WO2004058798A2 - Antimicrobial peptides - Google Patents

Abstract

Brevispirin peptides are small antimicrobial agents with potent activity against microbes. A pharmaceutical composition comprising brevispirin as an active agent is administered therapeutically to a patient suffering from a microbial infection, or to an individual facing exposure to a microbial infection.

Description

ANTIMICROBIAL PEPTIDES INTRODUCTION

[01] Antimicrobial peptides are effector molecules of the innate immune system with microbicidal and proinflammatory activities. Natural polycationic antimicrobial peptides have been found in many different species of animals and insects and shown to have broad antimicrobial activity. In mammals, these antimicrobial peptides are represented by two families, the defensins and the cathelicidins. Nearly all of these peptides have membrane affinity, and can permeate and permeabilize bacterial membranes, resulting in injury, lysis, and/or death to the microbes.

[02] While defensins have a complex, largely β-sheet structure; the cathelicidin LL-37/hCAP-

18 is a 37 residue peptide with a simple α-helical structure. LL-37 lacks cysteine, making it different from all other previously isolated human peptide antibiotics of the defensin family, each of which contain 3 disulfide bridges. Like several other genes expressed late in polymorphonuclear leukocyte development, the LL-37 gene does not contain typical TATA box or CCAAT sequences. It is encoded by a compact gene of 1 ,963 bp with 4 exons. Exons 1-3 encode for a signal sequence and the cathelin region. Exon 4 contains the information for the mature antibacterial peptide. Potential binding sites for acute-phase-response factors were identified in the promoter and in intron 2.

[03] Proteolytic cleavage of cathelicidin proforms is required to free the antibacterial activity of the C-terminal peptide LL-37/hCAP-18. The uncleaved proform is devoid of antibacterial activity, although at sub-nanomolar concentrations, it is capable of blocking the bioactivity of endotoxins.

[04] LL-37/hCAP-18 is expressed in leukocytes such as neutrophils, monocytes, NK cells, γδ

T cells, and B cells, and in epithelial cells of the testis, skin, the gastrointestinal tract, and the respiratory tract. It is secreted into wound and airway surface fluid. It is chemotactic for neutrophils, monocytes, mast cells, and T cells; induces degranulation of mast cells; alters transcriptional responses in macrophages; and stimulates wound vascularization and re- epithelialization of healing skin.

[05] LL-37 is of particular interest for therapeutic purposes because it has a broad spectrum of antimicrobial activity, and it retains activity in the presence of normal or elevated concentrations of NaCI, which is of interest for the treatment of infections associated with cystic fibrosis. LL-37 is also well tolerated and active in the vaginal environment, and is tolerated at high levels by skin cells (keratinocytes) after they have been injured or exposed to bacterial or bacterial products.

[06] A main drawback to the use of LL-37 as a therapeutic is that it contains 37 amino acids, which makes it expensive to synthesize and obtain in high purity. Improved peptides are of great clinical interest to meet the clinical need for novel antiviral and antimicrobial agents that have low toxicity against mammalian cells. The present invention addresses this need.

Relevant literature [07] Endogenous antimicrobials are reviewed in Schonwetter et al. (1995) Science 267: 1645-

1648; Schroder (1999)Ce// Mol Life Sci. 56:32^6 (1999); and Harwig et al. (1994) FEBS Lett

342:281-285. U.S. Patent no. 6,040,291 is directed to an 18 amino acid peptide with antimicrobial activity. The human CAP18 peptide is described by Larrick et al. (1995) Infect.

Imm. 63(4): 1291 -1297. [08] Characterization of human cathelicidin may be found in Agerberth et al. (1995). Proc.

Nat. Acad. Sci. 92: 195-199; and Larrick et al. (1996) FEBS Lett. 398: 74-80. [09] Processing of the cathelin precursor to the antibacterial peptide LL-37 is described by

Gudmundsson etal. (1996) Europ. J. Biochem. 238: 325-332; and Sorensen etal. (2001) Blood

97: 3951-3959. [10] The biological activities of LL-37 are discussed by Nagaoka etal. (2001) J. Immun. 167:

3329-3338; Niyonsaba etal. (2002) Immunology 106: 20-26; and Nizetef al. (2001) Nature414:

454-457.

SUMMARY OF THE INVENTION

[11] Methods and compositions are provided for the use of brevispirins, which are short peptides derived from cathelicidins. These peptides have a broad-spectrum bactericidal activity, and have enhanced activity against certain pathogens. A pharmaceutical composition comprising a brevispirin; or a cocktail comprising a plurality of brevispirins as an active agent is administered to a patient suffering from a microbial infection. Alternatively, a pharmaceutical composition comprising brevispirin(s) is administered as a protective agent to an individual facing potential exposure to pathogenic microbes. Brevispirin(s) may be administered alone, or in combination with other bacteriocidal agents, e.g. antibiotics and/or other antiviral agents, etc.

[12] Brevispirins also have a high affinity for endotoxin, and find use as affinity reagents for the separation of endotoxin from compositions. Affinity chromatography may be used as a method of separating endotoxin from a composition, e.g. a pharmaceutical composition, using biochemical affinity of endotoxin and brevispirins. Methods known in the art include columns, gels, capillaries, etc.

BRIEF DESCRIPTION OF THE DRAWINGS [13] Fig. 1. Effects of human LL-37 and WS22-N-amide on T. pallidum viability. Suspensions containing 10⁷ treponemes per ml were incubated at 35 C for 2 h with various concentrations of LL-37 or WS22-N-amide in either Treponema pallidum-conditioned medium (TpCM) or in TpCM diluted 1 :3 with distilled water low salt). The samples were then scored for motility (viability) and fluorescence. Experiments were repeated three times.

[14] Fig. 2. Kinetics of human LL-37 and the WS22-N-amide peptide on T. pallidum viability.

Suspensions containing 10 treponemes per ml were incubated at 35° C for 2 h in TpCM with either 22.1 or 44.2 μM LL-37; with 2.4 or 4.8 μM WS22-N-amide peptide; or without peptide, control. At various times, samples were removed and then scored for fluorescence using flow cytometry.

[15] Fig. 3 shows the LPS binding properties of brevispirins.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS [16] Novel compositions and methods are provided for the use of brevispirins as therapeutic and/or prophylactic agents. The peptides are effective at killing a variety of microbial organisms, and are active at high salt concentration. Brevispirin(s) are administered alone or in combination with other active agents to a patient suffering from an infection in a dose and for a period of time sufficient to reduce the patient population of pathogenic microbes. Alternatively, a pharmaceutical composition comprising brevispirin is administered as a protective agent to a individual facing potential exposure to pathogenic microbes. [17] Specific treatments of interest include, without limitation: using a brevispirin to prevent or treat infection, for example aerosol administration to the lungs of patients with cystic fibrosis to combat infection or forestall the emergence of resistance to other inhaled antibiotics; instillation into the urinary bladder of patients with indwelling catheters to prevent infection; application to the skin of patients with serious burns; opthalmic instillation, directly or in ophthalmic solutions, to treat or prevent infection; intravaginal application to treat bacterial vaginosis and/or prevent sexually transmitted disease such as syphilis; oral application to treat or prevent infections of the teeth, gums or mucous membranes. Brevispirins may be administered alone or in conjunction with other antimicrobial therapy.

ANTIMICROBIAL COMPOSITIONS

[18] For use in the subject methods, a brevispirin is a peptide of at least about 14 amino acids in length, more usually at least about 15 amino acids in length, and not more than about 17 amino acids in length, preferably of 16 amino acids. The carboxy terminus pf the peptide may be in the form of a free acid, or an amide, preferably an amide.

[19] Brevispirins comprise an amino acid sequence as follows:

[20] As set forth above, amino acid residues 1 , 4, 8, 11 , 12, 15 and 16 may be any hydrophobic amino acid, including Y, A, V, L, I, and W. Preferred are I, L, F and V. Amino acid residues 2 and 9 may be K, R or H. Amino acid residues 3 and 7 may be R, K or H. Amino acid residues 6, 10 and 14 may be Q or N.

[21] In one embodiment of the invention, the brevispirin I comprises the amino acid sequence

(SEQ ID NO:1) VLNRLFNKIRQVIRKF, or (SEQ ID NO:2) VLNRLFDKIRQVIRKF; which peptides may be in the acid or amide form.

In one embodiment of the invention, the sequence of SEQ ID NO:1 or SEQ ID NO:2 comprises one, two three or four histidine substitutions at a position independently selected from residue 2, 3, 7, 9. This histidine substitution provides for a pH sensitive trigger, allowing the peptide to be activated under acidic conditions.

[22] The sequence of the brevispirin polypeptide may be altered from those provided herein in various ways known in the art to generate targeted changes in sequence. The altered peptide will usually be substantially similar to the sequences provided herein, i.e. will differ by one amino acid, and may differ by two or more amino acids. The sequence changes may be substitutions, insertions or deletions.

[23] The brevispirin peptide may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes. Various post-translational modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide farnesylation or prenylation. In this situation, the peptide will be bound to a lipid group at a terminus, so as to be able to be bound to a lipid membrane, such as a liposome.

[24] Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acetylation, orcarboxylation. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

[25] Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids.

[26] The subject peptides may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, CA, Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

[27] If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[28] The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.

[29] In one embodiment of the invention, the antimicrobial peptide consists essentially of a polypeptide sequence set forth herein. By "consisting essentially of in the context of a polypeptide described herein, it is meant that the polypeptide is composed of the sequence set forth in the seqlist, which sequence may be flanked by one or more amino acid or other residues that do not materially affect the basic characteristic(s) of the polypeptide.

BREVISPIRIN CODING SEQUENCES

[30] The invention includes nucleic acids encoding brevispirins; and fragments and derivatives thereof. Other nucleic acid compositions contemplated by and within the scope of the present invention will be readily apparent to one of ordinary skill in the art when provided with the disclosure here.

[31] Brevispirin coding sequences can be generated by methods known in the art, e.g. by in vitro synthesis, recombinant methods, etc. to provide a nucleotide sequence that encodes a brevispirin polypeptide. Using the known genetic code, one can produce a suitable coding sequence. Nucleic acids of the invention include coding of truncated forms of cathelicidins/ and variants thereof, as well as synthetically generated coding sequences. Variants include naturally occurring variants of the nucleotide sequences (e.g., degenerate variants, allelic variants, etc.). Variants of the nucleic acids of the invention are identified by hybridization of putative variants with nucleotide sequences disclosed herein, preferably by hybridization under stringent conditions. For example, by using appropriate wash conditions, variants of the nucleic acids of the invention can be identified where the allelic variant exhibits at most about 25-30% base pair (bp) mismatches relative to the selected nucleic acid probe. In general, allelic variants contain 15-25% bp mismatches, and can contain as little as even 5-15%, or 2-5%, or 1-2% bp mismatches, as well as a single bp mismatch.

[32] The invention also encompasses homologs corresponding to the nucleic acids of human cathelicidin (LL-37), where the source of homologous genes can be any mammalian species, e.g., primate species, particularly human; rodents, such as rats; canines, felines, bovines, ovines, equines, fish, yeast, nematodes, etc. Between mammalian species, e.g., human and mouse, homologs generally have substantial sequence similarity, e.g., at least 75% sequence identity, usually at least 90%, more usually at least 95% between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 contiguous nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as gapped BLAST, described in Altschul et al. Nucl. Acids Res. (1997) 25:3389-3402.

[33] The nucleic acid compositions of the subject invention can encode all or a part of the subject polypeptides. Double or single stranded fragments can be obtained from the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. Isolated nucleic acids and nucleic acid fragments of the invention comprise at least about 18, about 50, about 100 contiguous nt selected from the nucleic acid sequence encoding a brevispirin polypeptide. For the most part, fragments will be of at least 18 nt, usually at least 25 nt, and up to at least about 50 contiguous nt in length or more.

[34] Probes specific to the nucleic acid of the invention can be generated using the disclosed nucleic acid sequence, or a DNA encoding a brevispirin polypeptide. The probes are preferably at least about 18 nt, 25 nt or more of the corresponding contiguous sequence. The probes can be synthesized chemically or can be generated from longer nucleic acids using restriction enzymes. The probes can be labeled, for example, with a radioactive, biotinylated, or fluorescent tag. Preferably, probes are designed based upon an identifying sequence of one of the provided sequences. More preferably, probes are designed based on a contiguous sequence of one of the subject nucleic acids that remain unmasked following application of a masking program for masking low complexity (e.g., BLASTX) to the sequence, i.e., one would select an unmasked region, as indicated by the nucleic acids outside the poly-n stretches of the masked sequence produced by the masking program.

[35] The nucleic acids of the invention are isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the nucleic acids, either as DNA or RNA, will be obtained substantially free of other naturally-occurring nucleic acid sequences, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant," e.g., flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

[36] Brevispirin encoding nucleic acids can be provided as a linear molecule or within a circular molecule, and can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences. Expression of the nucleic acids can be regulated by their own or by other regulatory sequences known in the art. The nucleic acids of the invention can be introduced into suitable host cells using a variety of techniques available in the art, such as transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and the like.

[37] Expression vectors may be used to introduce a brevispirin coding sequence into a cell.

Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks.

[38] The gene or brevispirin peptide may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth er al. (1992) Anal Biochem 205:365-368. The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang er al. (1992) Nature 356:152-154), where gold microprojectiles are coated with the peptide, then bombarded into skin cells.

METHODS OF USE [39] Formulations of brevispirins are administered to a host suffering from an ongoing microbial infection or who faces exposure to a microbial infection. Administration may be topical, localized or systemic, depending on the specific microorganism. Generally the dosage will be sufficient to decrease the microbial population by at least about 50%, usually by at least 1 log, and may be by 2 or more logs. The compounds of the present invention are administered at a dosage that reduces the pathogen population while minimizing any side-effects. It is contemplated that the composition will be obtained and used underthe guidance of a physician for in vivo use. Brevispirins are particularly useful for killing Treponema pallidum; Pseudomonas aeruginosa, Staphylococcus aureus, etc. [40] Brevispirins are also useful for in vitro formulations to kill microbes, particularly where one does not wish to introduce quantities of conventional antibiotics. For example, brevispirins may be added to animal and/or human food preparations, or to blood products intended for transfusion to reduce the risk of consequent bacterial or viral infection. Brevispirins may be included as an additive for in vitro cultures of cells, to prevent the overgrowth of microbes in tissue culture.

[41] The susceptibility of a particular microbe or virus to killing or inhibition by brevispirins may be determined by in vitro testing, as detailed in the experimental section. Typically a culture of the microbe is combined with brevispirins at varying concentrations for a period of time sufficient to allow the protein to act, usually ranging from about one hour to one day. The viable microbes are then counted, and the level of killing determined. Two stage radial diffusion assay is a convenient alternative to determining the MIC or minimum inhibitory concentration of an antimicrobial agent.

[42] Microbes of interest, but not limited to the following, include: Citrobactersp.;

Enterobacter sp.; Escherichia sp., e.g. E. coli; Klebsiella sp.; Morganella sp.; Proteus sp.; Providencia sp.; Salmonella sp., e.g. S. typhi, S. typhimurium; Serratia sp.; Shigella sp.; Pseudomonas sp., e.g. P. ae ginosa; Yersinia sp., e.g. Y. pestis, Y. pseudotuberculosis, Y enterocolitica; Franciscella sp.; Pasturella sp.; Vibrio sp., e.g. V. cholerae, V. parahemolyticus; Campylobactersp., e.g. C. jejuni; Haemophilus sp., e.g. H. influenzae, H. ducreyi; Bordetella sp., e.g. B. pertussis, B. bronchiseptica, B. parapertussis; Brucella sp., Neisseria sp., e.g. N. gonorrhoeae, N. meningitidis, etc. Other bacteria of interest include Legionella sp., e.g. L.. pneumophila; Listeria sp., e.g. L. monocytogenes; Staphylococcus sp., e.g. S. aureus Mycoplasma sp., e.g. M. hominis, M. pneumoniae; Mycobacterium sp., e.g. M. tuberculosis, M. leprae; Treponema sp., e.g. T pallidum; Borrelia sp., e.g. B. burgdorferi; Leptospirae sp.; Rickettsia sp., e.g. R. rickettsii, R. typhi; Chlamydia sp., e.g. C. trachomatis, C. pneumoniae, C. psittaci; Helicobacter sp., e.g. H. pylori, etc. Viral pathogens of interest include retroviral pathogens, e.g. HIV-1 ; HIV-2, HTLV, FIV, SIV, βte.

[43] Various methods for administration may be employed. For the prevention of sexually transmitted diseases, administration to mucosal surfaces is of particular interest, e.g. vaginal, rectal, etc. The polypeptide formulation may be given orally, or may be injected intravascularly, subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder, topically, efc. For example, methods of administration by inhalation are well-known in the art. The dosage of the therapeutic formulation will vary widely, depending on the specific brevispirin to be administered, the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered once or several times daily, semi- weekly, etc. to maintain an effective dosage level. In many cases, oral administration will require a higher dose than if administered intravenously. The amide bonds, as well as the amino and carboxy termini, may be modified for greater stability on oral administration.

Formulations

[44] The compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, lotions, and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, vaginal, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, etc., administration. The brevispirins may be systemic after administration or may be localized by the use of an implant or other formulation that acts to retain the active dose at the site of implantation.

[45] The compounds of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g., perforin, anti-inflammatory agents, antibiotics, etc.) In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are merely exemplary and are in no way limiting.

[46] For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[47] The compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[48] The compounds can be utilized in aerosol formulation to be administered via inhalation.

The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. [49] The compounds can be used as lotions, for example to prevent infection of burns, by formulation with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[50] Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

[51] Unit dosage forms for oral, vaginal or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[52] Implants for sustained release formulations are well-known in the art. Implants are formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well- tolerated by the host. The implant containing brevispirins is placed in proximity to the site of infection, so that the local concentration of active agent is increased relative to the rest of the body.

[53] The term "unit dosage form", as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with the compound in the host.

[54] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[55] Typical dosages for systemic administration range from 0.1 μg to 100 milligrams per kg weight of subject per administration. A typical dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.

[56] Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound.

[57] The use of liposomes as a delivery vehicle is one method of interest. The liposomes fuse with the cells of the target site and deliver the contents of the lumen intracellulariy. The liposomes are maintained in contact with the cells for sufficient time for fusion, using various means to maintain contact, such as isolation, binding agents, and the like. In one aspect of the invention, liposomes are designed to be aerosolized for pulmonary administration. Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus, etc. The lipids may be any useful combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipid will be normally be neutral or acidic lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.

[58] For preparing the liposomes, the procedure described by Kato er al. (1991) J. Biol.

Chem. 266:3361 may be used. Briefly, the lipids and lumen composition containing peptides are combined in an appropriate aqueous medium, conveniently a saline medium where the total solids will be in the range of about 1-10 weight percent. After intense agitation for short periods of time, from about 5-60 sec, the tube is placed in a warm water bath, from about 25-40° C and this cycle repeated from about 5-10 times. The composition is then sonicated for a convenient period of time, generally from about 1-10 sec. and may be further agitated by vortexing. The volume is then expanded by adding aqueous medium, generally increasing the volume by about from 1-2 fold, followed by shaking and cooling. This method allows forthe incorporation into the lumen of high molecular weight molecules.

Formulations with Other Active Agents [59] For use in the subject methods, brevispirins may be formulated with other pharmaceutically active agents, particularly other antimicrobial agents. Other agents of interest include a wide variety of antibiotics, as known in the art. Classes of antibiotics include penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in combination with β-lactamase inhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc. [60] Cytokines may also be included in a brevispirin formulation, e.g. interferon γ, tumor necrosis factor α, interleukin 12, etc. [61] Antiviral agents, e.g. acyclovir, gancyclovir, etc., may also be included in brevispirin formulations.

ENDOTOXIN REMOVAL

[62] Brevispirins have a high affinity for endotoxins, and find use in the removal of endotoxins from compositions, e.g. from pharmaceutical compositions. For removal of endotoxins, the brevispirin peptide is generally immobilized to an insoluble carrier or substrate, for adsorption and removal of endotoxin. The shape of the insoluble carrier to which the peptide of the present invention is immobilized may be any convenient form as known in the art, e.g. membranes (filter type, hollow type, tube type, flat membrane type, etc.), column resin, granule, latex, chip, powder, microplate, etc. Various materials are known and used in the art for such purposes, e.g. polystyrene materials, polypropylene materials, polyamide materials, cellulose materials, agarose materials, polyacrylamide materials, dextran materials, vinyl polymer materials, etc.

[63] The peptide is conjugated to the substrate by any convenient method that provide for stable attachment as well as to provide good presentation of the peptide to the endotoxins. For example, the linkage may be a homo- or heterobifunctional linker having a group at one end capable of forming a stable linkage to the peptide, and a group at the opposite end capable of forming a stable linkage to the substrate. Illustrative entities include: azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N^y-maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl [4-azidophenyl]-1 ,3'- dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-MAL; succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate; 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) or4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N- hydroxysuccinimide ester (SMCC).

[64] The substrate and brevispirin conjugate is brought into contact with a solution in which removal of endotoxin is desired, to form a complex of the endotoxin in the solution with the peptide, whereby the endotoxin in the solution can be removed. For example, a method in which a solution is passed through a filter-shaped or hollow fiber-shaped insoluble carrier or over a flat membrane-shaped insoluble carrier, a method in which a solution is passed through a column charged with a granular insoluble carrier, a method in which a solution is charged in a microplate-shaped well and the solution is left to stand for a certain time and then the solution is separated, a method in which a solution is added onto an insoluble carrier of any shape and shaken or left to stand for a certain time and then usual solid-liquid separation means (filtration, centrifugation, aspiration, decantation, etc.) can be used to obtain a solution which is free of endotoxin, or the like.

EXPERIMENTAL [65] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

EXAMPLE 1 [66] Four brevispirin peptides were derived from the sequence of human cathelicidin: WS-

22, WS-22N, WS-22 amide, and WS-22N amide. Each contains 16 amino acids. The primary structures, along with the primary sequence of LL-37, their progenitor, are shown in Table 1 below. Also shown in this table are their bactericidal activities against both Gram-negative and- positive bacteria. These data confirm their broad-spectrum bactericidal activity. All four peptides are substantially more active than LL-37 against Staphylococcus aureus, a pathogen that now displays resistance to multiple antibiotics.

Table 1. Amino acid sequences and bactericidal activities of truncated peptides of human LL-37

a: amino acids shown in single letter code b: minimal effective concentration shown in micrograms per ml c: Ec, Escherichia coli ML-35p; Pa, Pseudomonas aemginosa MR3007: Lm, Listeria monocytogenes EGD; Sa, Staphylococcus aureus 930918-3 d: *C-terminus is in the amidated form e: replacement of replacement of aspartic acid (D) with asparagine (N) [67] The affinity of LL-37 and brevispirins were measured against 011 :B4 lipopolysaccharide (LPS) by a previously described chromogenic procedure. The term EC₅₀ signifies the concentration of peptide that binds half of the added LPS. This value provides an estimate of each peptide's binding affinity for LPS (the lower the value, the higher the affinity). On a weight basis, WS-22amide and WS-22N amide bound LPS nearly as effectively as full length LL-37. Hence, these peptides can serve as not only antimicrobial agents but are effective in removing LPS from infected sites and fluids.

Table 2 LPSBinding Properies of LL-37 and the WS-22 Peptides

Other variants of LL-37 include the following:

Table 3

Name Residues Number of Formula Sequence Residues Weight lull length peptide and variants f i ϋδ- II

LL-37 1-37 37 4493.3 LLGDF FRKSK EKIGK EFKRI VQRIK DFLRN LVPRT ES

Pentamide 1-37(PAM) 37 4489.4 LLGNF FRKSK QKIGK QFKRI VQRIK NFLRN LVPRT QS

WS-9 1-37(Y₂₇) 37 4509.3 LLGDF FRKSK EKIGK EFKRI VQRIK DYLRN LVPRT ES

WS-30 1-37 37 4492.3 LLGDF FRKVK EKIGG (LL-37N) GLKKV GQKIK NFLGN LVPRT AS

WS-1 1-17 17 2042.4 LLGDF FRKSK EKIGK EF

WS-2 9-37 29 3516.1 SK EKIGK EFKRI VQRIK DFLRN LVPRT ES

WS-3 17-33 17 2142.6 FKRI VQRIK DFLRN LVP

WS-6 26-37 12 1446.6 DFLRN LVPRT ES

WS-7 17-28 12 1562.9 FKRI VQRIK DFL

[68] These peptides were tested by performing radial diffusion assays under low salt conditions (1% agarose, 10 mM sodium phosphate buffer, 30 mg/ml trypticase soy broth powder) and physiological salt conditions (the same + 100 mM NaCI), obtaining the following results

Table 4 Antimicrobial Activity of the WS-Peptides in the RDA Antimicrobial activity (μq/ml) in low salt: pH 7.4

Antimicrobial activity (μM) in low salt: pH 7.4

Antimicrobial activity (ug/ml) in high salt 100 mM NaCI. pH 7.4

Antimicrobial activity (ug/ml) in high salt 100 mM NaCI. pH 7.4

Antimicrobial activity (μM) in high salt 100 mM NaCI. pH 7.4

Antimicrobial activity (μM) in high salt 100 mM NaCI. pH 7.4

[69] These studies identified WS-22 as the most active peptide, and showed that removing one additional amino acid from its C-terminus (WS-23) resulted in a great loss of activity. Also of note, WS-22 was more active than WS-3 (residues 17-33).

TABLE 5

[70] Having established this, the potency of WS-22, was enhanced by amidating tits C-terminus and/or by replacing its only acidic residue, an aspartic acid, by an asparagine (hence WS-22 amide, WS-22N, and WS 22N amide). These peptides were tested against L. monocytogenes, S. aureus , E. coli, P. aeruginosa and N. gonorrhoeae, with the following results. All of the WS- 22 peptides were very effective against these strains of N. gonorrhoeae, but WS 22N amide was the most active. TABLE 6

[71] WS-22 amide and WS-22N amide are more potent than the free acid forms of the peptide. WS-22N amide was 2-3 fold more active than WS-22N two Gram positives (L. mono and S. aureus), and slightly more potent against P. aeruginosa. [72] Results for some additional truncated variants of LL-37, none of which were as active as the WS-22 group.

EXAMPLE 2

[73] In this study, we examined the capacity of human LL-37 and the similar CAP-18-derived peptide from rabbits to exert antimicrobial activity against the causative agent of syphilis, Treponema pallidum. We found that both peptides, as well as a truncated version of human LL- 37 that contains its bactericidal domain, could exert rapid, but salt-sensitive antimicrobial activity against T. pallidum. Infectivity of T. pallidum in a rabbit model could effectively be blocked with the synthetic truncated LL-37-derived peptide WS22-N-amide.

[74] T. pallidum, the etiologic agent of syphilis, is an atypical bacterium that cannot be cultivated with typical laboratory media. However, we and others have successfully cultivated T pallidum in the presence of rabbit skin (Sf1 Ep) cells. In this model, cultures of T pallidum can be grown for 17-21 days with >80% viability. This spirochete has an outer membrane similar to that of Gram-negative organisms but lacks lipopolysaccharide. Furthermore, the cell envelope structure is not typical of Gram-negative bacteria and the cell wall structure is more related to Gram- positive bacteria. However, the T pallidum lacks teichioic acid, a potential binding site for cationic antimicrobial peptides. Between the cell wall and the outer membrane is a periplasmic space that contains endoflagella for motility. Most importantly, the surface of this spirochete is almost devoid of intramembranous proteins, and is very lipid rich. This unusual cell architecture presents a unique set of parameters that the host immune system likely encounters during infection. [75] T pallidum has a limited range of hosts. Humans are the only natural host, although rabbits can be used for propagation of the spirochete in the testis and in the skin. In fact, the "gold standard" laboratory test to determine treponemal viability is the rabbit infectivity test (RIT), in which serial dilutions of a suspension are injected intra-dermally into the backs of shaved rabbits. Furthermore, in the natural course of the disease, the primary route of infection of the syphilis spirochete is via dermal tissues, both keratinized and non-keratinized. Antimicrobial peptides, such as human LL-37 and rabbit CAP-18, have been demonstrated to be synthesized by epithelial and testicular tissues during infection. With respect to LL-37, recent evidence implicated it as a major contributor to the innate host defense response against the Group A Streptococcus. However, the role that LL-37 and other antibacterial peptides may play in the innate host defense during the initial and subsequent stages of syphilis and in its transmission are largely unknown.

[76] We examined the sensitivity of T. pallidum to human and rabbit LL-37 as well as to the truncated peptide derivative, WS22-N-amide (Fig. 1), which represents the antimicrobial core of LL-37. The antimicrobial activity of these cathelicidins was further demonstrated by examining the viability of the treated suspensions of T pallidum using the rabbit infectivity test (RIT).

Materials and methods

[77] Spirochetes. T pallidum subsp. pallidum (Nichols strain) was propagated in rabbit testes. Briefly, 10 8 treponemes were injected into each testis of 4 kg male New Zealand white rabbits. At the peak of orchitis (approximately 10 days later), the animals were sacrificed, and the testes excised. The testes were minced and placed in a 50 ml screw-cap flask with 10 ml of 7. pallidum culture medium (TpCM) [5]. The flask was briefly gassed with 95% N2/5% C02 and then placed on a reciprocal shaker for 20 min. The medium was then removed from the flask and placed in a 15 ml conical centrifuge tube. The gross tissue debris was removed by centrifugation at 500 *g for 15 min. For the antimicrobial assays, spirochetes were diluted in TpCM containing 3 mg/dl dithiothreitol (DTT) and 2% (v/v) fetal bovine serum (FBS).

[78] Antimicrobial peptides. The four peptides, human LL-37, rabbit CAP-18, WS22- 103 N- amide, and Tp-47 were synthesized by solid phase chemistry and purified using reverse-phase high pressure liquid chromatography. Briefly, solid phase Fmoc methodology for synthesis of peptides was performed on 0.02 mM of Wang Resin reacted sequentially in an Advanced Chemtech 396 Peptide Synthesizer with the appropriately Fmoc amino acids using double coupling cycles (0.5 h each) with HOBT, DIC, and two cycles of deprotection (5 and 30 min) with 25% piperdine in DMF. The resuling peptido-resin was deprotected and then cleaved from the resin using TFA (90%), phenol (2%) triisopropylsilan (2%), thioanisol (2%), dithioethane (2%), and water (2%) at room temperature for 2.5 h. They were then precipitated in cold ether, centrifuged and washed four times with cold ether. The residue was dried in vacuo and the structures were confirmed by matrix-associated laser desorption time-of-flight mass spectroscopy (MALDITOF-MS). Tp-47 is a truncated peptide consisting of the first 20 amino acids of the 47 kDa lipoprotein (gene sequence Tp0574) of T pallidum.

[79] Bactericidal assay. To assess the activity of these three peptides, the appropriate amounts of peptide were added directly to the modified TpCM containing the spirochetes and 2% fetal bovine serum (FBS). Control samples containing no or an irrelevant peptide were simultaneously established. In low salt experiments, the TpCM was diluted 1 :3 by adding two volumes of distilled water before adding 10 7 treponemes per ml. One-half milliliter portions of the cell suspensions were placed in 1.5 ml microfuge tubes and the appropriate amount of peptide was added. The tubes were gassed with 95% N2/5% CO2, and incubated at 35.C for 2 h unless other-wise noted. The Ecso (Effective Concentration-50%) is the amount of antimicrobial peptide required to kill 50% of the spirochetes during the 2 h incubation.

[80] Viability measurement. To ascertain the bactericidal effects of the peptides, three microliters of the Live/Dead Bacϋght TM (Molecular Probes, Eugene, OR) was added to each suspension and mixed by gentle mixing. Five to ten minutes later, 10 μl of each suspension was placed on a slide and observed by both dark-field and fluorescence microscopy (Nikon, Melville, NY). Viability was monitored by counting both the number of motile organisms, and the number of organisms exhibiting green (live) and red (dead) fluorescence. For each condition analyzed by microscopy, two or three samples were scored; in each sample approximately 100 organisms were observed.

[81] Flow cytometry. In addition to microscopy, samples were analyzed by flow cytometry using a Bruker ACS 100 equipped with a mercury arc lamp. The wavelength of light used to excite the samples was 488 nm and the fluorescence signals were measured at 520 nm. The instrument was set to trigger on fluorescent particles rather than forward light scatter because of the small cell volume of the spirochetes. The flow rate was maintainedat 500 events per second. Samples were spiked with a small quantity of 0.7 μM green beads (Duke Scientific, Palo Alto, CA). The signal from these beads permitted monitoring the alignment of the instrument and ensured quality control of the collected data. The fluorescence of approximately 25,000 spirochetes was recorded per determination and determinations were repeated three or four times per sample.

[82] Rabbit infectivity tests (RIT). The ability of treated organisms to establish infections was determined by the RIT [7], Briefly, suspensions containing 10⁷ T pallidum per ml were incubated at 35° C with either cathelicidins, a sham protein, or alone for 2 h. Organisms were diluted to 10³, 10², 10 and 1 organism per ml. Mature ( ≥4 kg) New Zealand white rabbits were anesthetized with 50 mg of ketamine and 1 mg of acepromazine per kg body weight and 0.1 ml of the above dilutions was injected intra-dermally into their shaved backs. Ten sites were injected in each animal. The animals were housed in a room equipped with a refrigeration unit that maintained the air temperature between 62 and 65 F. The animals were also re-shaved at least once a week and observed on a daily basis for 30 days for the production of a chancre at each site treated. A site was considered positive only if it produced an ulcerative chancre that contained motile spirochetes.

Results

[83] Selection of anti-treponemal assay. Conventional methods for determining the bactericidal activity of peptides against typical bacteria are not applicable to studies with 7^". pallidum because this pathogen is not readily cultivated in vitro; it has very long generation time (>35 h); and methods for plating the organism on semi-solid media have not been developed. Therefore, we explored alternative methods. Since these organisms possess endoflagella, motility is typically used to ascertain treponemal viability. However, we also wanted to develop a method that would reflect the ability of the cathelicidins to compromise cell membrane integrity. Therefore, staining the spirochetes with a vital dye was another option chosen. When we compared the data from motility and fluorescence analysis of peptide-treated organisms, the values for the percentage of killed spirochetes were virtually identical (data not shown).

[84] Effects of cathelicidins on the viability of T pallidum In the first series of experiments, we varied the concentration of human LL-37, rabbit CAP-18, and WS22-N-amide during a 2 h incubation. The results measuring activity of human LL-37 against 7^". pallidum can be seen in Fig. 1. The Ecβoof LL-37 was determined to be slightly above 66 μM (300 μg/ml) for human LL- 37 in physiological salt concentrations and 11 μM (50 μg/ml) for the peptide under low salt conditions (Table 7). During these incubations, the viability of the control (no peptide) bacteria remained at or above 99%. We did not attempt to test concentrations of any peptide above 500 μg/ml, and therefore 100% of the spirochetes could not be killed with LL-37 in TpCM. However, after 2 h, there were no viable organisms in suspensions treated with 55 μM of the LL-37 under low salt conditions. This result was due to either enhanced activity of the peptide under these conditions, osmotic stress on the organisms, or a combination of both. The activity of the truncated peptide WS22-N-amide is also reported in Fig. 1. The Ecsofor WS22-N-amide was approximately 4.4 μM in TpCM and 1.5 μM under low salt conditions. In both assays, we did observe 100% killing. The Ecsofor CAP-18 was determined to be nearly 90 μM (Table 7). In this case as well, 100% killing was not achieved during the 2 h incubation period. The activity of these peptides was both dose- and time-dependent. To examine the kinetics of these killing reactions, we selected several concentrations and performed time-course assays. Fig.2 shows the results of four of these assays using two different concentrations each of LL-37 (22 and 44 μM) and WS22-N-amide (2.4 and 4.8 μM). The kinetics of the action of whole LL-37 were much slower than that for WS22-N-amide. Nevertheless, most of the activity of both occurred in the first 30 min. Similar results were seen at other concentrations. [85] Rabbit infectivity tests To determine and further demonstrate that the peptides could impart anti-treponemal activities, we conducted bactericidal assays and then diluted some of the suspension for use in the RIT to test for both the viability of treated organisms and the infectivity of those surviving. It is important to note that the sensitivity of this assay is between 1 and 10 organisms per injection site and a single organism can theoretically produce a chancre. However, the probability that a site injected with this concentration of organisms will produce a lesion is determined by Poisson distribution. The experiments were repeated three times and the cumulative data is reported in Table 2. At 66μM, which is near the Ecso of LL-37, exactly half of the infected sites produced lesions when the samples were diluted to one organism per site. At a concentration of 10 organisms per site, 6 of 7 produced lesions. In contrast, when 9.7 μM WS22-N-amide was used in the assay, no lesions developed even at 100 organisms per site. This finding correlates well with the data presented in Fig. 1 showing no survival at this peptide concentration. When an irrelevant peptide, a 20-merof the 47 kDa antigen, was used in the assay, about half of the injection sites produced lesions at 1 organism per site and 67% at 10 organisms per site. When the antimicrobial peptide was absent from the assay, 33% of the injections produced a lesion at 1 organism per site and 100% at 10 and 100 organisms per site, as would be expected. Thus, these RITs demonstrated that the activity of these peptides was bactericidal and could reduce the infectivity of 7. pallidum exposed to them.

[86] Under the conditions of the above experiments, 7^". pallidum can replicate in TpCM and survive for 14-17 days. It was found that T pallidum is much more sensitive to LL-37 under low salt conditions than in complete TpCM. However, with respect to T pallidum, it is not clear whether this was due to the increased activity of the peptide or to an increased vulnerability of the spirochete to low osmotic pressure. This result was most likely due to a combination of both.

[87] These studies demonstrate that the full length LL-37 peptide is less active against T. pallidum than its truncated synthetic variant, WS22-N-amide. This decreased sensitivity to LL- 37 activity may be explained by the architectural structure of the spirochete's cell envelope. The outer membranes (OMs) of spirochetes are unique and different from those of typical Gram- negative bacteria. First, lipopolysaccharides are absent from species of Treponema and Borrelia. Second, typical Gram-negative OMs have numerous porins which appearto be lacking in spirochetal OMs. In 7. pallidum, it is uncertain whether their OMs are totally devoid of porins or whether porins are present in very small numbers. In either case, the OMs of these spirochetes certainly possess far less porins than typical Gram-negative bacteria. Third, typical spirochetal OMs are very lipid rich, which tends to make the surface less charged and more hydrophobic than typical Gram-negative bacteria. Finally, the amount of surface-exposed proteins which also carry a charge is greatly diminished when compared with other bacteria. Freeze-fracture electron microscopy of its outer membrane reveals that the organism has only a few hundred intramembranous particles (IMPs) present per cell, compared to several thousand for Escherichia coli. 7 pallidum is an extreme example of this characteristic. [88] Cathelicidins are positively charged peptides and part of their action depends upon binding to targets on bacterial surfaces. Typical Gram-negative bacteria tend to be negatively charged and thus the initial process of cathelicidin binding involves ionic bonding to these charged targets. Two of these targets are LPS and lipoteichoic acids, neither of which are found in the cell architecture of 7 pallidum. Other targets may include negatively charged surface proteins. Again, those targets are either nonexistent or very scarce on the surface of 7 pallidum. Thus, the unique architecture of the OM of 7 pallidum confers its resistance to cathelicidin activity because of its low surface charge and more hydrophobic nature. 7 pallidum was shown to be much more sensitive to the truncated peptide, WS22-N-amide. The Ecsowas over 30 times less than that with the whole peptide. The explanation for this increased sensitivity lies in two differences between the truncated peptide and whole LL-37. First, WS22- N-amide consists of the 17 residues of the activity core of LL-37. Second, asparagine was substituted for the aspartic acid residue in this core sequence, thus removing a negatively charged amino acid residue and replacing it with a positively charged residue. This substitution gives WS22-N-amide an even higher charge to residue ratio than native LL-37. In fact, this would make most of the entire peptide either hydrophobic or positively charged. The antibacterial activity of short peptides is increased as the net charge of the peptide increases. Furthermore, the large number of uncharged residues that could be attracted to the lipid rich OM of 7 pallidum because of hydrophobic interaction may explain why this derivative is much more potent than the native peptide.

Table 7 Effectiveness of antimicrobial peptides against Treponema pallidum Cathelicidin ECso _a normal (μM) ECso low salt (μM)

Human LL-37 66^4 ϊϊ

WS22-N-amide 4.37 1.4

Rabbit CAP-18 89.3 ND

[89] To prove that the nonmotile organisms observed in the bactericidal assays were killed, infectivity assays using the RIT were performed. This test is considered the "gold standard" to determine viability of 7 pallidum. The rabbit is the most practical animal because (1) a local lesion is produced, (2) the tissues remain infective for the life of the animal, and (3) the sensitivity is between 1 and 10 viable spirochetes. Furthermore, the RIT is much less complicated than tests using hamsters where internal organs need to be excised and examined. The animals do not need to be sacrificed to determine if organisms are in the lesions, and therefore the incubation periods for sites containing very small numbers of viable organisms can be extended as long as needed. The treponemes were pre-treated with specific peptides before injection in order to tightly control the peptide concentration and incubation conditions, and to ensure reproducibility of the assays.

[90] The data in Table 7 shows that the truncated peptide WS22-N-amide was very effective in killing the treponemes at a concentration of 10 μM. None of the injected sites produced lesions, even with 100 spirochetes per site injected. In contrast, 66 μM of the whole peptide produced <50% killing according to the RIT. It should be noted that the difference between the

LL-37-treated and untreated organisms would have been more pronounced if concentrations above the Ecso had been used.

Table 8 Rabbit infectivity tests with 7 pallidum Organisms/site hLL-37 WS22-N Tp-47 None

100 6/6 0/6 6/6 3/3

10 6/7 0/7 4/6 4/4

1 4/8 0/8 2/6 1/3

0.1 0/6 0/6 0/4 0/2

[91] In conclusion, this study demonstrated that viable 7 pallidum was resistant to the bactericidal action of human LL-37 and rabbit CAP-18, but was much more sensitive to a truncated peptide of LL-37, WS22-N-amide. We suggest that this resistance is most likely due to the unique cell architecture of the spirochete that renders it less susceptible to the bactericidal mechanism of LL-37 than other bacteria. This resistance could be overcome by the removal of the N- and C-terminal amino acids of full length LL-37. Hence, the bactericidal domain of LL-37 might be less available to the targets on the surface due to the presence of these extensions.

EXAMPLE 3

[92] Variants of brevispirin peptides that contain a pH-dependent "switch" that will enhance their activity under acid conditions. Such a switch would focus the activity of these peptides on acidogenic bacteria, such as S. mutans, and minimi^ effects on other oral flora.

[93] Most antimicrobial peptides contain between 16 and 50 amino amino acid residues, and are cationic (i.e., positively charged) and amphipathic (their polar and apolar side-chains are clustered and separated in space). The net positive charge results from an abundance of arginine and/or lysine residues, augmented in some peptides by histidines. We describe below how histidine residues allow the introduction of a pH-responsive element into selected antimicrobial peptides. Our intent is to create novel antimicrobial peptides whose activity will be triggered by a local fall in pH, such as that caused by S. mutans, an acid-forming dental pathogen. [94] Ws-22N is a 16-amino acid fragment of LL-37 that has been slightly modified to enhance its antimicrobial properties. Histidine's imidazole group has a pKa of approximately 6.1 in free solution, however the pKa of a histidine residue in a protein can be much higher if it interacts with nearby negatively charged groups.

[95]

[96] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[97] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An isolated brevispirin peptide.

2. The isolated brevispirin peptide according to Claim 1 , wherein said peptide comprises the amino acid sequence:

wherein residues 1 , 4, 5, 8, 11 , 12, 15 and 16 are any hydrophobic amino acid; residues 2 and 9 are K, R or H; residues 3 and 7 are be R, K or H; residues 6, 10 and 14 are Q or N; residue 13 is R.

3. The isolated brevispirin peptide according to Claim 2, wherein said peptide is amidated at the carboxy terminus.

4. The isolated brevispirin peptide of Claim 2, wherein said brevispirin comprises the amino acid sequence (SEQ ID NO:1) VLNRLFNKIRQVIRKF or (SEQ ID NO:2) VLNRLFDKIRQVIRKF.

5. The isolated brevispirin peptide of Claim 4, comprising at least one histidine substitution at a position independently selected from residue 2, 3, 7, 9.

6. The isolated brevispirin peptide of Claim 2, and a pharmaceutically acceptable excipient.

7. A method for killing microbial organisms, the method comprising: administering an effective dose of brevispirin to said microbial organisms.

8. A method for treating microbial infection in a patient, the method comprising: administering brevispirin as a therapeutic agent to said patient.

9. A method for preventing microbial infection in a patient, the method comprising: administering brevispirin as a therapeutic agent to said patient.

10. The method according to any of claims 7-9, wherein said microbial organism is 7 pallidum.

11. The method according to any of claims 7-10, wherein said microbial organism is a Staphylococcus species.

12. A method of removing endotoxins from a composition, the method comprising: contacting said composition with an insoluble substrate conjugated to a brevispirin; allowing a complex to form between said brevispirin and said endotoxins; removing said complex from said composition.