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WO2002000839A2 - Novispirines: peptides antimicrobiens - Google Patents

Novispirines: peptides antimicrobiens Download PDF

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Publication number
WO2002000839A2
WO2002000839A2 PCT/US2001/019094 US0119094W WO0200839A2 WO 2002000839 A2 WO2002000839 A2 WO 2002000839A2 US 0119094 W US0119094 W US 0119094W WO 0200839 A2 WO0200839 A2 WO 0200839A2
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WIPO (PCT)
Prior art keywords
antimicrobial
peptide
seq
isoleucine
novispirin
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PCT/US2001/019094
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English (en)
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WO2002000839A3 (fr
Inventor
Robert I. Lehrer
Alan J. Waring
Brian F. Tack
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The Regents Of The University Of California
The University Of Iowa A Research Foundation
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Priority claimed from US09/840,009 external-priority patent/US6492328B2/en
Application filed by The Regents Of The University Of California, The University Of Iowa A Research Foundation filed Critical The Regents Of The University Of California
Priority to AU2001268421A priority Critical patent/AU2001268421A1/en
Priority to CA002415236A priority patent/CA2415236A1/fr
Priority to BRPI0112035-2A priority patent/BR0112035A/pt
Priority to IL15355601A priority patent/IL153556A0/xx
Priority to JP2002505955A priority patent/JP2004526663A/ja
Priority to EP01946359A priority patent/EP1359930A4/fr
Publication of WO2002000839A2 publication Critical patent/WO2002000839A2/fr
Publication of WO2002000839A3 publication Critical patent/WO2002000839A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/08—Antibacterial agents for leprosy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/04—Amoebicides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • NOVISPIRINS ANTIMICROBIAL PEPTIDES
  • Natural polycationic antibiotic 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 mainly 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. For example, the human peptides termed alpha-defensins are produced by neutrophils and intestinal Paneth cells.
  • defensins manifest an amphiphilic, largely beta- sheet structure, with a polar face formed largely by its arginines and with N- and C-terminal residues playing an important role in defining antimicrobial potency and spectrum.
  • Antimicrobial peptides are reviewed by Hancock and Lehrer (1998) Trends in Biotechnology 16:82.
  • Cystic fibrosis (CF) is an inherited disorder that occurs in one of every 3,300 U.S. newborns. It affects some 30,000 Americans today.
  • antimicrobial peptides including cecropin P1 , indolicidin, magainin
  • nisin and ranalexin has been tested against clinical isolates of P. aeruginosa.
  • the peptides were found to have a varied range of inhibitory values, and showed some synergy when combined with conventional antibiotics (Giacometti et al. (1999) J Antimicrob Chemother.
  • Novispirin peptides are small antimicrobial agents with potent activity against Gram-negative bacteria, including Chlamydia trachomatis, Pseudomonas aeruginosa, Escherichia coli and Stenotrophomonas maltophilia.
  • the peptides are nonhemolytic, exhibit reduced in vitro cytotoxicity relative to other antimicrobial peptides, and are well-tolerated in vivo after intravenous injection.
  • Novispirins are equally effective against growing and stationary phase P. aeruginosa, and they retain activity in the presence of high concentrations of salt or human serum.
  • Novispirins also bind lipopolysaccharide (LPS), a property that may mitigate symptoms associated with Gram-negative bacterial infection.
  • LPS lipopolysaccharide
  • a pharmaceutical composition comprising novispirin as an active agent is administered to a patient suffering from a microbial infection, particularly bacterial infections.
  • the protein is also effective at killing a variety of microbial organisms in vitro.
  • Novispirin may be administered alone, or in combination with other bacteriocidal agents, e.g. antibiotics, as a cocktail of effective peptides, etc.
  • Novispirin mediated killing of microbes is also useful for modeling and screening novel antibiotics.
  • Figures 1A and 1 B show the relative activity of ovispirin and representative novispirins against two Gram-negative bacteria.
  • Figure 2 shows the kinetics of antimicrobial peptides against P. aeruginosa.
  • Figure 3 shows the hemolytic activity of antimicrobial peptides against human red blood cells.
  • Figures 4A, 4B and 4C show the relative cytotoxicity of antimicrobial peptides against human cells.
  • Figure 5 shows the binding of LPS by novispirins.
  • Figures 6A and 6B show the permeabilization of bacterial membranes by novispirins.
  • novispirins as antimicrobial agents.
  • the peptides are effective at killing a variety of microbial organisms in vitro and in vivo by direct microbicidal activity.
  • Novispirin(s) are administered alone or in combination with other active agents to a patient suffering from or predisposed to an infection, in a dose and for a period of time sufficient to reduce the patient population of microbial pathogens.
  • novispirins also find use in the treatment of plant-pathogenic pseudomonads, in agricultural applications designed to prevent disease in and spoilage of food crops.
  • novispirins provides a basis for further therapeutic development, by modification of the polypeptide structure to yield modified forms having altered biological and chemical properties.
  • the native or modified forms are formulated in a physiologically acceptable carrier for therapeutic uses, or are otherwise used as an antimicrobial agent.
  • Novispirin Compositions For use in the subject methods, any of the provided novispirins, modifications thereof, or a combination of one or more forms may be used.
  • Novispirins include peptides of the formula as follows: SEQ ID NO:1 KNLRRX ⁇ RI ⁇ HIIKKYG wherein ⁇ , X 2 , X 3 and X 4 are independently selected from the group consisting of the D or L forms of glycine, threonione, serine and isoleucine, preferably glycine, threonine, serine, glutamic acid, aspartic acid, isoleucine, D-alanine and D-isoleucine, with the proviso that not more than 3 of the X residues are isoleucine.
  • Preferred amino acids for the non-isoleucine residues are glycine, serine and threonine.
  • the cytotoxicity of the antimicrobial peptide ovispirin is related to its high degree of rigidity and amphipathicity.
  • the substitution of glycine, threonine, serine, glutamic acid, aspartic acid, isoleucine, D-alanine or D-isoleucine for one of the isoleucine resides at positions 6, 7, 10 or 11 in ovispirin adds flexibility (glycine), breaks the alpha-helix (D-alanine (DA) or D-isoleucine (Dl)) or adds a polar residue to disrupt the overly hydrophobic region.
  • DA alpha-helix
  • Dl D-isoleucine
  • Table I shows the primary sequences of ovispirin and exemplary novispirin peptides. Bolded letters indicate the residues that distinguish these novispirins from ovispirin.
  • sequence of the novispirin polypeptides may also be altered in various ways known in the art to generate targeted changes in sequence.
  • the polypeptide will usually be substantially similar to the sequences provided herein, /. e. will differ by one amino acid, and may differ by two amino acids.
  • the sequence changes may be substitutions, insertions or deletions.
  • the protein may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes.
  • various post- translational modifications may be achieved.
  • 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.
  • the carboxy terminus of the peptide is amidated, thereby increasing the positive charge of the peptide.
  • the antimicrobial peptide consists essentially of the polypeptide sequence set forth in any one of SEQ ID NO:1 , or SEQ ID NO:3 to SEQ ID NO:30.
  • 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.
  • novispirins are administered to a host suffering from or predisposed to a microbial infection. Administration may be topical, localized or systemic, depending on the specific microorganism, preferably it will be localized. Generally the dose of novispirin 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 of killing.
  • the compounds of the present invention are administered at a dosage that reduces the microbial population while minimizing any side-effects. It is contemplated that the composition will be obtained and used under the guidance of a physician for in vivo use.
  • Novispirins are particularly useful for killing gram negative bacteria, including Pseudomonas aeruginosa, and Chlamydia trachomatis.
  • Novispirins are also useful for in vitro formulations to kill microbes, particularly where one does not wish to introduce quantities of conventional antibiotics.
  • novispirins may be added to animal and/or human food preparations.
  • Novispirins may be included as an additive for in vitro cultures of cells, to prevent the overgrowth of microbes in tissue culture.
  • the susceptibility of a particular microbe to killing with novispirins may be determined by in vitro testing, as detailed in the experimental section. Typically a culture of the microbe is combined with novispirins at varying concentrations for a period of time sufficient to allow the protein to act, usually between about one hour and one day. The viable microbes are then counted, and the level of killing determined.
  • Microbes of interest include, but are not limited to Gram-negative bacteria, for example:
  • Y. pseudotuberculosis Y enterocolitica
  • Franciscella sp. Franciscella sp.
  • Pasturella sp. Vibrio sp., e.g.
  • 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; Mycoplasma sp., e.g. M. hominis, M. pneumoniae; Mycobactehum sp., e.g. M. tuberculosis, M. leprae;
  • Treponema sp. e.g. T. pallidum
  • Borrelia sp. e.g. B. burgdorfeh
  • Leptospirae sp. e.g.
  • 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.
  • Non bacterial pathogens of interest include fungal and protozoan pathogens, e.g. Plasmodia sp., e.g. P. falciparum, Trypanosoma sp., e.g. T. brucei; shistosomes; Entaemoeba sp., Cryptococcus sp., Candida sp, e.g. C. albicans; etc.
  • Plasmodia sp. e.g. P. falciparum
  • Trypanosoma sp. e.g. T. brucei
  • shistosomes Entaemoeba sp.
  • Cryptococcus sp. e.g. C. albicans
  • Candida sp e.g. C. albicans
  • the polypeptide formulation may be given orally, or may be injected intravascularly, subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder, topically, etc.
  • methods of administration by inhalation are well-known in the art.
  • the dosage of the therapeutic formulation will vary widely, depending on the specific novispirin 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.
  • 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.
  • the carboxy terminus may be amidated.
  • 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, creams, foams, solutions, suppositories, injections, inhalants, gels, microspheres, lotions, and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration.
  • the novispirins 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.
  • a formulation for topical use comprises a chelating agent that decreases the effective concentration of divalent cations, particularly calcium and magnesium.
  • agents such as citrate, EGTA or EDTA may be included, where citrate is preferred.
  • the concentration of citrate will usually be from about 1 to 10 mM.
  • 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.
  • 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.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • 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.
  • an aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
  • solubilizers isotonic agents
  • suspending agents emulsifying agents
  • stabilizers and preservatives emulsifying agents
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • 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.
  • the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • 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.
  • Unit dosage forms for oral 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.
  • 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.
  • 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 novispirins 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.
  • unit dosage form 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.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • 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.
  • Typical dosages for systemic administration range from 0.1 ⁇ g 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.
  • 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.
  • 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 intracellularly.
  • 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.
  • 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.
  • 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.
  • an appropriate aqueous medium conveniently a saline medium where the total solids will be in the range of about 1-10 weight percent.
  • an appropriate aqueous medium conveniently a saline medium where the total solids will be in the range
  • novispirins 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 ⁇ -lactamase inhibitors, cephalosporins, e.g.
  • Anti-mycotic agents are also useful, including polyenes, e.g. amphotericin B, nystatin; 5- flucosyn; and azoles, e.g. miconazol, ketoconazol, itraconazol and fluconazol.
  • Antituberculotic drugs include isoniazid, ethambutol, streptomycin and rifampin. Cytokines may also be included in a novispirins formulation, e.g. interferon ⁇ , tumor necrosis factor , interleukin 12, etc.
  • 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.
  • synthesizers Naturally occurring amino acids may be substituted with unnatural amino acids, particularly D isomers, e.g. D-alanine and Disoleucine, diastereoisomers, side chains having different lengths or functionalities, and the like.
  • D isomers e.g. D-alanine and Disoleucine, diastereoisomers, side chains having different lengths or functionalities, and the like.
  • the particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
  • Chemical linking may be provided to various peptides or proteins comprising convenient functionalities for bonding, such as amino groups for amide or substituted amine formation, e.g. reductive amination, thiol groups for thioether or disulfide formation, carboxyl groups for amide formation, and the like.
  • various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface.
  • 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.
  • 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.
  • 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.
  • Antimicrobial activity was tested in media with low, normal or high salinity. In radial diffusion assays, this was accomplished by using a low salt "basic medium" containing 0.3mg of trypticase soy broth powder/ml of 10 mM sodium phosphate buffer, pH 7.4.
  • Media with "normal” or “high” salinity were prepared by supplementing the basic medium with 100 mM NaCI to obtain media with normal salinity, or with 175-200 mM NaCI to obtain high salt media. Retention of activity against P. aeruginosa in high salt media is important because airway fluids from cystic fibrosis patients are reported to show hypersalinity, and other sites - including skin surfaces and the urinary bladder -may have locally high salt concentrations.
  • Microorganisms used in this study included 13 different Pseudomonas aeruginosa strains.
  • the AML-654 and LZ-1 strains are recent clinical isolates from the UCLA Clinical Microbiology laboratory.
  • Nine strains (5 mucoid, 4 nonmucoid) were recent CF isolates provided by Dr. Lisa Saiman.
  • All of the P. aeruginosa strains except PAO-1 strain were resistant to multiple conventional antibiotics, and all 9 CF isolates were resistant to 250 ⁇ g/ml of tobramycin.
  • Atetrazolium reduction assay (Boehringer-Mannheim, Indianapolis IN) was used to study cytotoxicity.
  • ME-180 human cervical epithelial cells (ATCC HTB-33) and A549 human lung epithelial cells (ATCC CCL-185) were grown to confluency in RPMI Medium (ME- 180) or Ham's F12K medium with 2 mM L-glutamine (A-549) containing 10% fetal bovine serum, 2 mM L-glutamine and 50 ⁇ g/mL gentamicin ("medium").
  • the cells were harvested with trypsin-EDTA, washed with medium, and after their concentration and viability (trypan blue exclusion) was determined, they were diluted to 5 x 10 4 cells/mL. Aliquots (100 ⁇ l) were dispensed into 96 well tissue culture plates (Costar) and incubated for 5 hr. at 37 °C in room air with 5% CO 2 . Then, serially diluted peptides were added and after 20 hr. of additional incubation, 10 ⁇ l of MTT solution was added. Four hours later, 100 ⁇ l of solubilization buffer was added and left overnight. The following day, MTT reduction was determined by optical density measurements at 600 and 650 nm, using a Spectramax 250 Spectrophotometer (Molecular Devices, Sunnyvale, CA).
  • Hemolytic Activity This was tested by incubating various concentrations of peptide with a suspension (2.8% v/v) of washed human, murine, sheep or bovine red cells in PBS. After 30 min at 37C, the tubes were centrifuged and the percentage of total hemoglobin released to the supernatant was measured.
  • Ovispirin and novispirins manifested potent antimicrobial activity against Gram-negative bacteria, including 13/13 strains of Pseudomonas aeruginosa.
  • Novispirin-mediated bactericidal activity occurred within 5-15 minutes, most likely from permeabilization of the inner and outer membranes of the bacteria. Antimicrobial activity was maintained under high salt (175-200 mM NaCI) conditions and was minimally impacted by the presence of 20% serum. Novispirins were equally effective against growing or quiescent P. aeruginosa.
  • Table 1 compares the activity of novispirins against these Gram-negative bacteria with its activity against Listeria monocytogenes, Staphylococcus aureus and C. albicans.
  • novispirins show activity against these Gram-positive bacteria when tested in physiological (100 mM NaCI) salt concentrations, and also that Novispirin G10 is quite active against C. albicans.
  • neither ovispirin nor novispirins retained activity against C. albicans or the Gram positives under very high salt conditions (200 mM NaCI), they remained active against the Gram-negatives under these conditions. Consequently, even if the airways' NaCI concentrations are exceptionally high in CF patients, this factor will not impair the ability of ovispirin or novispirins to kill P. aeruginosa.
  • P. aeruginosa within the airways of patients with cystic fibrosis can differ in many ways from free-living planktonic members of the species. Among these differences are: their entrapment in an alginate-rich biofilm, selection for auxotrophy and antibiotic resistance, various modifications to their lipid and LPS structures, and metabolism characteristic of high density, nutrient-limited cultures. Many conventional antibiotics (e.g., inhibitors of cell wall or protein synthesis) and many antimicrobial peptides act preferentially against rapidly growing organisms and might therefor be relatively inactive against high mass/low turnover populations of bacteria. We therefor tested the activity of ovispirin and novispirins against 3 strains of P.
  • aeruginosa MR3007 (a serum-resistant strain) and E. coli ML-35P (sensitive to >5% serum). Ovispirin and novispirins lost some activity against P. aeruginosa in the presence of serum, but were affected much less than LL-37.
  • the underlay gel contained 60% RPMI-1640, ⁇ 2.5 or 20% normal human serum (NHS). The remainder of the solution was phosphate-buffered saline
  • FIG 3 shows the effect of ovispirin or novispirins in lysing red blood cells.
  • ovispirin was about as hemolytic for human red blood cells as protegrin PG-1 , the novispirins were non hemolytic. Similar results were obtained with murine red blood cells. In contrast, none of these peptides were appreciably hemolytic towards sheep or bovine red blood cells.
  • Figure 5 compares the cytotoxicity of these peptides for MRC-5 human lung fibroblasts.
  • the G10 and T10 novispirins were virtually devoid of cytotoxicity, demonstrating that they would be unlikely to retard tissue repair or wound healing.
  • mice In vivo toxicity. Acute toxicity studies were performed in mice. 3/3 mice survived an intravenous dose of 10 mg novispirin G10 / kg body weight, and 1/3 survived a dose of 20 mg/kg. If the blood volume of a mouse constitutes 5% of its mass, then the 10 mg/kg dose would yield a peak level of approximately 200 micrograms/ml - some 20 to 50 fold higher than its MIC concentrations.
  • LPS Binding We measured the ability of ovispirin and novispirins to bind LPS in two assays. One assay is spectrophotomentric, and uses a quantitative Limulus chromogenic assay. The other assay is physical, and measures surface plasmon resonance changes.
  • the apparent binding constant of ovispirin for LPS was approximately 6.7 x 10 "6 M.
  • the affinity of novispirins was lower by a factor of 4-5, i.e., between 2.5 and 3.3 x 10 "5 M.
  • the affinity of novispirins for LPS increased.
  • novispirins Effects of novispirins on bacterial membranes. All of the novispirins, when tested at 5 ⁇ g/ml, rapidly permeabilized the outer and inner membranes of E. coli M-35p. The onset of membrane permeabilization is rapid, beginning within 2 minutes and reaching maximal extent within 5 minutes. This experiment was performed with stationary phase E. coli, suspended in PBS (100 mM NaCI). Outer membrane permeabilization was measured by monitoring the hydrolysis of PADAC, a cephalosporin, and inner membrane permeabilization was measured by monitoring hydrolysis of ONPG (this E. coli strain has no lac permease.
  • Table 5 shows the activity of novispirins against S. maltophila.
  • Protegrin PG-1, polymyxin B and PGG were used as controls.
  • PGG is an ⁇ -helica peptide, whose sequence is: GLLRRLRKKIGEIFKKYG. It was designed and reported by Tossi, A., Tarantino, C. and Romeo, D. 1997. Design of synthetic antimicrobial peptides based on sequence analogy and amphipathicity. Eur. J. Biochem. 250: 549-58.
  • G-10 a novel 18-residue a-helical peptide was assayed in this study.
  • G-10 is nonhemolytic and has minimal cytotoxicity when tested against epithelial cells in vitro. It is relatively selective for Gram-negative organisms.
  • C. trachomatis serovar L2 causes invasive LGV
  • serovars D and E represent typical genital strains.
  • the effect of G-10 on Chlamydia trachomatis serovars (L2, D and E) was determined using standard and modified McCoy cell shell vial assays and varying concentrations of peptide.
  • the CWamy ⁇ Va/peptide mixture was pre- incubated for 2 hr., then removed from the host cell monolayer before the 48-hr. incubation.
  • the pre-incubation mixture was left on the monolayer for the 48 hr. incubation.
  • IFUs Inclusion-forming units
  • Example 4 Three new variants of novispirin G10 were prepared and tested to determine if increasing the net positive charge of the peptide renders it more effective in the presence of divalent cations. Amidation (-NH2) of the carboxy terminus in some of the novispirin G10 variants was designed to increase overall positive charge. The sequences of these peptides and their net charge is shown below in Table 7. The fractional (and pH dependent) charge on histidine- 12 was ignored in this calculation.
  • MIC signifies minimal inhibitory concentration, as determined by two stage radial diffusion assays. These were done under standard conditions, except that 10 mM HEPES buffer was used in place of 10 mM phosphate to avoid solubility problems when working with calcium.
  • Bold boxes show results that represent a significant improvement of activity (p ⁇ 0.01 by t-test), relative to novispirin G10. Note that G10, R10 novispirin amide showed improved activity against all four test organisms in the presence of calcium and magnesium and that G10, R10 novispirin was more active against the three Gram negative organisms (P. aeruginosa, E. coli and K. pneumoniae) but was not more effective against S. aureus.
  • Example 5 Four variants of G10 novispirin were synthesized, to see if increasing the positive charge of the N-terminus can also increase resistance to calcium and magnesium. Amino acid sequence of these G10 variants are shown below in Table 9 with their respective net charge.

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Abstract

Les peptides de novispirine sont des agents antimicrobiens avec une activité efficace contre les bactéries Gram négatif. Ces peptides sont non hémolytiques, présentent une cytotoxicité in vivo réduite par rapport à d'autres peptides antimicrobiens, et ils ont été bien tolérés in vivo après une injection intraveineuse. Ces novispirines se lient aussi avec la lipopolysaccharide (LPS), propriété qui peut atténuer les symptômes associés à l'infection par la bactérie Gram négatif. On administre une composition pharmaceutique comprenant de la novispirine comme principe actif à un patient souffrant d'une infection microbienne ou prédisposé à cette infection, et plus particulièrement aux infections par la bactérie Gram négatif.
PCT/US2001/019094 2000-06-28 2001-06-13 Novispirines: peptides antimicrobiens WO2002000839A2 (fr)

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CA002415236A CA2415236A1 (fr) 2000-06-28 2001-06-13 Novispirines: peptides antimicrobiens
BRPI0112035-2A BR0112035A (pt) 2000-06-28 2001-06-13 peptìdeos antimicrobianos de novispirin
IL15355601A IL153556A0 (en) 2000-06-28 2001-06-13 Novispirins: antimicrobial peptides
JP2002505955A JP2004526663A (ja) 2000-06-28 2001-06-13 ノビスピリン抗菌ペプチド
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Cited By (4)

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EP1501530A2 (fr) * 2002-03-21 2005-02-02 Cayman Chemical Company Analogues de la prostaglandine f2 alpha en combinaison avec des antimicrobiens pour le traitement du glaucome
WO2005092365A2 (fr) * 2004-03-29 2005-10-06 Novozymes A/S Utilisation pharmaceutique des novispirines
WO2005105831A1 (fr) 2004-05-04 2005-11-10 Novozymes A/S Polypeptides antimicrobiens
US9745348B2 (en) 2011-09-14 2017-08-29 The University Of Manchester Short designed peptides possessing selective actions against bacteria and cancer cells

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US7071293B1 (en) * 1999-08-18 2006-07-04 The University Of Iowa Research Foundation Alpha helical peptides with broad spectrum antimicrobial activity that are insensitive to salt
WO2008096814A1 (fr) * 2007-02-09 2008-08-14 Genomidea Inc. Nouveau polypeptide et agent antibactérien le renfermant en tant que principe actif

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WO2001012668A1 (fr) * 1999-08-18 2001-02-22 University Of Iowa Research Foundation Peptides derives de la cathelicidine ayant une activite antimicrobienne a large spectre

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WO2001012668A1 (fr) * 1999-08-18 2001-02-22 University Of Iowa Research Foundation Peptides derives de la cathelicidine ayant une activite antimicrobienne a large spectre

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DATABASE GENBANK [Online] 15 May 2001 TACK ET AL.: 'Ovine SMAP 29 cathelicidin derived antimicrobial peptide', XP002963608 Database accession no. (AAB70648) & WO 01 12668 A1 22 February 2001 *
DATABASE PIR_71 [Online] 28 October 1996 MAHONEY ET AL.: 'Cathelin-related protein 2 precursor-sheep (fragment)', XP002963606 Database accession no. (S68411) & FEBS LETTERS vol. 376, 1995, pages 225 - 228 *
MAHONEY ET AL.: 'Molecular analysis of the sheep cathelin family reveals a novel antimicrobial peptide' FEBS LETTERS vol. 377, 1995, pages 519 - 522, XP002963607 *
See also references of EP1359930A2 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1501530A4 (fr) * 2002-03-21 2006-05-03 Cayman Chem Co Analogues de la prostaglandine f2 alpha en combinaison avec des antimicrobiens pour le traitement du glaucome
EP1501530A2 (fr) * 2002-03-21 2005-02-02 Cayman Chemical Company Analogues de la prostaglandine f2 alpha en combinaison avec des antimicrobiens pour le traitement du glaucome
WO2005092365A2 (fr) * 2004-03-29 2005-10-06 Novozymes A/S Utilisation pharmaceutique des novispirines
WO2005092365A3 (fr) * 2004-03-29 2006-04-27 Novozymes As Utilisation pharmaceutique des novispirines
JP2008510452A (ja) * 2004-05-04 2008-04-10 ノボザイムス アクティーゼルスカブ 抗菌ポリペプチド
US7148404B2 (en) 2004-05-04 2006-12-12 Novozymes A/S Antimicrobial polypeptides
WO2005105831A1 (fr) 2004-05-04 2005-11-10 Novozymes A/S Polypeptides antimicrobiens
US7504375B2 (en) 2004-05-04 2009-03-17 Novozymes A/S Antimicrobial polypeptides
EP2267000A3 (fr) * 2004-05-04 2011-07-06 Novozymes Adenium Biotech A/S Polypeptides antimicrobiens
AU2005238127B2 (en) * 2004-05-04 2011-08-18 Novozymes Adenium Biotech A/S Antimicrobial polypeptides
US8080390B2 (en) 2004-05-04 2011-12-20 Novozymes Adenium Biotech A/S Antimicrobial polypeptides
JP2012147783A (ja) * 2004-05-04 2012-08-09 Novozymes Adenium Biotech As 抗菌ポリペプチド
US9745348B2 (en) 2011-09-14 2017-08-29 The University Of Manchester Short designed peptides possessing selective actions against bacteria and cancer cells

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