WO2009005798A2 - Antifungal formulation and method of preparation - Google Patents

Abstract

A pharmaceutical preparation for treating a microbial infection such as a fungal or bacterial infection in an individual comprises a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence of at least 8 amino acids selected from the group of amino acid sequences consisting of the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 and various modifications thereof, wherein the buffer molarity of the pharmaceutical preparation is suitable to achieve greater than 99% killing of C. albicans in vitro. A pharmaceutical preparation for treating a microbial infection such as a fungal or bacterial infection in an individual comprises a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence of at least 8 amino acids selected from the group of amino acid sequences consisting of the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 and various modifications thereof, wherein the pharmaceutical preparation has a buffer molarity of about 25 mM or less. A method for treating a microbial infection such as a fungal or bacterial infection in an individual comprises administering to the individual a therapeutically effective amount of a preparation comprising a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence of at least 8 amino acids selected from the group of amino acid sequences consisting of the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 and various modifications thereof, wherein the buffer molarity of the preparation is suitable to achieve greater than 99% killing of C. albicans in vitro. A method for treating a microbial infection such as a fungal or bacterial infection in an individual comprises administering to the individual a therapeutically effective amount of a preparation comprising a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an ami

Description

ANTIFUNGAL FORMULATION AND METHOD OF PREPARATION

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 60/958,216, filed July 3, 2007, the entire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Candidiasis is an infection with a fungus of the genus Candida, especially C. albicans, that usually occurs in the skin and mucous membranes of the mouth, respiratory tract, or vagina but may invade the bloodstream, especially in immunocompromised individuals. The antifungal agents commonly used to treat oral candidiasis subjects target either the high ergosterol content of Candida plasma membranes or the enzymes involved in ergosterol biosynthesis. The membrane- active polyenes nystatin and amphotericin B are fungicidal and for oral candidiasis are usually administered as a suspension or as lozenges, while ergosterol biosynthesis inhibitors (imidazoles and triazoles) are administered as tablets (miconazole, ketonazole, and fluconazole), as a gel (miconazole), or as troches (clotrimazole) (Budtz-Jδrgensen, 1990; Martin, 1990). Fluconazole, a fungistatic triazole that has superior pharmacokinetic properties and greater solubility than the imidazoles, is sometimes used for the oral candidiasis seen in AIDS subjects. Fluconazole and amphotericin B dominate the treatment of disseminated or recurrent fungal infections. However, amphotericin B can cause undesirable side effects, including significant renal toxicity, while prophylactic exposure to fluconazole can lead to resistance or overgrowth by naturally resistant organisms like Candida krusei and Candida glabrata (Vanden Bossche et al., 1994). Itraconazole, another azole compound, has antifungal activity against invasive aspergillosis and has been indicated for treatment of candidiasis, cryptococcosis, histoplasmosis, and blastomyosis. In vitro studies have demonstrated that itraconazole inhibits the cytochrome P-450-dependent synthesis of ergosterol, a vital component of fungal cell membranes. Itraconazole can cause undesirable side effects including liver dysfunction (Budtz-Jorgensen & Lombardi, 1996).

Thus, because of the limitations and restricted target range of existing antifungals, there is a need for more effective antifungals for treatment of candidiasis, especially that of the oral cavity.

SUMMARY OF THE INVENTION

This invention is based on the findings that mouthrinse formulations containing certain antifungal peptides or a pharmaceutically acceptable salt thereof which are active in inhibiting the growth of and/or in killing fungi can show increased antifungal activities in a preparation with a lower buffer molarity (ionic concentration). These peptides represent defined portions of the amino acid sequences of naturally occurring human histidine-rich salivary proteins called histatins, which will be referred to herein as histatin-based peptides. The histatin- based peptides of this invention also include defined portions of the amino acid sequences of histatins with specific amino acid substitutions at specified positions of the sequences and/or with other modifications at the N-terminus, C-terminus, or both the N- and C-termini.

According to one aspect of the present invention, a pharmaceutical preparation for treating a microbial infection such as a fungal or bacterial infection in an individual comprises a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence, of at least 8 amino acids, selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18; b) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the glycine at position 6 is replaced by lysine, arginine or another basic amino acid; c) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the lysine at position 8 is replaced by histidine, phenylalanine or another hydrophobic amino acid; d) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least on of the histidines at position 4, 5 and 12 is replaced by phenylalanine, tyrosine, leucine or another hydrophobic amino acid; e) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the lysines at positions 2 and 10 are replaced by glutamine, arginine or by another basic amino acid; f) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the arginines at positions 3 and 9 is replaced by glutamine, lysine, or by another basic amino acid; and g) any combination of amino acid sequences of preceding sections a)-f) with the exception that glutamine or any other non-basic amino acid cannot simultaneously occupy positions 2, 3, 9 and 10 of the amino acid sequence, wherein the buffer molarity of the pharmaceutical preparation is suitable to achieve greater than 99% killing of C. albicans in vitro.

According to another aspect of the present invention, a pharmaceutical preparation for treating a microbial infection such as a fungal or bacterial infection in an individual comprises a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence, of at least 8 amino acids, selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18; b) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the glycine at position 6 is replaced by lysine, arginine or another basic amino acid; c) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the lysine at position 8 is replaced by histidine, phenylalanine or another hydrophobic amino acid; d) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least on of the histidines at position 4, 5 and 12 is replaced by phenylalanine, tyrosine, leucine or another hydrophobic amino acid; e) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the lysines at positions 2 and 10 are replaced by glutamine, arginine or by another basic amino acid; f) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the arginines at positions 3 and 9 is replaced by glutamine, lysine, or by another basic amino acid; and g) any combination of amino acid sequences of preceding sections a)-f) with the exception that glutamine or any other non-basic amino acid cannot simultaneously occupy positions 2, 3, 9 and 10 of the amino acid sequence, wherein the pharmaceutical preparation has a buffer molarity of about 25 mM or less.

The amino acid sequence of the peptide can be selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18; b) the amino acid sequence of histatin 11 as set forth in SEQ ID NO: 1 1 ; c) the amino acid sequence of peptide 129 as set forth in SEQ ID NO: 23; d) the amino acid sequence of peptide 117 as set forth in SEQ ID NO: 19; e) the amino acid sequence of peptide 118 as set forth in SEQ ID NO: 20; f) the amino acid sequence of peptide 119 as set forth in SEQ ID NO: 21; g) the amino acid sequence of peptide 120 as set forth in SEQ ID NO: 22; h) the amino acid sequence of peptide 113-F4 as set forth in SEQ ID NO: 24; i) the amino acid sequence of peptide 113-F5 as set forth in SEQ ID NO:25; j) the amino acid sequence of peptide 113-F12 as set forth in SEQ ID NO:26; k) the amino acid sequence of peptide 113-F4.5 as set forth in SEQ ID NO: 27;

1) the amino acid sequence of peptide 113-F4.5.12 as set forth in SEQ ID NO:28; m) the amino acid sequence of peptide 113-K6 as set forth in SEQ ID NO:29; n) the amino acid sequence of peptide 113-H8 as set forth in SEQ ID NO:30; o) the amino acid sequence of peptide 113-K6H8 as set forth in SEQ ID NO:31; p) the amino acid sequence of peptide 113-F8 as set forth in SEQ ID NO:32; q) the amino acid sequence of peptide 113-L4.5.12 as set forth in SEQ ID NO:33; r) the amino acid sequence of peptide 113-Y4.5. 12 as set forth in SEQ ID NO:34; s) the amino acid sequence of peptide 113-Q2. 10 as set forth in SEQ ID NO:35; and t) the amino acid sequence of peptide 113-Q3.9 as set forth in SEQ ID NO:36; and u) any combination of amino acid sequences of preceding sections a)-t).

The peptide can have a modification comprising addition of at least one substituent to either the N-terminus, the C-terminus, or both the N-terminus and the

C-terminus of the peptide. The peptide concentration can be between about 0.001% and about 0.75%. The pharmaceutical preparation can further comprise one or more adjuvants selected from the group consisting of one or more polyhydric alcohols, one or more surfactants, one or more flavoring agents, one or more chelating agents, one or more sweeteners, one or more pH adjustors, one or more coloring agents, one or more stabilizing agents, and any combinations thereof.

According to still another aspect of the present invention, a method for treating a microbial infection such as a fungal or bacterial infection in an individual comprises administering to the individual a therapeutically effective amount of a preparation comprising a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence of at least 8 amino acids selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18; b) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the glycine at position 6 is replaced by lysine, arginine or another basic amino acid; c) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the lysine at position 8 is replaced by histidine, phenylalanine or another hydrophobic amino acid; d) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least on of the histidines at position 4, 5 and 12 is replaced by phenylalanine, tyrosine, leucine or another hydrophobic amino acid; e) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the lysines at positions 2 and 10 are replaced by glutamine, arginine or by another basic amino acid; f) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the arginines at positions 3 and 9 is replaced by glutamine, lysine, or by another basic amino acid; and g) any combination of amino acid sequences of preceding sections a)-f) with the exception that glutamine or any other non-basic amino acid cannot simultaneously occupy positions 2, 3, 9 and 10 of the amino acid sequence, wherein the buffer molarity of the preparation is suitable to achieve greater than 99% killing of C. albicans in vitro. According to yet another aspect of the present invention, a method for treating a microbial infection such as a fungal or bacterial infection in an individual comprises administering to the individual a therapeutically effective amount of a preparation comprising a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence of at least 8 amino acids selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18; b) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the glycine at position 6 is replaced by lysine, arginine or another basic amino acid; c) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the lysine at position 8 is replaced by histidine, phenylalanine or another hydrophobic amino acid; d) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least on of the histidines at position 4, 5 and 12 is replaced by phenylalanine, tyrosine, leucine or another hydrophobic amino acid; e) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the lysines at positions 2 and 10 are replaced by glutamine, arginine or by another basic amino acid; f) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the arginines at positions 3 and 9 is replaced by glutamine, lysine, or by another basic amino acid; and g) any combination of amino acid sequences of preceding sections a)-f) with the exception that glutamine or any other non-basic amino acid cannot simultaneously occupy positions 2, 3, 9 and 10 of the amino acid sequence, wherein the preparation has a buffer molarity of about 25 mM or less.

The microbial infection can be selected from the group consisting of: a) an infection of the oral cavity; b) an infection of the vagina; c) an infection of the urethra; d) an infection of the ear; e) an infection of the skin; f) a respiratory infection; g) a mucosal infection; h) an ophthalmic infection; i) any combinations of the preceding sections a)-h); and j) a systemic infection. The fungus or bacterium can be selected from the group consisting of

Candida albicans; Actinomyces actinomycetemcomitans; Actinomyces viscosus;

Bacteroides forsythus; Bacteroides fragilis; Bacteroides graciclis; Bacteroides ureolyticus; Campylobacter concisus; Campylobacter rectus; Campylobacter showae; Campylobacter sputorum; Capnocytophaga gingivalis; Capnocytophaga ochracea; Capnocytophaga sputigena; Clostridium histolyticum; Eikenella corrodens; Eubacterium nodatum; Fusobacterium nucleatum; Fusobacterium periodonticum; Peptostreptococcus micros; Porphyromonas endodontalis;

Porphyromonas gingivalis; Prevotella intermedia; Prevotella nigrescens;

Propionobacterium acnes; Pseudomonas aeruginosa; Selenomonas noxia; Staphylococcus aureus; Streptococcus constellatus; Streptococcus gordonli;

Streptococcus intermedius; Streptococcus mutans; Streptococcus oralis;

Streptococcus pneumonia; Streptococcus sanguis; Treponoma denticola; Treponema pectinovorum; Treponoma socranskii; Veillonella parvula; Wolinella succinogenes;

Candida krusei; Candida kefyr; Candida par apsilosis; Candida tropicalis; Cryptococcus neoformans; and Aspergillus fumigatus.

According to yet another aspect of the present invention, A mouthrinse preparation comprises: a) between about 0.01% and about 0.2% peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is selected from the group consisting of: i) P-113 peptide; ii) modified P-113 peptide, wherein the modification comprises addition of at least one substituent to either the N-terminus, the C- terminus, or both the N-terminus and the C-terminus of the peptide; and iii) any combination of peptides of proceeding sections i)-ii); b) about 0.13% edetate disodium; c) about 0.170% sodium acetate trihydrate; d) about 0.025% sodium saccharin; e) about 0.002% FD&C Yellow #5; f) about 0.003% FD&C Red #40; g) about 0.300% polyxyl 40 hydrogenated castor oil; h) about 0.100% hydroxyethyl cellulose; i) about 0.300% orange flavoring agent; and j) about 7.000% sorbitol, wherein said mouthrinse preparation has a pH of between about 5.5 and about 7.0. According to yet another aspect of the present invention, a method for treating Candida infection of the oral cavity in an individual comprises administering to the individual a therapeutically effective amount of a mouthrinse preparation comprising: a) between about 0.01% and about 0.2% peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is selected from the group consisting of: i) P-113 peptide; ii) modified P-113 peptide, wherein the modification comprises addition of at least one substituent to either the N-terminus, the C- terminus, or both the N-terminus and the C-terminus of the peptide; and iii) any combination of peptides of proceeding sections i)-ii); b) about 0.13% edetate disodium; c) about 0.170% sodium acetate trihydrate; d) about 0.025% sodium saccharin; e) about 0.002% FD&C Yellow #5; f) about 0.003% FD&C Red #40; g) about 0.300% polyxyl 40 hydrogenated castor oil; h) about 0.100% hydroxyethyl cellulose; i) about 0.300% orange flavoring agent; and j) about 7.000% sorbitol, wherein said mouthrinse preparation has a pH of between about 5.5 and about 7.0. The concentration of the peptide can be between about 0.0375% and about 0.15%. The modification can comprise an amide addition at the C-terminus.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

FIG. 1 is a plot illustrating C. albicans growth inhibitory activity of different mouthrinse solutions according to the present invention.

FIGs. 2A-2B are bar graphs illustrating reduction in viable counts upon incubation of C. albicans cells with different mouthrinse solutions (1-8) according to the present invention or with phosphate buffered saline (9).

FIG. 3 is microscopic images illustrating Trypan blue staining of C. albicans cells incubated for 1.5 h at 37⁰C with various solutions according to the present invention. Dark cells indicate permeabilized cells and white cells indicate intact cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to antifungal formulations for the purpose of therapy and the prevention of disease. Particularly, this invention relates to mouthrinse formulations containing peptides, which have antimicrobial activity, in which the amino acid sequences represent defined portions of the amino acid sequences of naturally occuring human histidine-rich salivary proteins called histatins, or modifications thereof. More particularly, the invention is directed to stable mouthrinse formulations which include P-1 13, a 12 amino acid histatin-based peptide (SEQ ID NO: 18). The invention is also directed to methods for the preparation of such mouthrinses.

By way of example, the invention will be described as embodied in mouthrinses. It will be understood that the invention is applicable to other pharmaceutical forms, which include, but are not limited to: aerosol metered-dose, aerosol with propellants, bar chewable, bead, biscuit, bolus, capsule, cartridge, capsule delayed release, cement, cream, douche, drops, dressing, capsule enteric- coated, tablet enteric-coated, elixir, emulsion, enema, tablet effervescent, floss, gas, granule for suspension delayed release, granule effervescent, gel, gel controlled release, globules, granules, gum chewable, insert, insert extended release, jam, kit, leaf, liposome, liquid, lotion, lozenge, mouthwash gargle, ointment, ovules, pad, patch, powder enteric-coated, powder effervescent, pencil, piece chewable, plaster, metered dose pump, drug premix, pellet (oral), powder, powder sustained release, powder for suspension sustained release, paste, powder for suspension, powder for solution, ring slow release, suspension delayed release, shampoo, spray metered dose, solution, soap bar, sprinkle capsule, sponge, spray, suppository sustained release, sustained release capsule, disc sustained release, syrup sustained release, sustained release tablet, stick, strip, suppository, suspension, suture, swab, syrup, tablet, tablet chewable, tablet dispersible, tablet delayed release, tablet rapid dissolve, tablet sublingual, tape, toothpaste, wafer and wipe. To indicate the relative amount of a particular ingredient in a preparation of a given pharmaceutical form, it will be understood that either molar concentration or weight/weight percentage, or both, can be used, where appropriate.

The histatins are a group of small, cationic histidine-rich peptides secreted by human parotid and submandibular salivary glands (Oppenheim et al. 1986, 1988, Lamkin & Oppenheim 1993). To date, twelve histatin peptides have been isolated from human saliva and characterized. Of these, histatin 1 and histatin 3, which are 38 and 32 amino acids in length respectively, have been shown to be encoded by different genes, both located on chromosome 4 (Sabatini & Azen 1989; vanderSpeck et al. 1989 and 1990). All other histatin peptides are believed to arise from these two "parent" peptides by proteolytic processing (Troxler et al. 1990). Histatin 5, for example, is composed of 24 amino acids, and is derived from the N-terminal portion of histatin 3.

Collectively, the histatins have been shown to bind to hydroxyapatite, suggesting a role in the formation of the acquired enamel pellicle (Mayhall 1970; Hay 1973). However, it is currently believed that the histatins play a principal role in protecting the host oral cavity from etiologic pathogens. In particular, the histatins are antifungal and kill Candida albicans in both the yeast and mycelial forms (Xu et al. 1990 and 1991). It has been suggested that the histatins adopt a helical structure on cell membranes and alter permeability leading to these candidacidal and candidastatic effects (Raj et al. 1990; Pollack et al. 1984). Localization studies have shown that histatins are internalized and targets the energized mitochondrion as indicated by a granular intracellular distribution of the labeled peptide co-localized with a permeant mitochondrial-specific probe (Helmerhorst et al 1999a).

The histatins are, therefore, a group of novel peptides having low toxicity, minimal immunogenicity, and potential therapeutic and preventive applications, given their biological activities and endogenous function in the oral cavity. Microbes which are amenable to histatin-based peptide therapy include, but are not limited to: Candida albicans; Actinomyces actinomycetemcomitans; Actinomyces viscosus; Bacteroides forsythus; Bacteroides fragilis; Bacteroides graciclis; Bacteroides ureolyticus; Campylobacter concisus; Campylobacter rectus; Campylobacter showae; Campylobacter sputorum; Capnocytophaga gingivalis; Capnocytophaga ochracea; Capnocytophaga sputigena; Clostridium histolyticum; Eikenella corrodens; Eubacterium nodatum; Fusobacterium nucleatum; Fusobacterium periodontium; Peptostreptococcus micros; Porphyromonas endodontalis; Porphyromonas gingivalis; Prevotella intermedia; Prevotella nigrescens; Propionobacterium acnes; Pseudomonas aeruginosa; Selenomonas noxia; Staphylococcus aureus; Streptococcus constellatus; Streptococcus gordonli; Streptococcus intermedius; Streptococcus mutans; Streptococcus oralis; Streptococcus pneumonia; Streptococcus sanguis; Treponoma denticola; Treponema pectinovorum; Treponoma socranskii; Veillonella parvula; Wolinella succinogenes; Candida krusei; Candida kefyr; Candida parapsilosis; Candida tropicalis; Cryptococcus neoformans; and Aspergillus fumigatus.

The amino acid sequences of the anti-fungal and anti-bacterial peptides of this invention represent all or defined portions of the amino acid sequence of peptide 113 (SEQ ID NO: 18). In addition, the anti-fungal and anti-bacterial peptides of this invention include all or defined portions of peptide 113 (SEQ ID NO: 18) with amino acid substitutions at particular positions of the peptide.

Preferred embodiments of peptide of this invention are peptide 113 itself (SEQ ID NO: 18); fragments of peptide 113 containing at least an 8 amino acid sequence from this peptide; an amino acid sequence of at least 8 amino acids from peptide 113 where the glycine at position 6 is replaced by lysine, arginine or another basic amino acid; an amino acid sequence of at least 8 amino acids from peptide 113 where the lysine at position 8 is replaced by histidine, phenylalanine or another hydrophobic amino acid; an amino acid sequence of at least 8 amino acids from peptide 113 where one or more of the histidines at positions 4, 5 and 12 is (are) replaced by phenylalanine, tyrosine, leucine or another hydrophobic amino acid; an amino acid sequence of at least 8 amino acids from peptide 113 where one or both of the lysines at positions 2 and 10 is (are) replaced by glutamine, arginine or a combination of glutamine and arginine (when both lysines are replaced); and an amino acid sequence of at least 8 amino acids from peptide 113 where one or both of the arginines at positions 3 and 9 is (are) replaced by glutamine, lysine or a combination of glutamine and lysine (when both arginines are replaced). Combinations of these amino acid replacements in an amino acid sequence of at least 8 amino acids from peptide 113 are all preferred embodiments of the invention provided that a combination of 4 glutamines or any other group of 4 non-basic amino acids at positions 2, 3, 9 and 10 does not occur.

Specific preferred embodiments of peptide of this invention are peptide 113 itself (SEQ ID NO: 18), histatin 11 (SEQ ID NO: 11), peptide 129 (SEQ ID NO: 23), peptide 117 (SEQ ID NO: 19), peptide 118 (SEQ ID NO: 20), peptide 119

(SEQ ID NO: 21), peptide 120 (SEQ ID NO: 22), peptide 113-F4 (SEQ ID NO: 24), peptide 113-F5 (SEQ ID NO: 25), peptide 113-Fl 2 (SEQ ID NO: 26), peptide 113- F4.5 (SEQ ID NO: 27), peptide 113-F4.5.12 (SEQ ID NO: 28), peptide 113-K6 (SEQ ID NO: 29), peptide 113-H8 (SEQ ID NO: 30), peptide 113-K6H8 (SEQ ID NO: 31), peptide 113-F8 (SEQ ID NO: 32), peptide 113-L4.5.12 (SEQ ID NO: 33), peptide 113-Y4.5.12 (SEQ ID NO: 34), peptide 113-Q2.10 (SEQ ID NO: 35), and peptide 113-Q3.9 (SEQ ID NO: 36).

Combinations of two or more of these peptides are also effective as anti- fungal or anti-bacterial compositions. However, the combination of these peptides where glutamine occurs at positions 2, 3, 9 and 10, i.e. peptide 113-Q2.3.9.10 (SEQ ID NO: 37) is not a specifically preferred embodiment.

The peptides can be obtained from a naturally occurring source of histatin or they can be chemically synthesized or obtained by recombinant DNA techniques as expression products from cellular sources. These peptides can be altered by minor chemical modifications, such as by adding small substituents or by modifying one or more of the covalent bonds within or between the amino acid residues, without significantly diminishing the anti-fungal or anti -bacterial activities of the peptides. Quite useful modifications are the addition of a substituent to either the amino terminus, the carboxyl terminus or to both ends of the peptide. These substituent addition modifications appear to stabilize the peptide in its active form and to aid in the prevention of enzymatic degradation of these peptides. These substituent groups are added to the amine, at the amino terminus, or to the carboxyl group, at the carboxyl terminus. The substituent groups can be somewhat bulky and may include one or more natural or modified amino acids. Particularly useful modifications are acetylation or carbamylation of the amino terminus of the peptide or amidation of the carboxyl terminus of the peptide. A combination of both modifications is especially useful. Such modifications appear to increase the biological half-life of the peptides before degradation, encapsulation, internalization or excretion occurs. Preferably the peptide of the present invention is amidated at the C-terminus. P-113 peptide containing a C-terminal amide group was found almost twofold more potent in killing C. albicans than the unamidated peptide of the same amino acid sequence (Rothstein et al. 2001).

Histatin-based peptides can be produced in a variety of expression vector/host systems, which are available commercially or can be reproduced according to recombinant DNA and cell culture techniques. The vector/host expression systems can be prokaryotic or eucaryotic, and can include bacterial, yeast, insect, mammalian, and viral expression systems. The construction of expression vectors encoding histatin-based peptides, transfer of the vectors into various host cells, and production of peptides from transformed host cells can be accomplished using genetic engineering techniques, as described in manuals such as Molecular Cloning and Current Protocols in Molecular Biology, whose teachings are incorporated herein by reference.

Modified histatin based peptides, such as particular amino acid substitutions of peptide 113, can be synthesized chemically, or be produced from cloned DNAs containing mutated nucleotide sequences. Histatin-based peptides encoded by expression vectors may be modified due to post-translational processing in a particular expression vector/host cell system. (See, e.g., Wold, F., 1981). Histatin- based peptides may also be modified by chemical alteration of amino acid side-chain groups, or by other covalent modification. (See, e.g., Glazer, A. N. et al., 1975; Katre, N. V. et al., 1987)

Preferably, the peptide of the present invention is in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salt of a peptide can be made by mixing the peptide with an acid or a base that provides an appropriate counter ion. A peptide, in a salt form, has greater solubility than the pure peptide and is easier to isolate. Preferably, the peptide of the present invention is included in the preparation as an acetate salt.

The antimicrobial activities of the peptides have been tested in chemically defined buffers and broths (Oppenheim et al., U.S. Pat. No. 5,631,228; Oppenheim et al., U.S. Pat. No. 5,912,230; Rothstein et al. 2001; Helmerhorst et al. 1999b; Helmerhorst et al. 2005). P-113-containing mouthrinse formulations have been proven effective in clinical and microbial evaluation with experimental gingivitis (Mickels et al. 2001; Van Dyke et al. 2002).

Mouthrinses containing histatin-based peptides preferably comprise between about 0.001% and about 0.75% peptide (all percentages herein are on a weight/weight basis unless indicated otherwise). More preferably, the mouthrinse comprises between about 0.005% and about 0.2% peptide. Preferably, the peptide is P-113. More preferably, the peptide is a modified P-113 selected from the group consisting of a) an acetyl addition at the N-terminus of P-113; b) a carbamyl addition at the N-terminus of P- 113 ; c) an amide addition at the C-terminus of P-

113; d) a combination of preceding sections a) and c); e) a combination of preceding sections b) and c); and f) a pharmaceutically acceptable salt of any modified P-113 of proceeding sections a)-e).

The present inventors made the surprising findings that a lower buffer molarity (ionic concentration) of a mouthrinse containing P-113 and/or modified P- 113 could increase its antifungal activities. The buffer molarity of the mouthrinse is about 25 mM or less. Preferably, the buffer molarity of the mouthrinse is between about 3 mM and about 20 mM. More preferably, the buffer molarity of the mouthrinse is between about 10 mM and about 15 mM. Even more preferably, the buffer molarity of the mouthrinse is about 12.5 mM.

Buffer molarity of the mouthrinse can be conferred by one or more salts. Preferably, the mouthrinse comprises about 25 mM or less sodium acetate. More preferably, the mouthrinse comprises between about 3 mM and about 20 mM sodium acetate. Even more preferably, the mouthrinse comprises between about 10 mM and about 15 mM sodium acetate. Most preferably, the mouthrinse comprises about 12.5 mM sodium acetate. It is generally desirable, and often preferred to include various adjuvants and

/or excipients to the basic compositions of this invention. These can include, but are not limited to, one or more polyhydric alcohols such as glycerol and sorbitol, one or more surfactants, especially nonionics, one or more flavors such as orange flavor, one or more chelating agents, one or more sweeteners, one or more pH adjustors, one or more coloring agents and one or more stabilizing agents.

The polyhydric alcohol can range from about 1 to 20% by weight, with 3- 15% being preferred, and about 7% being more preferred. Sorbitol is preferred as the polyhydric alcohol component, which serves humectant and viscosity-control agent. Without being bound to any theory, the surfactant components are thought to serve as solubilizing, dispersing, emulsifying, wetting and viscosity-control agents and when used in certain combinations, are especially effective to solubilize the flavor.

Polyoxyl 40 hydrogenated castor oil is a surfactant and solubilizer for fat- soluble vitamins, essential oils and other hydrophobic pharmaceuticals. The main constituent of polyoxyl 40 hydrogenated castor oil is glycerol polyethylene glycol oxystearate, which, together with fatty acid glycerol polyglycol esters, forms the hydrophobic part of the product. The hydrophilic part consists of polyethylene glycols and glycerol ethoxylate. Particular features are that it has very little odor and in aqueous solutions is almost tasteless. Preferably, the mouthrinse formulation comprises about 0.30% polyoxyl 40 hydrogenated castor oil.

Hydroxyethyl cellulose is another surfactant that is nonionic, water-soluble polymer and can thicken, suspend, bind, emulsify, form films, stabilize, disperse, retain water, and provide protective colloid action. It is readily soluble in hot or cold water and can be used to prepare solutions with a wide range of viscosities.

For the aforementioned functions of solubilizing, dispersing, emulsifying, wetting and viscosity-control, it is preferred to use from about 0.1% to about 10% by weight of surfactant; more preferred range is about 0.1% to about 6% and a most preferred range is from about 0.1% to about 0.3%.

Preferably the mouthrinse comprises about 0.3% flavoring agent, preferably orange flavor.

The mouthrinse can also contain an appropriate chelating agent. Edetate disodium is a reagent that chelates calcium cation, magnesium cation and other divalent and trivalent metal ions. Preferably the mouthrinse comprises between about 0.01% and about 1% chelating agent, more preferably about 0.130%. Preferably the chelating agent in the mouthrinse is edetate disodium.

The mouthrinse of the present invention can optionally contain any of a variety of sweetening agents alone or in combination. Sweetening agents can include, for example, saccharin, dextrose, levulose, xylitol, sodium cyclamate, perillartine, D-tryptophan, aspartame, dihydrochalcones and the like. The sweetening agent content of the mouthrinse is variably dependent on the taste sought to be imparted and the strength of the selected sweetener. A preferred sweetener component compound is saccharin, especially sodium saccharin, but other known orally acceptable sweetener compounds can be employed, typically in concentrations of about 0.01% to about 5%, preferably at about 0.025%.

Sodium hydroxide is a typical pH adjustor. Preferably the mouthrinse has a pH between about 5.5 and about 7.5. More preferably, the mouthrinse has a pH between about 6.0 and about 7.0. Even more preferably, the mouthrinse has a pH between about 6.0 and about 6.5.

A coloring agent is also often desirable for enhanced appearance and acceptability, but should be carefully selected for compatibility with the other named components. FD&C Red #40 and Yellow #5 have been found to satisfy the requirements of this invention, employed in effective coloring amounts as desired, typically in concentrations of about 0.0002 to 0.003% by weight in the solution. A stabilizing agent is also often desirable for enhanced stability and longer storage. Parabens are a group of chemicals widely used as preservatives in the cosmetic and pharmaceutical industries. They are esters of pαrα-hydroxybenzoic acid, from which the name is derived. Common parabens include methylparaben, ethylparaben, propylparaben and butylparaben. Less common parabens include isobutylparaben, isopropylparaben, benzylparaben and their sodium salts. The general chemical structure of a paraben is shown below, where R symbolizes an alkyl group such as methyl, ethyl, propyl or butyl. Parabens are effective preservatives in many types of formulas. These compounds, and their salts, are used primarily for their bacteriocidal and fungicidal properties. They can be found in shampoos, commercial moisturizers, shaving gels, cleansing gels, personal lubricants, topical/parenteral pharmaceuticals and toothpaste. They are also used as food additives. The mouthrinse of the present invention can optionally contain about 0.17% methyparaben and about 0.03% propylparaben.

General chemical structure of a paraben (ajrørø-hydroxybenzoate) where R = an alkyl group

Mouthrinses prepared in accordance with the present invention are characterized by superior chemical stability and, consequently, shelf life. The mouthrinses are thus stable, i.e. they remain clear, with no visible formation of precipitation or detectable evolution of gases after prolonged periods of storage, i.e. at least six months of storage at room temperature.

The following examples illustrate the invention.

EXAMPLE 1 100 g of a 70% sorbitol solution was propeller mixed with 600 g of purified water to provide a 10% sorbitol solution in a main vessel.

1.30 g edetate disodium, 6.81 g sodium acetate trihydrate, 0.25 g sodium saccharin, 0.003 g FD&C Red #40, 0.002 g FD&C Yellow #5 were added to the main vessel and dissolved by continued mixing. In a separate beaker, 3.O g polyoxyl 40 hydrogenated castor oil was mixed with 3.00 g orange flavor to provide a solubilized flavor solution. The solubilized flavor solution was added to the main vessel and continuously mixed. 10Og purified water was used to rinse the beaker and the rinse added to the main vessel.

In another separate beaker, 1.50 g P-113 or a modified P-113 peptide was mixed with 50 g purified water to provide a peptide solution. The peptide solution was transferred to the main vessel and continuously mixed. 50 g purified water was used to rinse the beaker and the rinse added to the main vessel. pH of the mixed solution in the main vessel was adjusted to 5.5-6.5 with sodium hydroxide.

1.0 g hydroxyethyl cellulose was dispersed in the main vessel. Mixing was continued until the polymer was completely hydrated.

Pure water was added to the solution in the main vessel to form 1 L of a stabilized mouthrinse containing P-113 or a modified P-113.

EXAMPLE 2

A mouthrinse having the composition as set forth below (Mouthrinse Formulation 1) was prepared by the method as set forth in Example 1.

* 0.681% sodium acetate trihydrate is 50 mM buffer molarity.

** A modified P-113 with an amide addition at the C-terminus and as acetate salt. Another mouthrinse having the composition as set forth below (Mouthrinse Formulation 2) was prepared by the method as set forth in Example 1 , with the following modifications: instead of mixing 3.O g polyoxyl 40 hydrogenated castor oil with 3.00 g orange flavor to provide a solubilized flavor solution, which was then added to the main vessel, 3.0 g polyoxyl 40 hydrogenated castor oil was added to the main vessel without addition of any flavoring agent.

* 0.681% sodium acetate trihydrate is 50 mM buffer molarity.

** A modified P-113 with an amide addition at the C-terminus and as acetate salt.

Still another mouthrinse having the composition as set forth below (Mouthrinse Formulation 3) was prepared by the method as set forth in Example 1, except that the orange flavor was replaced with orange/papaya flavor.

* 0.681% sodium acetate trihydrate is 50 mM buffer molarity.

** A modified P-113 with an amide addition at the C-terminus and as acetate salt.

EXAMPLE 3

Mouthrinses having compositions as set forth below were prepared by the methods as set forth below.

Solution 1 (PAC-113 0.15% mouthrinse 5OmM buffer molarity) - 1Og of Mouthrinse Formulation 2.

Solution 2 (PAC-113 0.15% mouthrinse 12.5mM buffer molarity) - prepared by using 2.5g of Mouthrinse Formulation 2, 7.48875g of deionized water and 0.01125gram of PAC-113 peptide. Solution 3 (PAC-113 0.075% mouthrinse 5OmM buffer molarity) - prepared by using 5g of Mouthrinse Formulation 2, 4.96595g of deionized water and 0.03405g of Sodium Acetate Trihydrate, USP.

Solution 4 (PAC-113 0.075% mouthrinse 12.5mM buffer molarity) - prepared by using 2.5g of Mouthrinse Formulation 2, 7.49625g of deionized water and 0.00375g of PACl 13 peptide.

Solution 5 (PAC-113 0.0375% mouthrinse 5OmM buffer molarity) - prepared by using 2.5g of Mouthrinse Formulation 2, 7.44893g of deionized water and 0.05108g of Sodium Acetate Trihydrate, USP Solution 6 (PAC-113 0.0375% mouthrinse 12.5mM buffer molarity) - prepared by using 2.5g of Mouthrinse Formulation 2 and 7.5g of deionized water. Solution 7 (mouthrinse at 5OmM buffer molarity without PAC-113) - prepared by using 0.0681 gram of sodium acetate trihydrate and 9.9319 gram of deionized water. Solution 8 (mouthrinse at 12.5mM buffer molarity without PAC-113) - prepared by using 0.0170gram of sodium acetate trihydrate and 9.983 gram of deionized water.

EXAMPLE 4

Mouthrinses having the compositions of Example 3 were evaluated in a blind study for inhibition of fungal growth. C. albicans ATCC 10231 cells were cultured on Sabouraud dextrose agar (SDA) for 48 hr at 30°C. A single colony was suspended in 10% Sabouraud dextrose broth (10% SDB), and cells were cultured to mid log-phase (OD₆₂₀ 0.6-0.8). The cells were subsequently diluted in 50% SDB to a final OD₆₂₀ of 0.01. A two-fold serial dilution series of each of solutions 1-8 (in triplicate) was prepared in deionized water in polypropylene microtiter plates, to a final volume of 80 μl/well. To each well, 20 μl of the diluted fungal suspension was added. Plates were covered with plastic and a lid and incubated for 24 hr at 30°C. The cells were resuspended using a multichannel pipet and the OD_62O of the suspensions in each well were read using a microtiter plate reader The test results obtained are presented in FIG. 1 and Table I. The results demonstrate that tested peptide P-113 solutions are effective in inhibiting C. albicans growth even if diluted 1000 fold. Solutions 7 and 8, not containing any peptide P-113, were devoid of growth inhibitory activity, indicating that the observed activity in solutions 1-6 is attributable to peptide 1 13.

TABLE I

* Concentration required to inhibit 50% of growth.

EXAMPLE 5 To gain a better insight into differences in efficacy between solutions 1-6 with regard to their activity toward C. albicans, mouthrinses having the compositions of Example 3 were evaluated for fungal cell killing activity. C. albicans ATCC 10231 cells were cultured to log phase in 10% SDB as described above. Cells were harvested by centrifugation and concentrated to an OD₆20 of 15. From this suspension, 20 μl was added to 1 ml of each of solutions 1-8 (in triplacate), or to 1 ml of phosphate-buffered saline (PBS; in triplicate). The 27 suspensions were subsequently incubated for 1.5 hr at 37°C. After this incubation period cells were diluted and plated according to two methods: 1. To determine the percent cell killing, a 50 μl aliquot was diluted 18Ox in

PBS and a 25 μl aliquot was plated on SDA. After 48 hr of incubation at 30⁰C colonies were counted, and the percent killing was calculated as: (1 -(number of colonies of cells incubated in each solution/number of colonies of cells incubated in PBS))* 100%. 2. To determine the log reduction in viable counts a 25 μl aliquot was diluted

10x, 10Ox and 100Ox in PBS. From the undiluted, 10x, 10Ox, and 100Ox diluted suspensions, a 25 μl aliquot was plated on SDA. After 48 hr of incubation at 30°C, the plates were counted and the number of colony forming units per ml in each of the original undiluted suspensions was calculated taking into account the dilution factor and the volume of the cells plated. 25 μL of each dilution was plated on

Sabouraud dextrose agar. Colonies were counted after incubation for 48 h at 30°C.

The percentage killing results obtained are presented in Table II. The log reduction results obtained are presented in FIGs. 2A-2B.

Following the cell killing assay described above, the remainder of the 1-mL suspensions were centrifuged and suspended in 100 μL PBS. To this suspension, 100 μL of Trypan blue was added to a final concentration of 25 mg/mL. Cells were analyzed for trypan exclusion (cell integrity) using a light microscope. Results of C. albicans permeabilization assay using Trypan blue are presented in FIG. 3.

Test solutions containing modified P-113 are effective in inhibiting C. albicans cell growth and in causing cell killing. The antifungal effects are directly attributable to the modified P-113 peptide and are dose-dependent. Solutions 2, 4 and 6, containing % of the buffer molarity were found to be superior in activity to Solutions 1, 3 and 5. Solutions 2, 4 and 6, with a lower buffer molarity compared to Solutions 1, 3 and 5, were able to achieve greater than 99% killing of C. albicans in vitro. The results show that the antifungal activity of the modified P-113 peptide formulation is critically dependent on the ionic composition.

TABLE II

* Values represent the average of two experiments, each performed in triplicate.

EXAMPLE 6

Mouthrinse having the composition of Mouthrinse Formulation 3 as set forth in Example 2 was evaluated for 1) safety and tolerance when administered orally in HIV-positive subjects with oral candidiasis; 2) efficacy in eliminating or reducing clinical signs and symptoms of oral Candida infections; and 3) microbiological response of Candida to P-113, in a randomized, examiner-blinded, parallel design clinical trial, which features two treatment arms, conducted at the University of California at San Francisco, the University of Southern California at Los Angeles and various sites in the Republic of South Africa.

At least forty-four (44) HIV positive subjects per treatment arm were recruited for a minimum of 88 subjects total. The study design includes a screening visit, a 14-day treatment phase visit, and a 14 day follow-up period with a 28 day follow-up visit. During the screening visit subjects were assessed for study eligibility. Eligible subjects were randomized to one of the following treatment arms:

A. 0.15% P-1 13 mouthrinse (Mouthrinse Formulation 3 ; 5 mL q.i.d.);

B. nystatin oral suspension (5 mL q.i.d.).

Subjects were evaluated clinically for safety and severity of clinical signs and symptoms of oral candidiasis (dysphagia and oral pain/burning of mouth) and extent of lesions at baseline (Day 1), Day 3, Day 7, Day 14 (end of treatment), and

Day 28 (follow-up visit), with clinical response being assessed on Days 7, 14 and

28. Subjects also had a microbiological analysis performed at screening, Day 7, Day

14 and Day 28, with microbiological response assessed on Days 7, 14 and 28. Each symptom of oral candidiasis (dysphagia and oral pain/burning of mouth) were given a score using the grading scale below. These scores were then averaged to give an overall score for clinical symptoms ranging from 0 (absent) to 3

(severe).

Grade 0 = absent Grade 1 = mild

Grade 2 = moderate

Grade 3 = severe

The extent of lesions due to oral candidiasis were assessed through an examination of the oral cavity and recorded using the grading scale below. Confluent lesions refers to lesions whose borders have clearly merged. The WHO map of the oral cavity were used to record the number, size and location of lesions.

Grade 0 = absent

Grade 1 = scant disease (< 5 lesions that are not confluent or < 10% oral cavity surface involvement)

Grade 2 = mild disease (5 - 10 lesions that are not confluent or > 10% to < 25% oral cavity surface involvement)

Grade 3 = moderate disease (> 10 lesions that are not confluent or 25 - 50% oral cavity surface involvement)

Grade 4 = severe disease (> 10 confluent lesions or > 50% oral cavity surface involvement) Clinical responses were assessed using the criteria below: Complete Response: complete resolution of clinical disease (grade 0 for symptoms and lesions);

Partial Response: grade 0 for lesions with mild symptoms remaining (grade < 1 for symptoms) or grade 0 for symptoms with minimal lesions remaining (grade < 2 for extent of lesions);

Failure: no improvement or worsening of grade severity, or grade >

1 for both lesions and symptoms; and

Relapse: a grade > 0 for lesions and symptoms after achieving a grade of 0 for both at the Day 14 assessment, or any subject who requires therapy for OPC

(Oro-pharengeal candidiasis) after the Day 14 visit.

Microbiological responses were assessed using the criteria below: Eradication: negative fungal cultures on Day 14;

Colonization: positive cultures post-therapy where the quantity of yeast is scant to light, and the fungal species is the same as at baseline, and MIC values have not increased more than four fold;

Persistence/Failure: continued positive culture with the same number or an increase in number from baseline, and the fungal species is the same as at baseline and MIC values are the same or higher that those at baseline;

New Infection: a positive fungal culture after a negative fungal culture was obtained; the species in the positive culture is different than the species that was isolated at the baseline sample; the quantity of yeast and the MIC values should be determined but does not need to be compared to the baseline values, as different species will be tested;

Relapse: positive fungal culture after a prior negative fungal culture; the same species is present in both the baseline and post-therapy samples; the post-therapy isolate may or may not have higher MIC values compared to the baseline isolate. Patient populations on which efficacy and/or safety analyses were performed are defined below:

Intent-to-Treat (ITT2) population: subjects receiving at least 80% of the planned total dosage of study treatment. There were 41 patients in the PAC-113 ITT2 group and 31 patients in the Nystatin ITT2 group.

Intent-to-Treat (ITTl) population: subjects receiving at least one dose of study mouthrinse and providing at least one post-baseline efficacy assessment. There were 54 patients in the PAC-113 ITTl group and 46 patients in the Nystatin ITTl group. Safety population: subjects receiving at least one dose of study mouthrinse.

There were 55 patients in the PAC-113 safety population and 48 patients in the Nystatin safety population.

Efficacy analyses were performed on the Intent-to-Treat (ITTl) population and on Intent-to-Treat (ITT2) population. Safety analyses were performed on the Safety population.

The primary efficacy analysis compared the active treatment group (Mouthrinse Formulation 3) with the nystatin oral suspension group (the control group) with respect to the complete clinical response rate at Day 14 for the ITT2 population (see above for definition of complete clinical response). Specifically, the Mouthrinse Formulation 3 treatment group was compared to the nystatin oral suspension group with respect to the percentage of subjects experiencing a complete clinical response at Day 14 using the Cochran-Mantel-Haenszel test controlling for study center at the alpha = 0.05 level of significance. The significance of the treatment-by-study center interaction was assessed using the Breslow-Day test. If it was deemed that certain demographic and baseline covariates were highly related to response rate, a logistic regression comparing treatment groups on clinical response rate adjusting for such covariates was performed as a secondary analysis. Note that any subject prematurely withdrawing due to lack of efficacy was considered a failure for purposes of the primary efficacy analysis. .The following secondary efficacy analyses were performed on the ITT2 population: 1. A comparison of Mouthrinse Formulation 3 with nystatin with respect to the individual clinical response categories at Day 14 using the Cochran-Mantel- Haenszel test;

2. Complete and partial responders (i.e. subjects who at least improved from pre- treatment with respect to clinical response, or non-failures) at Day 14 were analyzed using the same statistical plan outlined for complete responders;

3. A comparison of Mouthrinse Formulation 3 with nystatin with respect to the overall score for extent of lesions was performed at Day 14 using the Cochran- Mantel-Haenszel test; 4. A comparison of Mouthrinse Formulation 3 with nystatin with respect to the overall score for symptoms of oral candidiasis at Day 14 was carried out using the Wilcoxon Rank Sum test;

5. The two groups were compared with respect to the overall microbiological response rate and the response rate of the individual fungal species in a similar manner as was described for complete clinical response (any subject with a microbiological response of eradication will be considered a microbiological responder; see above); and

6. The relapse rates were calculated among the responders and the two groups were compared using the Fisher's Exact test. All aforementioned secondary efficacy analyses were repeated (a) at each study visit at which disease severity is assessed; and (b) for subjects in the ITTl population. Note that for the analysis on clinical and microbiological response at Day 28, subjects in the "Relapse" category were categorized as failures.

Preliminary clinical trial results obtained are presented in Table III. TABLE III

Complete Response Rates at Day 14 for PAC-113* (N = 41) 36.6%

ITT2 (primary efficacy analysis) Nystatin (N = 31) 35.5%

Complete and Partial Response Rates at PAC-113* (N = 41) 95.1%

Day 14 for ITT2 Nystatin (N = 31) 87.1%

Complete and Partial Response Rates at PAC-113* (N = 54) 85.2%

Day 14 for ITTl Nystatin (N = 46) 89.1%

Complete Response Rates at Day 28 for PAC-113* (N = 41) 36.6%

ITT2 Nystatin (N = 31) 32.6%

Complete and Partial Response Rates at PAC-113* (N = 41) 82.9%

Day 28 for ITT2 Nystatin (N = 31) 74.2% Complete and Partial Response Rates at PAC-113 (N = 54) 72.2%

Day 28 for ITTl Nystatin (N = 46) 65.2%

Microbiological Responders at Day 14 PAC-113 (N = 41) 22.0% for ITT2 Nystatin (N = 31) 35.5%

Microbiological Responders at Day 14 PAC-113* (N = 54) 18.5% for ITTl Nystatin (N = 46) 28.3%

Microbiological Responders at Day 28 PAC-113* (N = 41) 14.6% for ITT2 Nystatin (N = 31) 12.9%

Microbiological Responders at Day 28 PAC-113* (N = 54) 16.7% for ITTl Nystatin (N = 46) 13.0%

* Mouthrinse Formulation 3

The relevant teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

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Claims

CLAIMSWhat is claimed is:

1. A pharmaceutical preparation for treating a microbial infection such as a fungal or bacterial infection in an individual, comprising a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence of at least 8 amino acids selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18; b) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the glycine at position 6 is replaced by lysine, arginine or another basic amino acid; c) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 where the lysine at position 8 is replaced by histidine, phenylalanine or another hydrophobic amino acid; d) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least on of the histidines at position 4, 5 and 12 is replaced by phenylalanine, tyrosine, leucine or another hydrophobic amino acid; e) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the lysines at positions 2 and 10 are replaced by glutamine, arginine or by another basic amino acid; f) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the arginines at positions 3 and 9 is replaced by glutamine, lysine, or by another basic amino acid; and g) any combination of amino acid sequences of preceding sections a)-f) with the exception that glutamine or any other non-basic amino acid cannot simultaneously occupy positions 2, 3, 9 and 10 of the amino acid sequence, wherein the buffer molarity of the pharmaceutical preparation is suitable to achieve greater than 99% killing of C. albicans in vitro.

2. A pharmaceutical preparation for treating a microbial infection such as a fungal or bacterial infection in an individual, comprising a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence of at least 8 amino acids selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18; b) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 where the glycine at position 6 is replaced by lysine, arginine or another basic amino acid; c) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 where the lysine at position 8 is replaced by histidine, phenylalanine or another hydrophobic amino acid; d) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least on of the histidines at position 4, 5 and 12 is replaced by phenylalanine, tyrosine, leucine or another hydrophobic amino acid; e) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the lysines at positions 2 and 10 are replaced by glutamine, arginine or by another basic amino acid; f) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 where at least one of the arginines at positions 3 and 9 is replaced by glutamine, lysine, or by another basic amino acid; and g) any combination of amino acid sequences of preceding sections a)-f) with the exception that glutamine or any other non-basic amino acid cannot simultaneously occupy positions 2, 3, 9 and 10 of the amino acid sequence, wherein the preparation has a buffer molarity of about 25 mM or less.

3. The preparation of Claim 2, wherein the pharmaceutical preparation has a buffer molarity between about 3 mM and about 20 mM.

4. The preparation of Claim 3, wherein the pharmaceutical preparation has a buffer molarity between about 10 mM and about 15 mM.

5. The preparation of Claim 4, wherein the pharmaceutical preparation has a buffer molarity of about 12.5 mM.

6. The pharmaceutical preparation of Claims 1 or 2, wherein the amino acid sequence of the peptide is selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18; b) the amino acid sequence of histatin 1 1 as set forth in SEQ ID NO: 11 ; c) the amino acid sequence of peptide 129 as set forth in SEQ ID NO: 23; d) the amino acid sequence of peptide 1 17 as set forth in SEQ ID NO: 19; e) the amino acid sequence of peptide 1 18 as set forth in SEQ ID NO: 20; f) the amino acid sequence of peptide 1 19 as set forth in SEQ ID NO: 21 ; g) the amino acid sequence of peptide 120 as set forth in SEQ ID NO: 22; h) the amino acid sequence of peptide 1 13-F4 as set forth in SEQ ID NO:24; i) the amino acid sequence of peptide 1 13-F5 as set forth in SEQ ID NO:25; j) the amino acid sequence of peptide 1 13-Fl 2 as set forth in SEQ ID NO:26; k) the amino acid sequence of peptide 113-F4.5 as set forth in SEQ ID

NO:27;

1) the amino acid sequence of peptide 113-F4.5.12 as set forth in SEQ ID

NO:28; m) the amino acid sequence of peptide 113-K6 as set forth in SEQ ID

NO:29; n) the amino acid sequence of peptide 113-H8 as set forth in SEQ ID NO:30; o) the amino acid sequence of peptide 113-K6H8 as set forth in SEQ ID

NO:31; p) the amino acid sequence of peptide 1 13-F8 as set forth in SEQ ID NO:32; q) the amino acid sequence of peptide 1 13-L4.5.12 as set forth in SEQ ID

NO:33; r) the amino acid sequence of peptide 1 13-Y4.5. 12 as set forth in SEQ ID

NO:34; s) the amino acid sequence of peptide 113-Q2. 10 as set forth in SEQ ID

NO:35; and t) the amino acid sequence of peptide 113-Q3.9 as set forth in SEQ ID

NO:36; and u) any combination of amino acid sequences of preceding sections a)-t).

7. The pharmaceutical preparation of Claims 1 or 2, wherein the peptide has a modification comprising addition of at least one substituent to either the N- terminus, the C-terminus, or both the N-terminus and the C-terminus of the peptide.

8. The pharmaceutical preparation of Claim 7, wherein the modification of the peptide is selected from the group consisting of: a) an acetyl addition at the N-terminus; b) a carbamyl addition at the N-terminus; c) an amide addition at the C-terminus; d) a combination of preceding sections a) and c); and e) a combination of preceding sections b) and c).

9. The pharmaceutical preparation of Claim 8, comprising a pharmaceutically acceptable salt of the peptide with an amide addition at the C-terminus.

10. The pharmaceutical preparation of Claims 1 or 2, wherein the peptide concentration is between about 0.001% and about 0.75%.

1 1. The pharmaceutical preparation of Claims 1 or 2, further comprising one or more adjuvants selected from the group consisting of one or more polyhydric alcohols, one or more surfactants, one or more flavoring agents, one or more chelating agents, one or more sweeteners, one or more pH adjustors, one or more coloring agents, one or more stabilizing agents, and any combinations thereof.

12. A method for treating a microbial infection such as a fungal or bacterial infection in an individual comprising administering to the individual a therapeutically effective amount of a preparation comprising a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence of at least 8 amino acids selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18; b) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 where the glycine at position 6 is replaced by lysine, arginine or another basic amino acid; c) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 where the lysine at position 8 is replaced by histidine, phenylalanine or another hydrophobic amino acid; d) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 where at least on of the histidines at position 4, 5 and 12 is replaced by phenylalanine, tyrosine, leucine or another hydrophobic amino acid; e) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the lysines at positions 2 and 10 are replaced by glutamine, arginine or by another basic amino acid; f) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 where at least one of the arginines at positions 3 and 9 is replaced by glutamine, lysine, or by another basic amino acid; and g) any combination of amino acid sequences of preceding sections a)-f) with the exception that glutamine or any other non-basic amino acid cannot simultaneously occupy positions 2, 3, 9 and 10 of the amino acid sequence, wherein the buffer molarity of the preparation is suitable to achieve greater than 99% killing of C. albicans in vitro.

13. A method for treating a microbial infection such as a fungal or bacterial infection in an individual comprising administering to the individual a therapeutically effective amount of a preparation comprising a peptide or a pharmaceutically acceptable salt thereof, wherein said peptide has an amino acid sequence of at least 8 amino acids selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18; b) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the glycine at position 6 is replaced by lysine, arginine or another basic amino acid; c) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where the lysine at position 8 is replaced by histidine, phenylalanine or another hydrophobic amino acid; d) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 where at least on of the histidines at position 4, 5 and 12 is replaced by phenylalanine, tyrosine, leucine or another hydrophobic amino acid; e) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18 where at least one of the lysines at positions 2 and 10 are replaced by glutamine, arginine or by another basic amino acid; f) the amino acid sequence of peptide P-113 as set forth in SEQ ID NO: 18 where at least one of the arginines at positions 3 and 9 is replaced by glutamine, lysine, or by another basic amino acid; and g) any combination of amino acid sequences of preceding sections a)-f) with the exception that glutamine or any other non-basic amino acid cannot simultaneously occupy positions 2, 3, 9 and 10 of the amino acid sequence, wherein the preparation has a buffer molarity of about 25 mM or less.

14. The method of Claim 13, wherein the preparation has a buffer molarity between about 3 mM and about 20 mM.

15. The method of Claim 13, wherein the preparation has a buffer molarity between about 10 mM and about 15 mM.

16. The method of Claim 13, wherein the preparation has a buffer molarity of about 12.5 mM.

17. The method of Claims 12 or 13, wherein the amino acid sequence of the peptide is selected from the group of amino acid sequences consisting of: a) the amino acid sequence of peptide P-1 13 as set forth in SEQ ID NO: 18; b) the amino acid sequence of histatin 1 1 as set forth in SEQ ID NO: 11 ; c) the amino acid sequence of peptide 129 as set forth in SEQ ID NO: 23; d) the amino acid sequence of peptide 1 17 as set forth in SEQ ID NO: 19; e) the amino acid sequence of peptide 1 18 as set forth in SEQ ID NO: 20; f) the amino acid sequence of peptide 119 as set forth in SEQ ID NO: 21; g) the amino acid sequence of peptide 120 as set forth in SEQ ID NO: 22; h) the amino acid sequence of peptide 113-F4 as set forth in SEQ ID NO:24; i) the amino acid sequence of peptide 1 13-F5 as set forth in SEQ ID NO:25; j) the amino acid sequence of peptide 1 13-F12 as set forth in SEQ ID NO:26; k) the amino acid sequence of peptide 1 13-F4.5 as set forth in SEQ ID

NO:27;

1) the amino acid sequence of peptide 1 13-F4.5.12 as set forth in SEQ ID NO:28; m) the amino acid sequence of peptide 1 13-K6 as set forth in SEQ ID

NO:29; n) the amino acid sequence of peptide 1 13-H8 as set forth in SEQ ID NO:30; o) the amino acid sequence of peptide 113-K6H8 as set forth in SEQ ID NO:31 ; p) the amino acid sequence of peptide 1 13-F8 as set forth in SEQ ID NO:32; q) the amino acid sequence of peptide 1 13-L4.5.12 as set forth in SEQ ID

NO:33; r) the amino acid sequence of peptide 113-Y4.5. 12 as set forth in SEQ ID NO:34; s) the amino acid sequence of peptide 1 13-Q2. 10 as set forth in SEQ ID NO:35; and t) the amino acid sequence of peptide 1 13-Q3.9 as set forth in SEQ ID

NO:36; and u) any combination of amino acid sequences of preceding sections a)-t).

18. The method of Claims 12 or 13, wherein the peptide has a modification comprising addition of at least one substituent to either the N-terminus, the

C-terminus, or both the N-terminus and the C-terminus of the peptide.

19. The method of Claim 18, wherein the modification of the peptide is selected from the group consisting of: a) an acetyl addition at the N-terminus; b) a carbamyl addition at the N-terminus; c) an amide addition at the C-terminus; d) a combination of preceding sections a) and c); and e) a combination of preceding sections b) and c).

20. The method of Claim 19, comprising a pharmaceutically acceptable salt of the peptide with an amide addition at the C-terminus.

21. The method of Claims 12 or 13, wherein the peptide concentration is between about 0.001% and about 0.75%.

22. The method of Claim 12 or 13, wherein the preparation further comprises one or more adjuvants selected from the group consisting of one or more polyhydric alcohols, one or more surfactants, one or more flavoring agents, one or more chelating agents, one or more sweeteners, one or more pH adjustors, one or more coloring agents, one or more stabilizing agents, and any combinations thereof.

23. The method of Claims 12 or 13, wherein the microbial infection is selected from the group consisting of: a) an infection of the oral cavity; b) an infection of the vagina; c) an infection of the urethra; d) an infection of the ear; e) an infection of the skin; f) a respiratory infection; g) a mucosal infection; h) an ophthalmic infection; i) any combinations of the preceding sections a)-h); and j) a systemic infection.

24. The method of Claims 12 or 13, wherein the fungus or bacterium is selected from the group consisting of Candida albicans; Actinomyces actinomycetemcomitans; Actinomyces viscosus; Bacteroides forsythus;

Bacteroides fragilis; Bacteroides graciclis; Bacteroides ureolyticus;

Campylobacter concisus; Campylobacter rectus; Campylobacter showae; Campylobacter sputorum; Capnocytophaga gingivalis; Capnocytophaga ochracea; Capnocytophaga sputigena; Clostridium histolyticum; Eikenella corrodens; Eubacterium nodatum; Fusobacterium nucleatum;

Fusobacterium periodonticum; Peptostreptococcus micros; Porphyromonas endodontalis; Porphyromonas gingivalis; Prevotella intermedia; Prevotella nigrescens; Propionobacterium acnes; Pseudomonas aeruginosa;

Selenomonas noxia; Staphylococcus aureus; Streptococcus constellatus;

Streptococcus gordonli; Streptococcus intermedius; Streptococcus mutans;

Streptococcus oralis; Streptococcus pneumonia; Streptococcus sanguis;

Treponoma denticola; Treponoma pectinovorum; Treponoma socranskii; Veillonella parvula; Wolinella succinogenes; Candida krusei; Candida kefyr;

Candida par apsilosis; Candida tropicalis; Cryptococcus neoformans; and

A spergillus fum igatus.

25. A mouthrinse preparation, comprising: a) between about 0.01% and about 0.2% peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is selected from the group consisting of: i) P-113 peptide; ii) modified P-113 peptide, wherein the modification comprises addition of at least one substituent to either the N-terminus, the C- terminus, or both the N-terminus and the C-terminus of the peptide; and iii) any combination of peptides of proceeding sections i)-ii); b) about 0.13% edetate disodium; c) about 0.170% sodium acetate trihydrate; d) about 0.025% sodium saccharin; e) about 0.002% FD&C Yellow #5; f) about 0.003% FD&C Red #40; g) about 0.300% polyxyl 40 hydrogenated castor oil; h) about 0.100% hydroxyethyl cellulose; i) about 0.300% orange flavoring agent; and j) about 7.000% sorbitol, wherein said mouthrinse preparation has a pH of between about 5.5 and about 7.0.

26. The mouthrinse preparation of Claim 25, wherein the concentration of the peptide is between about 0.0375% and about 0.15%.

27. The mouthrinse preparation of Claim 25, wherein the modification comprises an amide addition at the C-terminus.

28. A method for treating Candida infection of the oral cavity in an individual comprising administering to the individual a therapeutically effective amount of a mouthrinse preparation comprising: a) between about 0.01% and about 0.2% peptide or a pharmaceutically acceptable salt thereof, wherein the peptide is selected from the group consisting of: i) P-113 peptide; ii) modified P-113 peptide, wherein the modification comprises addition of at least one substituent to either the N-terminus, the C- terminus, or both the N-terminus and the C-terminus of the peptide; and iii) any combination of peptides of proceeding sections i)-ii); b) about 0.13% edetate disodium; c) about 0.170% sodium acetate trihydrate; d) about 0.025% sodium saccharin; e) about 0.002% FD&C Yellow #5; f) about 0.003% FD&C Red #40; g) about 0.300% polyxyl 40 hydrogenated castor oil; h) about 0.100% hydroxyethyl cellulose; i) about 0.300% orange flavoring agent; and j) about 7.000% sorbitol, wherein said mouthrinse preparation has a pH of between about 5.5 and about 7.0.

29. The method of Claim 28, wherein the concentration of the peptide is between about 0.0375% and about 0.15%.

30. The method of Claim 28, wherein the modification comprises an amide addition at the C-terminus.