Priority of U.S. provisional application 60/952,059 filed on 26/7/2007, which is incorporated herein by reference in its entirety.
Summary of The Invention
The present invention provides antimicrobial peptides that exhibit broad antimicrobial activity against gram-positive and gram-negative bacteria as well as fungi, molds, and certain viruses. The peptides of the invention are cationic, comprising positively charged amino acids such as lysine, arginine and histidine, and other uncharged amino acids such as glycine or glutamine. The peptides of the invention are safe, effective and sufficiently stable to allow long-term or routine use.
The present invention further provides antimicrobial compositions comprising the antimicrobial peptides. The compositions are stable and therefore can be formulated in a variety of forms, including conventionally used forms. The compositions are particularly useful for topical application to the skin, hair, nails, vagina, urethra, ear, mouth, nasal passages, respiratory system, eye, various mucosal areas and other affected areas to treat or prevent microbial infections, or to prevent recurrence of microbial infections. The composition of the invention improves the condition and/or appearance (apearance) of the treated site by reducing, inhibiting or preventing microbial infections, even for long-term or routine use.
The invention further provides kits for improving the condition or appearance of skin, nails, or other treatment areas. These kits may contain components that aid in the dispensing and administration of the compositions of the invention, and may be designed for convenient administration and long-term storage of the compositions.
Detailed Description
The HIV-TAT peptide fragments are used in combination with botulinum toxin to produce a more stable, more efficacious and safer botulinum toxin composition for therapeutic, aesthetic and/or cosmetic purposes. Compositions comprising these Botulinum Toxins are disclosed in U.S. provisional Application entitled "Compositions and Methods of Topical Application of Botulinum toxin Delivery of Botulinum toxin Stabilized treated polypeptide Fragments Derived From HIV-TAT", filed on 29.12.2006, which is incorporated herein by reference in its entirety. The inventors of the present invention have found that peptide fragments of HIV-TAT and related peptides have antimicrobial activity and are useful as active ingredients in pharmaceutical and cosmetic compositions. Such peptides are safe and effective for long term or routine use (e.g., absence of botulinum toxin).
Thus, one aspect of the invention is a cationic peptide comprising an HIV-TAT sequence or an inverted HIV-TAT sequence at the N-or C-terminus, or at both the N-and C-terminus. For example, the antimicrobial peptide may have an HIV-TAT sequence at the N-or C-terminus, or both the N-and C-terminus, such as Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO: 1), or an inverted HIV TAT sequence, such as Arg-Arg-Arg-Gln-Arg-Arg-Lys-Lys-Arg (SEQ ID NO: 2).
In one embodiment, the cationic peptide comprises an N-terminal portion (the N-terminal portion being an HIV-TAT or reverse HIV-TAT sequence), a C-terminal portion (the C-terminal portion being an HIV-TAT or reverse HIV-TAT sequence), and one or more cationic residues (e.g., Lys or Arg) between the N-terminal portion and the C-terminal portion. For example, the peptide may have 5 to 20 cationic residues (e.g., Lys) between the N-terminal portion and the C-terminal portion, e.g., about 12, about 15, or about 17 cationic residues.
In an exemplary embodiment, the cationic peptide of the invention has the following sequence: Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Gly- (Lys) n-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO: 3), or Arg-Arg-Gln-Arg-Arg-Lys-Arg-Gly- (Lys) n-Gly-Arg-Arg-Arg-Gln-Arg-Arg-Lys-Arg-Gly- (Lys) (SEQ ID NO: 4), wherein n is from about 5 to about 20, for example from about 10 to about 20.
In one embodiment of the invention, the N-terminal part of the peptide is an HIV-TAT sequence and the C-terminal part of the peptide is an HIV-TAT sequence. In another embodiment, the N-terminal portion is a reverse HIV-TAT sequence and the C-terminal portion is a reverse HIV-TAT sequence. For example, the antimicrobial peptide may have the following amino acid sequence: Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Gly- (Lys)15-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO: 5).
In certain embodiments, the peptide comprises a naturally occurring amino acid sequence, such as an HIV-TAT sequence, but may alternatively comprise modifications of the naturally occurring sequence to enhance its potency. Antimicrobial peptides with improved efficacy can be identified using the assays described or exemplified herein. In certain embodiments, the peptides of the invention are readily biodegradable.
In general, antimicrobial derivatives of the HIV-TAT and reverse HIV-TAT sequences contemplated by the present invention are characterized by having particularly high levels of Arg and Lys residues. For example, a peptide of the invention may comprise at least about 50% of the total Arg and Lys amino acid residues, may comprise at least about 75%, or at least about 80% Arg and Lys residues. In these or other embodiments, such derivatives may have the amino acid sequence of SEQ ID NO: 3 or 4, and having 1 to 5 amino acid substitutions, insertions or deletions (in total), including substitutions to the amino acid sequence of SEQ ID NO: 3 or 4, 1, 2, 3 or 4 amino acid substitutions, insertions or deletions. In certain embodiments, such substitutions, insertions, or deletions are within the HIV-TAT or reverse HIV-TAT sequence.
The antimicrobial peptides of the present invention have a length of about 15 amino acids to about 100 amino acids. In certain embodiments, the cationic peptide is from about 25 to about 50, or from about 25 to about 40 amino acids in length. In an exemplary embodiment of the invention, the antimicrobial peptide is about 35 amino acids in length.
The present invention is effective in inhibiting, killing and/or lysing a wide variety of microorganisms, and can have a broad antimicrobial spectrum. Exemplary bacteria that may be inhibited or killed by the present invention include gram-negative and gram-positive bacteria such as: staphylococcus aureus (Staphylococcus aureus), Streptococcus pyogenes (Streptococcus group) (group a), Streptococcus species (Streptococcus spp.) (viridome), Streptococcus agalactiae (Streptococcus agalactiae) (group B), Streptococcus bovis (Streptococcus bovis), Streptococcus (anaerobic species), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus mutans (Streptococcus mutans), Enterococcus species (Enterococcus spp.), Neisseria gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), trichoderma mucosae (branhamellaralis), Bacillus (Bacillus subtilis), Bacillus subtilis (Clostridium difficile), Clostridium difficile (Clostridium difficile), Clostridium (Clostridium difficile), Streptococcus pneumoniae (Clostridium sp.), Streptococcus agalactiae (Clostridium difficile), Streptococcus lactis (Streptococcus pneumoniae), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus (Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus (Streptococcus pneumoniae), Streptococcus (Streptococcus pneumoniae), Streptococcus (, proteus mirabilis (Proteusmirabilis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Klebsiella pneumoniae (Klebsiella pneumoniae), Salmonella species (Salmonella spp.), Shigella species (Shigella spp.), Campylobacter jejuni (Campylobacter jejuni), Actinobacillus actinomycetemcomitans (Actinobacillus comatus), Porphyromonas gingivalis (Porphyromonas gingivalis), Bacteroides fusciparum (Bacteriodes forCyticus), Treponema dentis (Treponema pallidus), Prevotella intermedia (Prevotella intermedia), and Eubacterium nodatum (Bacillus nodatus).
Thus, the antimicrobial peptides of the invention may be used to inhibit such bacteria to ameliorate microbial infection or reduce the likelihood of microbial infection, including: bacteremia, pneumonia, meningitis, osteomyelitis, endocarditis, caries, periodontal disease, sinusitis, rhinitis, pink eye, urinary tract infection, tetanus, gangrene, colitis, acute gastroenteritis, impetigo, acne, acneposacue, wound infection, burn infection, fasciitis, bronchitis and various abscesses, hospital infection, and opportunistic infection.
The antimicrobial peptide of the present invention is also effective in inhibiting the growth and survival of fungi such as dermatophytes (e.g., Microsporum spp.) such as Microsporum canis (Microsporum canis), Microsporum austenitis (Microsporum audouini), and Microsporum gypseum (Microsporum gypseum), and Trichophyton spp (Trichophyton spp.) such as Trichophyton rubrum (t.rubrum), Trichophyton mentagrophytes (t.mentagrophytes), t.trichophyton, Trichophyton schoenophytum (t.schoenleinii) and Trichophyton sechii (t.tonsurnosuroides)), acremon spp (Fusarium oxysporum), scoparia glaucopias (scoparia), Trichophyton (t.tonsuroides), Trichophyton sechiza (t.tonsuroides), Trichophyton sechii (t.e), Trichophyton sp.sp.; yeasts (e.g. Candida albicans, Candida tropicalis, or other Candida species, and Saccharomyces cerevisiae), Torulopsis glabrata (Torulopsis glabrata), Epidermophyton floccosum (Epidermophyton florum), Malassezia furfur (Malassezia furur), Pityropson orbiculare or ovale, Cryptococcus neoformans (Cryptococcus neoformans), Aspergillus fumigatus (Aspergillus fumigatus), Aspergillus nidulans (Aspergillus nidulanus), Aspergillus niger (Aspergillus niger) and other Aspergillus species of the genus (Aspergillus spp.), Zygomyces conjugalis (e.g. Rhizopus and Mucor), Paracoccus neospora (Paracoccus bractensis), Candida albicans (Bacillus sporogenes), Candida tropicalis (Bacillus sporogenes), and Bacillus sporogenes (Bacillus sporogenes), and Bacillus sporogenes (Bacillus sporogenes, Sphings).
Thus, antimicrobial peptides can be used to inhibit fungi to treat or prevent infections such as: aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, Paracoccidiomycosis (Paracoccidiomycosis), sporotrichosis, zygomycosis. In certain embodiments of the invention, the antimicrobial peptides are effective in treating or preventing tinea pedis (Tineapedis), tinea versicolor, and Onychomycosis (onymus). Other fungal infections against which the antimicrobial peptides of the invention are effective include: tinea barbae, Lobomycosis (Lobomycosis), bystosis, trichosporosis, pityriasis versicolor, tinea capitis, tinea corporis, tinea cruris, tinea flavum, tinea nigra, otomycosis, hyphomycosis and nosema.
The invention is also effective against certain viruses such as HIV, herpes simplex virus, cytomegalovirus, and human papilloma virus. Thus, the antimicrobial peptides of the present invention are also effective in preventing and treating infections (e.g., cold sores, genital herpes and warts), and in preventing HIV infection.
The present invention provides a pharmaceutical or cosmetic composition comprising an antimicrobial peptide of the invention together with a pharmaceutically or cosmetically acceptable carrier and/or diluent. Typically, the Compositions of the present invention do not contain a botulinum toxin, as disclosed in U.S. provisional Application entitled "Compositions And methods of Topical Application And Transdermal Delivery of Botulinum toxin Stabilized With Polypeptide Fragments derived from HIV-TAT", filed on 29.12.2006.
The compositions of the invention may comprise, consist of, or consist essentially of an antimicrobial peptide of the invention in an amount effective for antimicrobial activity and a carrier and/or diluent. In this regard, the term "consisting essentially of. For example, when formulated for topical administration, the compositions of the present invention may optionally comprise benzoyl peroxide, clindamycin, erythromycin, tetracycline, bacitracin, neomycin, mupirocin, polymyxin B, miconazole, and/or clotrimazole. In certain embodiments, the carrier and/or diluent is an aqueous carrier or diluent, such as a buffered saline solution.
The pharmaceutical or cosmetic compositions of the present invention may be formulated for topical administration, which may be a welcome alternative to systemic treatments for treating or preventing many microbial infections. In certain embodiments, topical treatment with the compositions of the present invention is administered in conjunction with systemic or other topical treatment to provide an additive or synergistic effect. For example, the antimicrobial peptides of the present invention may act synergistically with one or more of benzoyl peroxide, clindamycin, erythromycin, tetracycline, bacitracin, neomycin, mupirocin, polymyxin B, miconazole, clotrimazole, and/or equivalent antimicrobial agents.
Treatment with the compositions of the present invention can not only eliminate various microbial infections, but also effectively prevent the initial occurrence of such infections and prevent the recurrence of such infections. In the latter aspect, the present compositions are administered to the affected area after local or systemic treatment. To prevent recurrence of an effectively treated infection, or to prevent initial onset of infection in a susceptible area, the compositions may be administered routinely (e.g., daily) for a period of time, including days, weeks, or even years.
When formulated for topical administration, the compositions of the present invention may comprise ingredients commonly used in topical pharmaceutical or cosmetic compositions, such as carriers, vehicles, or vehicles. In particular, the carrier, vehicle, or medium is compatible with the tissue to which it is applied, e.g., skin, hair, nails, vagina, urethra, ear, mouth, nasal passages, respiratory system, eye, and/or mucous membranes. The compositions and components of the present invention are suitable for contact with infected tissue or use in patients, and are generally not overly toxic, incompatible, unstable, allergic, and the like. The compositions of the present invention may comprise any ingredient generally contemplated for use in the art, as appropriate.
In their form, the compositions of the present invention may include solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders, or other solid or liquid compositions for application to the skin and other tissues to which they may be applied. Such compositions may comprise: additional antimicrobial agents, moisturizers and hydrating agents, penetrants, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents, emollients (emollients), antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, colorants, powders, viscosity modifiers and water, and optionally anesthetics, antipruritic actives, botanical extracts, conditioners (conditioning agents), deepening or whitening agents (darker lightening agents), glitter, moisturizers, micas, minerals, polyphenols, silicones or derivatives thereof, sunscreens, vitamins and phytomedicinals. In certain embodiments, the compositions of the present invention are formulated with the above-described ingredients to render them stable for long periods of time, which may be beneficial when sustained or long-term treatment is desired.
The compositions of the present invention may be in the form of controlled or sustained release compositions in which the antimicrobial peptides are encapsulated or contained within a material with additional active agents so that they are released in a controlled manner over time onto the skin or affected area. The compositions of the present invention may be contained within or on a matrix, liposome, vesicle, microcapsule, microsphere, or the like, or within or on a solid particulate material.
The compositions of the invention may be applied to any affected or susceptible area, for example, to the legs, shoulders, back (including the waist), axilla, palm, foot, neck, groin, back or hands or feet, elbow, upper arm, knee, thigh, hip, trunk, pelvis, or any other part of the body for which treatment or prevention of infection may be desired. Such treatments are also contemplated for use in treating and/or bandaging wounds (e.g., cuts, abrasions, and burns of the skin) to treat or prevent infection of the injured area.
The compositions of the present invention are suitable for use in physiological environments having a pH of about 4.5 to about 6.3, and thus, the compositions may be formulated at similar or identical pH. The compositions according to the invention can be stored at room temperature or under refrigerated conditions.
The compositions of the present invention comprise an antimicrobial peptide in an amount effective for antimicrobial action. Typically, the composition comprises about 0.01% (wt./vol.) to about 20% antimicrobial peptide. In certain embodiments, the composition comprises from about 0.5% to about 10% antimicrobial peptide, for example about 0.5%, about 1%, about 5%, or about 10% antimicrobial peptide.
In one embodiment, the pharmaceutical composition of the invention is a cleanser or moisturizer. In particular, the cleansers and moisturizers of the present invention are useful for conditioning the skin when treating and/or preventing microbial infections, such as acne, including Propionibacterium acne. This aspect of the invention is also effective in preventing the recurrence of acne after the acne has effectively resolved. In this regard, the cleansing or moisturizing agent may be applied to the affected area on a regular basis (e.g., on an approximately daily basis), and on a continuous or regular basis. For example, treatment may last for days, weeks, months or even years.
In another embodiment, the composition of the present invention is a topical solution effective against Athlete's foot (tinea pedis) and/or onychomycosis. In this regard, the composition may be an aqueous solution for soaking or painting the affected area. In this embodiment, the present invention is also effective in preventing the recurrence of the condition by continued daily treatment. Furthermore, when used to treat onychomycosis, the compositions of the invention may be used in combination with systemic treatments such asTogether to provide a more effective action and improve the condition and/or appearance of the nail.
In other embodiments, the compositions of the present invention are formulated as a mouthwash, oral spray, or oral gel. In this regard, the mouth rinse or oral spray of the present invention is effective in preventing dental caries, treating and/or preventing periodontal disease, treating sore throat, or treating or preventing halitosis that may be caused by the presence or action of microorganisms. When formulated as an oral gel, the compositions of the present invention are effective in treating and preventing ulcers (e.g., cold sores and mouth ulcers) around the mouth. When formulated as a mouthwash, oral spray, or oral gel, the composition typically comprises from about 0.01% to about 10% antimicrobial peptide, for example about 1%, about 2%, about 3%, or about 5% antimicrobial peptide.
In another embodiment, the present invention is formulated as a cream or lotion. In particular, such formulations are useful for the treatment and/or prevention of herpes simplex virus infection or outbreak. Thus, in certain embodiments, the present invention is suitable for treating genital herpes, cold sores, or chickenpox.
The composition of the present invention may also be formulated as a nasal spray effective against rhinitis or as eye drops effective against red eye.
In another aspect, the present invention provides methods for treating or preventing a condition associated with a microbial infection. The method of the invention comprises administering a pharmaceutical or cosmetic composition of the invention comprising an antimicrobial peptide to a patient having or suspected of having a microbial infection. In certain embodiments, the composition is administered to the patient after treatment of the infection with the composition or replacement therapy of the present invention to prevent further relapse. The patient may be any veterinary or human patient.
The compositions of the present invention may be used for the treatment and/or prevention of local and systemic bacterial, fungal and viral infections via topical routes or systemic infections, affecting the skin, hair, nails, vagina, urethra, ear, oral cavity, respiratory system, eye, nose and various mucosal areas, by various routes (e.g. intravenous or subcutaneous). Such diseases may be, for example, acne (including propionibacterium acne), onychomycosis, tinea pedis, genital herpes, dental caries, periodontal disease, rhinitis and redness of the eye. In exemplary embodiments, the infection is an infection with staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, candida albicans, aspergillus niger, and/or herpes simplex virus.
To treat or prevent microbial infections or to prevent recurrence of microbial infections, the antimicrobial peptide may be administered at least once daily for at least about one week. Optionally, the composition is administered at least twice daily for at least two days. In certain embodiments, the composition is administered about once daily, at least once daily, twice weekly, or for about one month. In certain embodiments, the compositions of the present invention are administered for months, e.g., at least two months, six months, or about 1 year or more. The invention is further suitable for long term use, in particular it may be beneficial to prevent recurrent infections or to prevent infections or pathologies in patients at risk or susceptible (including immunocompromised patients). The long-term use may include at least two years, three years, four years, or even five or more years of treatment.
In another aspect, the compositions of the invention are kits comprising a peptide or composition of the invention packaged to facilitate distribution and/or administration of the composition to an affected or susceptible area. The package or dispenser may include a bottle, tube, spray bottle or other dispenser. In certain embodiments of the invention, the composition is packaged in concentrated form and diluted to the desired concentration at the time of use by the end user. In this or other aspects, the compositions are formulated and packaged in a form suitable for long-term storage to maintain the efficacy of the composition.
The kit may further include additional components (e.g., a brush, sponge, cotton swab, etc.) to facilitate application of the composition to the affected area.
Examples
Example 1 Effect of peptides on conditions characterized by microbial infection
The following experiments were designed to test the effect of the diluted formulations; however, it has surprisingly been found that cationic peptides are themselves effective antimicrobial agents. The peptides of the invention are found to be not only safe for long-term use, but also effective in the treatment and prevention of a variety of conditions associated with microbial infection (including prevention of recurrence thereof). Based on these results, the effect of the peptides on microbial growth was tested in example 2.
Example 1.1
A15 year old male with a two year history of acne disease was given a 5% peptide (SEQ ID NO: 5) formulation topically twice daily for 4 weeks. Cream formulations consist of the excipients cetyl alcohol, isopropyl myristate, stearyl alcohol, butylated hydroxytoluene (butylated hydroxytoluene), polyoxyethylene stearyl ether, edetate disodium and purified water. With the topical formulation, acne disappeared within two weeks. No new attacks or adverse events were observed with the formulation for two years.
Example 1.2
A36 year old female with tinea pedis (tinea pedis) (manifested by irritation between toes, itchy rash, and exudation) bathed their foot in a 5% peptide (SEQ ID NO: 5) formulation for 10 minutes prior to sleep and topically administered 1% peptide formulation twice daily to the affected area and the rest of the foot. Cream formulations consisted of excipients cetyl alcohol, isopropyl myristate, stearyl alcohol, butylated hydroxytoluene, polyoxyethylene stearyl ether, edetate disodium and purified water. At week 1, itching decreased and exudation ceased. By week 4, the scab of the skin was sloughed off and there was no evidence of abnormal skin at the feet. She continued to use the 0.05% peptide formulation for 5 years with no further seizures or adverse events.
Example 1.3
After laboratory confirmation of fungal infestation (based on microscopic examination and culture of shaved debris or clippings from the nails), 78 year old men were diagnosed with onychomycosis. He began painting the nail and surrounding nail bed twice daily with a 5% peptide (SEQ ID NO: 5) formulation. Cream formulations consisted of excipients cetyl alcohol, isopropyl myristate, stearyl alcohol, butylated hydroxytoluene, polyoxyethylene stearyl ether, edetate disodium and purified water. After 12 weeks, all the emerging nails had normal texture and color. He continued to use the peptide formulation for two years, while his nails were all free of fungal infection.
Example 1.4
After laboratory confirmation of fungal infestation (based on microscopic examination and culture of shaved debris or clippings from the nails), 65 year old men were diagnosed with onychomycosis. He started oral treatment for 1 month. He then started to paint the nail and surrounding nail bed once a day with a 10% peptide (SEQ ID NO: 5) formulation. Cream formulations consisted of excipients cetyl alcohol, isopropyl myristate, stearyl alcohol, butylated hydroxytoluene, polyoxyethylene stearyl ether, edetate disodium and purified water. After 12 weeks, all nails grew with normal structure and color. He continued to use the peptide formulation for two years, while all of his nails were free of fungal infection and there was no recurrence of additional fungal infection in the toenails.
Example 1.5
A10 year old boy with a two-year history of caries used a peptide (SEQ ID NO: 5) mouthwash for two weeks. The preparation consists of physiological saline and 1-5% of peptide. His symptoms were significantly reduced. He continued to use the peptide mouthwash for two years and no new cavities were observed.
Example 1.6
A36 year old female with an outbreak of cold sores in the mouth is administered a 1% peptide (SEQ ID NO: 5) formulation three times a day. The hydrogel formulation comprised 30% poloxamer. All cold sores disappeared within two days.
Example 1.7
A24 year old female with genital herpes applied 1% of the peptide (SEQ ID NO: 5) twice daily to the affected area for one week. Cream formulations consisted of excipients cetyl alcohol, isopropyl myristate, stearyl alcohol, butylated hydroxytoluene, polyoxyethylene stearyl ether, edetate disodium and purified water. At the end of the treatment period, all symptoms disappeared.
Example 1.8
A18 year old female with a three year history of rhinitis uses a nasal spray comprising a peptide of the invention (SEQ ID NO: 5) three times a day for one week. The preparation comprises physiological saline and 1-5% peptide. She immediately felt a reduction in symptoms (e.g., itching, sneezing, and nasal congestion).
Example 1.9
An 8-year-old boy with red eyes was administered a peptide (SEQ ID NO: 5) preparation consisting of physiological saline and 1-5% of peptide, and two days later, the symptoms disappeared.
Example 2 in vitro antimicrobial Activity
Exemplary peptides of the invention were used in a panel of gram-negative and gram-positive bacteria and fungi to test for antimicrobial activity. The sequence of the exemplary peptide is Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly- (Lys)15-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO: 5) and which has been formulated in 15% poloxamer 0.9% saline. Peptide concentrations were 3.3mg/ml, 11. mu.g/ml, and 11ng/ml, respectively. Antimicrobial activity is shown in tables 1-3. The peptides showed strong antimicrobial activity in a dose-dependent manner. Notably, pseudomonas aeruginosa, a gram-negative bacterium known for its resistance to antibiotics, can be inhibited with 0.33% of the peptides of the invention (table 1).
TABLE 1.3.3mg/ml (0.33% peptide) antimicrobial Activity
| Biological organisms | Initial counting | 14 days (CFU/ml) | 28 days (CFU/ml) |
| Staphylococcus aureus ATCC 6538 | 7.4x105 | 1253.8 logarithmic reduction | No increase of < 10 |
| Pseudomonas aeruginosa ATCC 9027 | 1.1x106 | Log reduction < 100 > 4.0 | < 100 No increase |
| Escherichia coli ATCC 8739 | 5.5x105 | < 10 > 4.7 log reduction | No increase of < 10 |
| Candida albicans ATCC 10231 | 1.0x105 | < 10 YunWith the increase of | No increase of < 10 |
| Aspergillus niger ATCC 16404 | 3.0x105 | 3.1x105Without increasing | 3.0x105Without increasing |
TABLE 2.11. mu.g/ml (0.0011% peptide) antimicrobial Activity
| Biological organisms | Initial counting | 14 days (CFU/ml) | 28 days (CFU/ml) |
| Staphylococcus aureus ATCC 6538 | 7.0x105 | < 10 > 4.8 log reduction | No increase of < 10 |
| Pseudomonas aeruginosa ATCC 9027 | 1.2x106 | 4.3x1060.6 logarithmic increase | 1.7x1070.6 logarithmic increase |
| Escherichia coli ATCC 8739 | 6.9x105 | 1.7x1032.6 log reduction | No increase of < 10 |
| Candida albicans ATCC 10231 | 1.6x105 | 2.1x103Without increasing | 1.0x103Without increasing |
| Aspergillus niger ATCC 16404 | 4.6x105 | 2.5x105Without increasing | 2.2x105Without increasing |
TABLE 3.11ng/ml (0.0000011% peptide) antimicrobial activity
| Biological organisms | Initial counting | 14 days (CFU/ml) | 28 days (CFU/ml) |
| Staphylococcus aureus ATCC 6538 | 7.0x105 | < 10 > 4.8 log reduction | No increase of < 10 |
| Pseudomonas aeruginosa ATCC 9027 | 1.2x106 | 3.9x1060.5 logarithmic increase | 5.6x106Without increasing |
| Escherichia coli ATCC 8739 | 6.9x105 | 4.0x1041.2 logarithmic reduction | 860 without increase |
| Candida albicans ATCC 10231 | 1.6x105 | 3.8x104Without increasing | 3.1x104Without increasing |
| Aspergillus niger ATCC 16404 | 4.6x105 | 2.9x105Without increasing | 2.6x105Without increasing |
<120> antimicrobial peptides, compositions and methods of use
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<151>2007-07-26
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