the teachings provided herein, it is apparent that other proteins may be used as carriers. Furthermore, the entire carrier protein need not be used, as long as the protective anionic region is present. To facilitate isolation of the peptide sequence, amino acids susceptible to chemical cleavage (e.g., CNBr) or enzymatic cleavage (e.g., V8 protease, trypsin) are used to bridge the peptide and fusion partner. For expression in E. coli, the fusion partner is preferably a normal intracellular protein that directs expression toward inclusion body formation. In such a case, following cleavage to release the final product, there is no requirement for renaturation of the peptide. In the present invention, the DNA cassette, comprising fusion partner and peptide gene, may be inserted into an expression vector, which can be a plasmid, virus or other vehicle known in the art. Preferably, the expression vector is a plasmid that contains an inducible or constitutive promoter to facilitate the efficient transcription of the inserted DNA sequence in the host. Transformation of the host cell with the recombinant DNA may be carried out by Ca++-mediated techniques, by electroporation, or other methods well known to those skilled in the art.
At minimum, the expression vector should contain a promoter sequence. However, other regulatory sequences may also be included. Such sequences include an enhancer, ribosome binding site, transcription termination signal sequence, secretion signal sequence, origin of replication, selectable marker, and the like. The regulatory sequences are operably linked with one another to allow transcription and subsequent translation. In preferred aspects, the plasmids used herein for expression include a promoter designed for expression of the proteins in bacteria. Suitable promoters, including both constitutive and inducible promoters, are widely available and are well known in the art. Commonly used promoters for expression in bacteria include promoters from T7, T3, T5, and SP6 phages, and the trp, lpp, and lac operons. Hybrid promoters (see, U.S. Pat. No. 4,551,433), such as tac and trc, may also be used. Examples of plasmids for expression in bacteria include the pET expression vectors pET3a, pET 11a, pET 12a-c, and pET 15b (see U.S. Pat. No. 4,952,496; available from Novagen, Madison, Wis.). Low copy number vectors (e.g., pPD100) can be used for efficient overproduction of peptides deleterious to the E. coli host (Dersch et al., FEMS Microbiol. Lett. 123: 19, 1994). Bacterial hosts for the T7 expression vectors may contain chromosomal copies of DNA encoding T7 RNA polymerase operably linked to an inducible promoter (e.g., lacUV promoter; see, U.S. Pat. No. 4,952,496), such as found in the E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS, HMS174(DE3) and BL21(DE3). T7 RNA polymerase can also be present on plasmids compatible with the T7 expression vector. The polymerase may be under control of a lambda promoter and repressor (e.g., pGP1-2; Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82:1074, 1985).
Antimicrobial cationic peptides, and analogs or derivatives thereof, of the present invention are assessed, either alone or in combination with an antimicrobial agent or another analog, for their potential as antibiotic therapeutic agents using a series of assays. Preferably, all peptides are initially assessed in vitro, the most promising candidates are selected for further assessment in vivo, and then candidates are selected for pre-clinical studies. The in vitro assays include measurement of antibiotic activity, toxicity, solubility, pharmacology, secondary structure, liposome permeabilization and the like. In vivo assays include assessment of efficacy in animal models, antigenicity, toxicity, and the like. In general, in vitro assays are initially performed, followed by in vivo assays.
Generally, cationic peptides are initially tested for (1) antimicrobial activity in vitro; (2) in vitro toxicity to normal mammalian cells; and (3) in vivo toxicity in an animal model. Cationic peptides that have some antimicrobial activity are preferred, although such activity may not be necessary for enhancing the activity of an antibiotic agent. Also, for in vivo use, peptides should preferably demonstrate acceptable toxicity profiles, as measured by standard procedures, where lower toxicity is preferred. Additional assays may be performed to demonstrate that the peptide is not immunogenic and to examine antimicrobial activity in vivo.
Briefly, a candidate antimicrobial cationic peptide in Mueller Hinton broth supplemented with calcium and magnesium is mixed with molten agarose. Other broths and agars may be used as long as the peptide can freely diffuse through the medium. The agarose is poured into petri dishes or wells, allowed to solidify, and a test strain is applied to the agarose plate. The test strain is chosen, in part, on the intended application of the peptide. Thus, by way of example, if an indolicidin or analog or derivative thereof with activity against S. aureus is desired, a S. aureus strain is used. It may be desirable to assay the candidate antimicrobial cationic peptide on several strains and/or on clinical isolates of the test species. Plates are incubated overnight and inspected visually for bacterial growth. A minimum inhibitory concentration (MIC) of a cationic peptide is the lowest concentration of peptide that completely inhibits growth of the organism. Peptides that exhibit good activity against the test strain, or group of strains, typically having an MIC of less than or equal to 16 μg/ml are selected for further testing. Preferred antimicrobial cationic peptides or analogs or derivatives thereof may be microbicidal or microbistatic.
Selection of antimicrobial cationic peptides and analogs or derivatives thereof as potential therapeutics is typically based on in vitro and in vivo assay results. In general, peptides that exhibit low immunogenicity, good in vivo stability, and high efficacy at low dose levels are preferred candidate antimicrobial cationic peptides and analogs or derivatives thereof.
The pharmaceutically acceptable excipients noted above are known in the art, yet the unexpected result of the present invention is that particular combinations of excipients afford the antimicrobial cationic peptides and analogs and derivatives thereof stability and prolonged activity when stored at ambient temperature, even in the presence of an alcoholic solvent. Solvents useful in the present compositions are well known in the art and include without limitation water, glycerin, propylene glycol, isopropanol, ethanol, and methanol. In some embodiments, the solvent is glycerin or propylene glycol, preferably at a concentration ranging from about 0.1% to about 20%, more preferably about 5% to about 15%, and most preferably about 9% to 11%. In other embodiments, the solvent is water or ethanol, preferably at a concentration up to about 99%, more preferably up to about 90%, and most preferably up to about 85%. (Unless otherwise indicated, all percentages are on a w/w basis.) In yet other embodiments, the solvent is at least one of water, glycerin, propylene glycol, isopropanol, ethanol, and methanol, preferably is glycerin or propylene glycol and ethanol, more preferably is glycerin and ethanol, and most preferably is glycerin and water. One embodiment is a composition comprising an antimicrobial cationic peptide, a viscosity-increasing agent, a solvent, wherein the solvent comprises at least one of water at a concentration up to 99%, glycerin at a concentration up to 20%, propylene glycol at a concentration up to 20%, ethanol at a concentration up to 99%, and methanol at a concentration up to 99%.
In certain applications, it may be desirable to maintain the pH of the antimicrobial cationic peptide composition contemplated by the present invention within a physiologically acceptable range and within a range that optimizes the activity of the peptide or analog or derivative thereof. The antimicrobial cationic peptides of the present invention function best in a composition that is neutral or somewhat acidic, although the peptides will still have antimicrobial and anti-inflammatory activity in a composition that is slightly basic (i.e., pH 8). Accordingly, a composition comprising an antimicrobial cationic peptide, a viscosity-increasing agent, and a solvent, may further comprise a buffering agent. In certain embodiments, the buffering agent comprises a monocarboxylate or a dicarboxylate, and more specifically may be acetate, fumarate, lactate, malonate, succinate, or tartrate. Preferably, the antimicrobial cationic peptide composition having the buffering agent has a pH ranging from about 3 to about 8, and more preferably from about 3.5 to 7. In another preferred embodiment, the buffering agent is at a concentration ranging from about 1 mM to about 200 mM, and more preferably from about 2 mM to about 20 mM, and most preferably about 4 mM to about 6 mM.
Other optional pharmaceutically acceptable excipients are those that may, for example, aid in the administration of the formulation (e.g., anti-irritant, polymer carrier, adjuvant) or aid in protecting the integrity of the components of the formulation (e.g., anti-oxidants and preservatives. Typically, a 1.0% antimicrobial cationic peptide composition may be stored at 2� C. to 8� C. In certain embodiments, the composition comprising an antimicrobial cationic peptide, a viscosity-increasing agent, and a solvent, may further comprise a humectant, preferably sorbitol and the like, or a preservative, preferably benzoic acid, benzyl alcohol, phenoxyethanol, methylparaben, propylparaben, and the like. In certain circumstances, the antimicrobial cationic peptide or analog or derivative thereof may itself function as a preservative of the final therapeutic composition. For example, a preservative is optional in the gel formulations described herein because the gels may be sterilized by autoclaving and, furthermore, show the surprising quality of releasing (i.e., making bioavailable) the antimicrobial cationic peptide at a more optimal rate than other formulations, such as a cream. In addition, particular embodiments may have in a single formulation a humectant, a preservative, and a buffering agent, or combinations thereof. Therefore, a preferred embodiment is a composition comprising an antimicrobial cationic peptide, a viscosity-increasing agent, a solvent, a humectant, and a buffering agent. Another preferred embodiment is a composition comprising an antimicrobial cationic peptide, a viscosity-increasing agent, a buffering agent, and a solvent. In yet another preferred embodiment, the composition comprises an antimicrobial cationic peptide, a buffering agent, and a solvent. Each of the above formulations may be used to treat or prevent infection or to reduce the microflora at a target site, such as a catheter insertion site on a subject (i.e., animal or human).
In yet other embodiments, the composition is in the form of an ointment comprising an antimicrobial cationic peptide (preferably in an amount sufficient to treat or prevent an infection) and an oleaginous compound. For example, oleaginous compound may be petrolatum. In one embodiment, the oleaginous compound is present at a concentration ranging from about 50% to about 100%, more preferably from about 70% to about 100%, even more preferably from about 80% to about 100%, and most preferably from about 95% to about 100%. In certain other embodiments, the ointment composition may further comprise at least one emollient. The emollients may be present at a concentration ranging from about 1% to about 40%, more preferably from about 5% to about 30%, and more preferably from about 5% to about 10%. In certain preferred embodiments, the emollient may be mineral oil, cetostearyl alcohol, glyceryl stearate, and a combination thereof. In another aspect the composition is in the form of a semi-solid emulsion (e.g., a cream) comprising an antimicrobial cationic peptide (preferably in an amount sufficient to treat or prevent an infection), a solvent, a buffering agent, at least one emollient, and at least one emulsifier. In a preferred embodiment, the semi-solid emulsion or cream further comprises at least one of a humectant (e.g., sorbitol and/or glycerin), an oleaginous compound (e.g., petrolatum), a viscosity increasing agent (e.g., dextran, polyvinylpyrrolidone, hydroxyethyl cellulose, and/or hydroxypropyl methylcellulose), an anti-oxidant (e.g., butylated hydroxytoluene and preferably at a concentration ranging from about 0.01% to about 0.1%), a preservative (e.g., benzoic acid, benzyl alcohol, phenoxyethanol, methylparaben, propylparaben, or a combination thereof), or a combination thereof. In certain preferred embodiments, the emollient may be one or more of stearyl alcohol, cetyl alcohol, and mineral oil. In certain other preferred embodiments, the emulsifiers may be one or more of stearyl alcohol, cetyl alcohol, polyoxyethylene 40 stearate, and glyceryl monostearate. In a preferred embodiment, the emulsifier is present at a concentration ranging from about 1% to about 20%, more preferably from about 5% to about 10, and most preferably from about 1% to about 1.5%. As noted above, the function of each of these emulsifiers and emollients is not mutually exclusive in that an emollient may function as an emulsifier and the emulsifier may function as an emollient, depending on the particular formulation, as is known in the art and is described herein. In certain preferred embodiments the solvent comprises water and the like, and the buffering agent comprises a monocarboxylate or dicarboxylate and the like, as described herein.
Uses of antimicrobial cationic peptide formulations of the present invention encompass numerous applications where a topical antimicrobial is useful in the treatment or prevention of infection. For example, burn wound infections remain the most common cause of morbidity and mortality in extensively burned patients. Moreover, infection is the predominant determinant of wound healing, incidence of complications, and outcome of burn patients. The main organisms responsible are Pseudomonas aeruginosa, S. aureus, Streptococcus pyogenes, and various gram-negative organisms. Frequent debridements and establishment of an epidermis or a surrogate, such as a graft or a skin substitute, is essential for prevention of infection. Preferably, the antimicrobial peptide formulations, alone or in combination with antibiotics, is applied to burn wounds as a gel, ointment or cream, and/or administered systemically. Topical application may prevent systemic infection following superficial colonization or eradicate a superficial infection. The antimicrobial peptide composition is preferably administered as a 0.5 to 2% gel, cream, or ointment. Application to the skin could be done once a day or as often as dressings are changed. Systemic administration could be via intravenous, intramuscular or subcutaneous injections or infusions. Other routes of administration known in the art could also be used.
Another use for the present compositions and methods would be in the treatment of surgical wounds, especially those associated with foreign material (e.g., sutures). Nosocomial infections may occur in as many as 71% of all surgical patients, and 40% of those are infections at the operative site. Despite efforts to prevent infection, it is estimated that between 500,000 and 920,000 surgical wound infections complicate the approximately 23 million surgical procedures performed annually in the United States. The infecting organisms are varied, but Staphylococci spp. are important organisms in these infections. Preferably, the antimicrobial peptide formulations, alone or in combination with antibiotics, is applied as an gel, ointment, cream or liquid to the wound site, or as a liquid in the wound prior to and during closure of the wound. Following closure, the antimicrobial peptide composition could also be applied at dressing changes. For surgical or trauma wounds that are infected, the antimicrobial peptide formulation described herein may be applied topically and/or systemically.
Additionally, the compositions and methods of the present invention may be used to reduce the risk of device-related infections by directly coating a medical device prior to insertion at a target site or by impregnating the external surface of a medical device at the time of manufacture. In yet another aspect of this invention, the formulation includes an antimicrobial cationic peptide suitable for impregnating or coating a medical device. Thus, antimicrobial cationic peptides may be formulated as a coating or impregnation material suitable for treating the surfaces of a medical device or its components. In certain embodiments, such coatings and impregnation materials may include covalent and/or non-covalent attachment of an antimicrobial cationic peptide and analog or derivative thereof, to the interior and/or exterior surfaces of a medical device or its components. In other embodiments, such a coating and impregnation material may include the entrapment of an antimicrobial cationic peptide in a hydrogel layer or a bioerodable layer.
By way of example and not limitation, both local and systemic infection may result from contaminated intravascular devices, such as a CVC, and the organisms typically responsible are coagulase-negative Staphylococci (CoNS), Staphylococcus aureus, Enterococcus spp, E. coli and Candida spp. Hence, the antimicrobial cationic peptide or analog or derivative thereof, preferably in the form of a gel or cream, may be applied to the catheter site prior to insertion of the catheter and then again at each dressing change. Preferably, the peptide is at a concentration ranging from about 0.85% to about 1.15%. Therefore, in a typical embodiment, a composition contains an antimicrobial cationic peptide at a concentration ranging from about 0.01% to about 10%; a viscosity-increasing agent selected of dextran, polyvinylpyrrolidone, hydroxyethyl cellulose, or hydroxypropyl methylcellulose; and a solvent of water, glycerin, propylene glycol, isopropanol, ethanol, or methanol; and at a pH ranging from about 3 to about 8.
In a preferred embodiment, the present invention is useful in a method for reducing microflora at a target site, comprising applying to the target site a composition comprising an antimicrobial cationic peptide, a viscosity-increasing agent, and a solvent. As used herein, a target site is any site on a subject where there is present, or there is a risk of, a primary or secondary or opportunistic infection (which infection is outside or inside the subject), and is any site where a formulation of the present invention may be administered or applied. In certain embodiments, the microflora being reduced at the target site may be prokaryotic, eukaryotic, or viral, and preferably is prokaryotic. In other embodiments, the method for reducing microflora at a target site, comprises applying to the target site a composition containing an antimicrobial cationic peptide, a viscosity-increasing agent, and a solvent, and further comprises inserting a medical device at the target site before and/or after applying the composition. In another embodiment, the composition may further contain a buffering agent as described above and may have a pH ranging from about 3.5 to about 7. In addition, the composition may further contain a preservative, such as benzoic acid, benzyl alcohol, phenoxyethanol, methylparaben, propylparaben, and the like. Preferably, the peptide is an indolicidin or analog or derivative thereof, as described herein.
The antimicrobial cationic peptides, particularly the labeled analogs and derivatives thereof, may be used in image analysis and diagnostic assays or for targeting sites in multicellular and single cellular organisms. As a targeting system, the analogues may be coupled with other peptides, proteins, nucleic acids, antibodies, chemical compounds (e.g., fluorescent tags), and the like.
Synthesis Purification and Characterization of Cationic Peptides and Analogues
Indolicidin analogs and derivatives thereof and Other Antimicrobial
Apidaecin IA
G N N R P V Y I P Q P R P P H P R I
Deber A2KA2
K K A A A K A A A A A K A A W A A K A A A K K K K
I L P W K W P W W P W R R
I L K K W P W W P W R R K
11CNR
K R R W P W W P W K K L I
11A1CN
I L K K F P F F P F R R K
11A2CN
I L K K I P I I P I R R K
11A3CN
I L K K Y P Y Y P Y R R K
11A4CN
I L K K W P W P W R R K
11A5CN
I L K K Y P W Y P W R R K
11A6CN
I L K K F P W F P W R R K
11A7CN
I L K K F P F W P W R R K
11A8CN
I L R Y V Y Y V Y R R K
11A9CN
I L R W P W W P W W P W R R K
11A10CN
W W R W P W W P W R R K
11B1CN
I L R R W P W W P W R R K
11B2CN
I L R R W P W W P W R K
11B3CN
I L K W P W W P W R R K
11B4CN
I L K K W P W W P W R K
11B5CN
I L K W P W W P W R K 11B7CN
I L R W P W W P W R R K
11B7CNR
K R R W P W W P W R L I
11B8CN
I L W P W W P W R R K 11B9CN
I L R R W P W W P W R R R
11B10CN
I L K K W P W W P W K K K
11B16CN
I L R W P W W P W R R K I M I L K K A G S
11B17CN
I L R W P W W P W R R K M I L K K A G S
11B18CN
I L R W P W W P W R R K D M I L K K A G S
11B19CN
I L R W P W R R W P W R R K
11B20CN
I L R W P W W P W R R K I L M R W P W W P W R R K M A A
11B32CNR
K R K W P W W P W R L I
11B36CN
I L K W V W W V W R R K
11C3CN
I L K K W A W W P W R R K
11C4CN
I L K K W P W W A W R R K
11C5CN
W W K K W P W W P W R R K
11D1CN
L K K W P W W P W R R K
11D3CN
P W W P W R R K
11D4CN
I L K K W P W W P W R R K M I L K K A G S
11D5CN
I L K K W P W W P W R R M I L K K A G S
11D6CN
I L K K W P W W P W R R I M I L K K A G S
11D9M8
W W P W R R K
11D10M8
I L K K W P W
I L K K W P W W P W R R K M
11D12H
I L K K W P W W P W R R M
11D13H
I L K K W P W W P W R R I M
11D14CN
I L K K W W W P W R K
11D15CN
I L K K W P W W W R K
11D18CN
W R I W K P K W R L P K W
11D19CN
C L R W P W W P W R R K
11E1CN
11E2CN
11E3CN
11F1CN
I L K K W V W W V W R R K
11F2CN
I L K K W P W W V W R R K
11F3CN
I L K K W V W W P W R R K
11F4CN
I L R W V W W V W R R K
11F4CNR
K R R W V W W V W R L I
11F5CN
I L R R W V W W V W R R K
11F6CN
I L R W W V W W V W W R R K
11F12CN
R L W V W W V W R R K
11F17CN
R L W V W W V W R R
11F50CN
R L G G G W V W W V W R R
11F56CN
R L W W V V W W R R
11F63CN
R L V V W W V V R R
11F64CN
R L F V W W V F R R
11F66CN
R L V V W V V W R R
11F67CN
11F68CN
11F93CN
W V R L W W R R V W
11G2CN
I K K W P W W P W R R K
11G3CN
I L K K P W W P W R R K
11G4CN
I L K K W W W P W R R K
11G5CN
I L K K W P W W W R R K
11G6CN
I L K K W P W W P R R K
11G7CN
I L K K W P W W P W R R
11G13CN
I L K K W P W W P W K
11G14CN
I L K K W P W W P W R
11G24CN
L W P W W P W R R K
11G25CN
L R W W W P W R R K
11G26CN
L R W P W W P W
11G27CN
W P W W P W R R K
11G28CN
R W W W P W R R K
11H1CN
A L R W P W W P W R R K
11H2CN
I A R W P W W P W R R K
11H3CN
I L A W P W W P W R R K
11H4CN
I L R A P W W P W R R K
11H5CN
I L R W A W W P W R R K
11H6CN
I L R W P A W P W R R K
11H7CN
I L R W P W A P W R R K
11H8CN
I L R W P W W A W R R K
11H9CN
I L R W P W W P A R R K
11H10CN
I L R W P W W P W A R K
11H11CN
I L R W P W W P W R A K
11H12CN
I L R W P W W P W R R A
11J01CN
R R I W K P K W R L P K R
11J02CN
W R W W K P K W R W P K W
11J02ACN
11J30CN
W R W W K V A W R W V K W
11136CN
W R W W K V W R W V K W
11J38CN
W R W W K V V W R W V K W
11J58CN
W (Orn) W W (Orn) V A W (Orn) W V (Orn) W
11J67CN
W (Orn) W W (Orn) P (Orn) W (Orn) W P (Orn) W
11J68CN
W (Dab) W W (Dab) P (Dab) W (Dab) W P (Dab) W
K K W W R R V L S G L K T A G P A I Q S V L N K
K K W W R R A L Q G L K T A G P A I Q S V L N K
K K W W R R V L K G L S S G P A L S N V
K K W W R R A L Q A L K N G L P A L I S
K W K S F I K K L T S A A K K V V T T A K P L I S S
K W K L F K K I G I G A V L K V L T T G L P A L I S
K W K L F K K I G I G A V L K V L T T G L P A L K L T K
K W K S F I K K L T T A V K K V L T T G L P A L I S
K W K S F I K N L T K V L K K V V T T A L P A L I S
K W K S F I K K L T S A A K K V L T T G L P A L I S
K W K L F I K K L T P A V K K V L L T G L P A L I S
G K P R P Y S P I P T S P R P I R Y
REWH 53A5
R L A R I V V I R V A R
M suffix = MAP branched peptide
Orn = ornithine
Dab = diamino butyric acid
Caprolactam modification. A purified peptide in DMF solution is cooled to 0� C. on ice with stirring. Added to the peptide solution is 2-(1H-benzotriazole-1-yl)-1,1,3,3-teramethyluronium hexafluorophosphate and N-methylmorpholine; the reaction mixture is removed from the ice bath and stirred for 1 h, until the reaction mix rises to room temperature. Water is added and the resulting caprolactam peptide solution is purified by C8 RP-HPLC.
Peptide modified
Acetylated α-N-terminus
11ACN
11CNW1
Fmoc-derivatized N-terminus
11CNX1
Polymethylated derivative
11CNY1
Peracetylated derivative
Four branch derivative
Eight branch derivative
11B1CNW1
11B4ACN
Acetylated N-terminus
11B7CN
11B7ACN
11B7CNF12
Formylated Lys[12]
11B7Cap12
Caprolactam Lys[12]
11B9CN
11B9ACN
11G6ACN
11G7ACN
Antimicrobial Activity of Cationic Peptides
In order to mimic in vivo conditions, calcium and magnesium supplemented Mueller Hinton broth is used in combination with a low EEO agarose as the bacterial growth medium. Agarose, rather than agar, is used as the charged groups in agar prevent peptide diffusion through the medium. The medium is autoclaved, then cooled to 50-55� C. in a water bath before aseptic addition of a peptide solution. The same volume of different concentrations of peptide solution is added to the cooled, molten agarose that is then poured into a petri plate to a depth of 3-4 mm and allowed to solidify.
The bacterial inoculum is adjusted to a 0.5 McFarland turbidity standard (PML Microbiological) and then diluted 1:10 before application on to the agarose plate. The final inoculum applied to the agarose is approximately 104CFU in a 5-8 mm diameter spot. The agarose plates are incubated at 35-37� C. for 16 to 20 hours.
Activity of antimicrobial cationic peptides as determined by agarose dilution susceptibility
11B32CN
KPN001
SEP010
11F27CN
11J36CN
Nt-Glucosyl
An In Vitro Drug Release Method for Topical Formulations Using Low-Flow Cells
An in vitro drug release method for topical formulations using a low-flow cells is used to examine the release of peptide from experimental formulations. The cell consists of an upper (donor) chamber physically separated from a lower (receptor) chamber by a permeable synthetic membrane (Tuffryn, Gelman). A total of 1 gram of a candidate formulation is placed in the donor chamber. The receptor fluid (distilled water, 37� C.) is pumped through the receptor chamber at 2 ml/h. Fractions were collected at various hourly intervals. Fractions were collected into vials, and the amount of receptor fluid collected in grams per fraction was recorded. The concentration of drug in the receptor fluid was determined by RP-HPLC on a Nova Pak C8 column. The column was eluted with a gradient from 20% to 40% acetonitrile over 10 min. using 0.1% aqueous trifluoroacetic acid, and 0.1% trifluoroacetic acid in acetonitrile, as solvents. The flow rate was 1 ml/min.
TABLE 4 Flow Cell Measurement of In Vitro Release Time Period (h) Gel 73A Gel 75A Gel 76A 0–1 974 789 992 2–3 760 707 777 4–6 722 503 671 7–9 590 414 435 10–12 541 499 455 13–15 396 400 376 16–18 303 300 249 19–21 312 204 220 22–24 309 276 372 The membrane is a 0.45 μm Tuffryn (hydrophilic polysulfone) membrane, the data represents μg of antimicrobial cationic peptide released during the time interval. The detection limit is 1 μg released per hour. The data in Table 4 shows that gel formulations show excellent initial release that should facilitate rapid antibiotic action upon application. The gel formulations also demonstrate good sustained release of drug. During the 22-24 hour collection, the average concentration of drug released into the receptor fluid ranged from 93 μgrams/ml (Gel 75A) to 124 μgrams/ml (Gel 76A)—well above the MIC values for sensitive organisms.
Gel 73A
Gel 75A
Gel 76A
Antimicrobial Cationic Peptide
0.1M Lactate buffer, pH 4
Dextran (40,000), USP
Antimicrobial Activity of an Aqueous 1.0% Cationic Peptide Gel
An inoculum of 1�108 CFU/ml for each of the above listed organisms was prepared. For each of the series of tubes designated for each specific organism, 10 μl of undiluted inoculum was added to the gel in each of the tubes giving a final bacterial concentration of 5�105 CFU/g of gel. The contents of the tube were mixed using the handle of a sterile swab.
Formulation without 11B7CN (48 hour counts)
11B7CN in Formulation (48 hour counts)
Avg. Colony
Colony Count (CFU/g)
10−1 1.5 � 105 TNTC
10−2 1.2 � 105 3.3 � 105 5.0 � 105 4.1 � 105 0
10−3 2.5 � 105 3.5 � 105 5.0 � 105 7.5 � 105 0
3.0 � 106 1.5 � 106 0
Formulation without Antimicrobial cationic peptide (48 h counts)
Antimicrobial cationic peptide in Formulation (48 h counts)
5.3 � 103 1.0 � 102 0
10−1 5.5 � 103 0
10−2 0
10−3 0
7 Daysa Time 0
10−1 TNTC
10−2 4.8 � 105 8.4 � 105 7.6 � 105 4.2 � 105 0
10−3 3.5 � 105 1.0 � 106 1.3 � 106 6.0 � 105 0
2.1 � 104 1.2 � 103 0
10−1 8.2 � 104 5.5 � 104 1.9 � 104 3.0 � 103 0
10−2 1.3 � 105 4.5 � 104 1.0 � 104 0
10−3 1.0 � 105 5.0 � 104 0
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