WO2015065916A1 - Anti microbial peptides incorporating cyclic alpha tetra-substituted unnatural amino acids - Google Patents

Anti microbial peptides incorporating cyclic alpha tetra-substituted unnatural amino acids Download PDF

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Publication number
WO2015065916A1
WO2015065916A1 PCT/US2014/062442 US2014062442W WO2015065916A1 WO 2015065916 A1 WO2015065916 A1 WO 2015065916A1 US 2014062442 W US2014062442 W US 2014062442W WO 2015065916 A1 WO2015065916 A1 WO 2015065916A1
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Prior art keywords
acid
seq
amide
infection
spp
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PCT/US2014/062442
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French (fr)
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Rickey P. Hicks
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East Carolina University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/524—Preservatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to synthetic antimicrobial peptides that include unnatural amino acids and their methods of use for preventing and treating an infection and/or disease.
  • MDROs drug resistant pathogenic microorganisms
  • Pseudomonas aeruginosa which is the leading cause of severe infections in cystic fibrosis patients ( Rowe et al. (2005) N. Engl. J. Med. 352, 1992- 2001). Pseudomonas aeruginosa is also the causative agent of over 200,000 cases o severe hospital-acquired infections each year in the United States (Marr et al. (2006) Curr. Opin. Pharmacol 6, 468-472; Gooderhamet al. (2008) J. Bacterial 190, 5624-5634; Lyczak et al. (2000) Microbes Infect. 2, 1051 -1060; Kapoor et al.
  • CRE carbapenem- resistant Enterobacteriaceae
  • E. coli and Klebsiella pneumonia carbapenem- resistant Enterobacteriaceae
  • the Enterobacteriaceae are a family of Gram-negative bacteria that inhabit the gastrointestinal tract of humans as well as some animals. (Altieri et al. (1995) J. Am. Chem. Soc. 1 17, 7566-7567). They are associated with infections in hospitals and other health-care facilities. (Prevention, C.
  • AMPs Antimicrobial peptides
  • Naturally occurring AMPs are produced by almost every class of living organism as a defense against invading microorganisms (e.g., bacteria, fungi, protozoa, viruses and the like). They are generally small (10-50 amino acid residues), highly positively charged (+2 to +9)
  • amphipathic molecules with well defined hydrophobic and hydrophilic regions. For most AMPs, it is their amphipathicity that allows them to interact with and disrupt bacterial membranes. (See, U.S. patent No: 8,1 88,033; Hoskin et al. Biochim Biophys Acta
  • AMPs are divided into two major classes, those that are membrane disruptive and those that are nonmembrane disruptive.
  • the membrane disrupting AMPs are further divided into those that are cell selective and those that are non-selective.
  • a cell selective AMP is active against bacterial cells, while being inactive against mammalian cells and non-selective membrane disrupting AMPs are active against both mammalian and bacterial cells. The selectivity is believed to be based on the differences in chemical composition between mammalian cells and bacterial cells. (Id.)
  • AMP's for prokaryotic versus eukaryotic cells is believed to be derived from the differences in the chemical compositions of their respective membranes.
  • Bacterial cells contain a high percentage of negatively charged phospholipids while mammalian cells contain a much higher concentration of zwitterionic phospholipids in addition to cholesterol (Papo, N., and Shai, Y. (2003) Biochemistry 42, 9346-9354; Findlay et al. (2010) Antimicrobial Agents and
  • AMPs have a reduced tendency to induce resistance in the target organisms or target cells as compared to traditional antibiotics and therefore present promise for assisting in overcoming the loss of traditional antibiotics.
  • the present invention overcomes the deficiencies in the art by providing new synthetic antimicrobial peptides and methods for their use in treating recalcitrant infections and other diseases, disorders and conditions.
  • One aspect of the invention is a synthetic antimicrobial peptide comprising the formula of: Acetyl-Gly-Phe-A-B-A-C-A-B-E-D-NH 2 (Formula I);
  • Acetyl -Gly-Phe-A-B-A-C-A-B-A-C-A-B-E-D-NH 2 (Formula III); or Acetyl -D-Gly-Phe-A-B-A-C-A-B-A-C-A-B-E-NH 2 (Formula IV);
  • A is a three amino acid sequence of Y-Z-Y, and
  • each Y is a cyclic C" tetra substituted amino acid, each Y can be the same or a different C u tetra substituted amino acid and
  • Z is Glycine, ⁇ -alanine, GABA or 6-aminohexanoic acid
  • each B is independently X, U-X, X-U, U-X-U, or X-X and
  • each X is independently lysine, arginine, histidine, ornithine, 2,3- diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dab), 4-aminopiperidine-4- carboxylic acid (Apc4), or 3-aminopiperidine-3-carboxylic acid (Apc3); and
  • each U is independently glycine, alanine, ⁇ -alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12- Adda), or 16-aminopalmitic acid (16-Apa);
  • each C is independently S, R-S, S-T or R-S-T, and
  • each S is independently phenylalanine, tyrosine, tryptophan, 4- flurophenylalanine (Fpa), 4-clorophenylalanine (Cph), 4-nitrophenylalanine (Nph), phenyl glycine (Phg), valine or isoleucine,
  • each R is independently alanine, ⁇ -alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa), and
  • each T is independently alanine, ⁇ -alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa);
  • each D is independently three to six of any combination of lysine, arginine, histidine, ornithine, 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dab), 4- aminopiperidine-4-carboxylic acid (Apc4), or 3-aminopiperidine-3-carboxylic acid (Apc3), and
  • E is a cyclic C" tetra substituted amino acid.
  • the synthetic antimicrobial peptide comprises the acid sequence of:
  • A6c is 1 -aminocycohexane carboxylic acid
  • A5c is 1 -aminocyclopentane carboxylic acid
  • GABA is Gamma aminobutyric acid
  • Oic is Octahydroindolecarboxylic acid
  • Tic is
  • Tetrahydroisoquinolinecarboxylic acid Dab is diaminobutionic acid, Dpr is diaminopropionic acid, G is glycine, F is phenyalanine, K is lysine, and Ac is acetyl.
  • Another aspect of the invention provides a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention and/or salt thereof, and a pharmaceutically acceptable carrier.
  • Further aspects of the invention provide methods of treating or preventing an infection, disease, and/or condition in a subject comprising, administering to a subject in need thereof an effective amount of at least one synthetic antimicrobial peptide of the invention, a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention, and/or a product comprising one or more synthetic antimicrobial peptides of the invention.
  • Embodiments of the invention include the use, for the preparation of a medicament for the treatment or prevention of an infection, disease, and/or condition in a subject, of a synthetic antimicrobial peptide as described herein.
  • kits comprising one or more synthetic antimicrobial peptides of the invention, and/or a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention, and/or a product comprising one or more synthetic antimicrobial peptides of the invention.
  • Figure 1 shows representative amino acids useful with this invention.
  • FIG. 2 shows the amino acid sequence of TSP-1 (SEQ ID NO: I).
  • the buffer only spectrum (— ) exhibits characteristics of a random coil or a disordered conformation.
  • the micelle spectra ((" " ) and (— )) exhibit characteristics of -helical conlbrmers.
  • Figure 4 shows the amide to alkyl region of the TOCSY spectrum of TSP-1 in buffer. Phe connectivities, Gly connectivities and Lys connectivities are shown.
  • Figure 5 shows the amide to alkyl region of the TOCSY spectrum of TSP-1 in the presence of SDS micelles. Phe connectivities, Gly connectivities and Lys connectivities are shown.
  • Figure 6 shows the amide to alkyl region of the TOCSY spectrum of TSP-1 in the presence of DPC micelles. Phe connectivities, Gly connectivities and Lys connectivities are shown.
  • phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y.
  • phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”
  • the terms “increase,” “increases,” “increased,” “increasing,” and similar terms indicate an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.
  • the terms “reduce,” “reduces,” “reduced,” “reduction,” and similar terms mean a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more. In particular embodiments, the reduction results in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.
  • Effective amount refers to an amount of a compound, composition and/or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect.
  • the effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art.
  • an "effective amount” in any individual case can be determined by one of skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.
  • treat By the term “treat,” “treating,” or “treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation, or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or condition, and/or delay o the onset of a disease or illness.
  • a disease or a condition the term refers to, e.g., a decrease in the symptoms or other manifestations of the infection, disease or condition.
  • treatment provides a reduction in symptoms or other manifestations of the infection, disease or condition by at least about 5%, e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.
  • the terms "treat,” “treating,” or “treatment of and the like refer to, e.g., elimination of or a decrease in the presence or amount of a microorganism (e.g., bacteria, fungi, virus) in said subject.
  • a microorganism e.g., bacteria, fungi, virus
  • the infection, disease, and/or condition is ameliorated, alleviated, severity reduced, symptoms reduced and the like as compared to a similar subject not treated with the synthetic peptides of this invention, thereby treating the infection, disease and/or condition.
  • the treatment of an infection by a bacteria or fungus as described herein can be, for example, bactericidal and/or bacteriostatic and/or fungicidal and/or fungistatic.
  • bacteria refers to killing the bacteria or fungi, respectively
  • bacteriostatic refers to inhibiting or retarding the growth of the bacteria or the fungi, respectively, without killing the microorganism.
  • the terms “eliminate,” “eliminated,” and/or “eliminating” refer to complete cessation of the specified acti vity.
  • the terms “retarding the growth” or “retardation of growth” refers to reducing, delaying, inhibiting, and/or hindering the activity contributing to the growth and multiplication of a microorganism.
  • a subject in need of treatment may be identified by, for example, well-established hallmarks of an infection, such as fever,modul, culture of organisms, and the like, or a subject may be treated prior to infection to prevent or reduce the likelihood of infection in said subject.
  • a “therapeutically effective” amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • prevent refers to prevention and/or delay of the onset of an infection, disease, condition and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the infection, disease, condition and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention.
  • the prevention can be complete, e.g., the total absence of the infection, disease, condition and/or clinical symptom(s).
  • the prevention can also be partial, such that the occurrence of the infection, disease, condition and/or clinical symptom(s) in the subject and/or the severity of onset is less than what would occur in the absence of the present invention.
  • prevention refers to any level of prevention or protection which is of some benefit to a subject, such that there is a reduction in the incidence and/or the severity of the disease in a treated subject, regardless of whether the protection or reduction in incidence and/or severity is partial or complete.
  • prevention refers to a decrease in the symptoms or other manifestations of the infection, disease or condition compared to the symptoms or other manifestations of the infection, disease or condition in the absence of administration of the synthetic peptides of the invention.
  • prevention effective amount is an amount of a synthetic peptide of the invention that is sufficient to prevent and/or delay the onset of an infection, disease, condition and/or associated clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of an infection, disease, condition and/or associated clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention.
  • level of prevention need not be complete, as long as some benefit is provided to the subject.
  • a "subject" of the invention includes any animal that has or is susceptible to an infection, disease or condition involving bacteria and/or fungi, or a disease such as cancer.
  • such subject can be mammals, avians, reptiles, amphibians, or fish.
  • Mammalian subjects include but are not limited to humans, non-human primates (e.g., gorilla, monkey, baboon, and chimpanzee, etc.), dogs, cats, goats, horses, pigs, cattle, sheep, and the like, and laboratory animals (e.g., rats, guinea pigs, mice, gerbils, hamsters, and the like).
  • Avian subjects include but are not limited to chickens, ducks, turkeys, geese, quail, pheasants, and birds kept as pets (e.g., parakeets, parrots, macaws, cockatoos, canaries, and the like).
  • Suitable subjects include both males and females and subjects of any age, including embryonic (e.g., i utero or in ovo), infant, juvenile, adolescent, adult and geriatric subjects
  • a "subject in need" of the methods of the invention can be a subject known to have, suspected of having, or having an increased risk of developing an infection, disease, or condition, including secondary infections, caused by, for example, bacteria or fungi, or a disease or condition such as cancer.
  • the research focus of the present inventor is to develop novel AMPs which incorporate unnatural amino acids at specific locations in the primary sequence as effective antibacterial therapeutic agents ( Bhonsle et al.. (2007) J. Med. Chem. 50, 6545-6553).
  • this invention is directed in part to the discovery that the novel synthetic antimicrobial peptides (AMPs) described herein, which incorporate unnatural amino acids, have physicochemical properties distinct from previously known AMPs. These properties allow the novel synthetic peptides of this invention to be designed to be selective for specific membranes such as those of bacteria, fungi and/or malignant cells.
  • the unnatural amino acids induce semi-rigid conformations onto the peptide backbone, as well as specific regions of hydrophobicity and charge, thus controlling the three-dimensional physicochemical properties of the resulting peptide.
  • the incorporation of unnatural amino acids into the AMPs of this invention offers several advantages over antimicrobial peptides consisting of solely natural amino acids.
  • the synthetic peptides of this invention can be tailored to kill not only bacteria, fungi, and/or malignant cells but can be tailored to the membranes of specific bacterial and/or fungal species and strains or specific cancer cell types by modulating the charge density and location, hydroplasticity, and molecular flexibility of the peptides.
  • one aspect of the invention is a synthetic antimicrobial peptide comprising, consisting essentially of, consisting of the formula of:
  • Acetyl -Gly-Phe-A-B-A-C-A-B-A-C-A-B-E-D-NH 2 (Formula III); or Acetyl -D-Gly-Phe-A-B-A-C-A-B-A-C-A-B-E-NH 2 (Formula IV); wherein
  • A is a three amino acid sequence of Y-Z-Y, and
  • each Y is a cyclic C" tetra substituted amino acid, each Y can be the same or a different C u tetra substituted amino acid and
  • Z is Glycine, ⁇ -alanine, GABA or 6-aminohexanoic acid
  • each B is independently X, U-X, X-V, U-X-V, or X-X and
  • each X is independently lysine, arginine, histidine, ornithine, 2,3- diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dab), 4-aminopiperidine-4- carboxylic acid (Apc4), or 3-aminopiperidine-3-carboxylic acid (Apc3);
  • each U is independently glycine, alanine, ⁇ -alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa); and
  • each V is independently glycine, alanine, ⁇ -alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid ( 16-Apa);
  • each C is independently S, R-S, S-T or R-S-T, and
  • each S is independently phenylalanine, tyrosine, tryptophan, 4- flurophenylalanine (Fpa), 4-clorophenylalanine (Cph), 4-nitrophenylalanine (Nph), phenyl glycine (Phg), valine or isoleucine,
  • each R is independently alanine, ⁇ -alanine, gamma-am i nobutyri c acid (GABA), c-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (l OAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid ( 16-Apa), and
  • each T is independently alanine, ⁇ -alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa);
  • each D is independently three to six of any combination of lysine, arginine, histidine, ornithine, 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dab), 4- aminopiperidine-4-carboxylic acid (Apc4), or 3-aminopiperidine-3-carboxylic acid (Apc3), and
  • the cyclic C a tetra substituted amino acid of the synthetic antimicrobial peptide can comprise, consisting essentially of, or consisting of one more of the followin :
  • a synthetic antimicrobial peptide comprises, consists essentially of, or consists of the amino acid sequence of:
  • A6c is 1 -aminocycohexane carboxylic acid
  • A5c is 1 -aminocyclopentane carboxylic acid
  • GABA is Gamma aminobutyric acid
  • Oic is Octahydroindolecarboxylic acid
  • Tic is Tetrahydroisoquinolinecarboxylic acid
  • Dab is diaminobutionic acid
  • Dpr is diaminopropionic acid
  • G is glycine
  • F is phenyalanine
  • K is lysine
  • Ac is acetyl.
  • Peptides of the present invention may be synthesized using any well-known technique in the art, including solid phase synthesis.
  • Means for synthesizing peptide libraries on a solid phase support are well known in the art. (See U.S. Patent No. 5,834,318; U.S. Patent No. 6,207,807; U.S. Patent No. 6,670,142; U.S. Patent No. 6,599,875; R. Merrifield, J. Am. Chem. Soc, 85:2149-2154 (1963); G. Baray and R. Merrifield, Solid phase peptide synthesis, In The Peptides, E. Gross and J. Meinhofer, eds., Academic Press, New York, l-284p (1980); G. Fields, Solid-Phase Peptide Synthesis, Academic Press, San Diego (1997), all of which are incorporated herein by reference).
  • compositions comprising, consisting essentially of, or consisting of one or more synthetic antimicrobial peptides of the invention (e.g., as defined by the Formulas I to IV and/or the amino acid sequence of any one of SEQ ID NOs:l-19, 23, 24) and/or a salt thereof, and a
  • compositions can comprise from 0.01 to 99.9 weight percent.
  • “Pharmaceutically acceptable carrier” as used herein means any carrier, diluents or excipient which is compatible with the other ingredients of the formulation and not deleterious to the recipient.
  • “Pharmaceutically acceptable” as used herein means that the compound or
  • composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
  • salts shall mean non-toxic salts of the compounds employed in this invention which are generally prepared by reacting the free acid with a suitable organic or inorganic base or the free base with a suitable organic or inorganic acid.
  • suitable organic or inorganic base or the free base with a suitable organic or inorganic acid.
  • suitable organic or inorganic acid examples include, but are not limited to, acetate, aluminum,
  • benzenesulfonate benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, diethanolamine, dihydrochloride, edetate, edisylate, estolate, esylate, iumarate, gluceptate, gluconate, glutamate,
  • administration of a compound, therapeutic agent or known drug with a synthetic peptide of the present invention means administration of a known medication or drug and, in addition, the one or more synthetic peptides of the invention at such time that both the known drug and the one or more synthetic peptides will have a therapeutic effect. In some cases this therapeutic effect will be synergistic.
  • Such concomitant administration can involve concurrent (i.e., at the same time), prior, or subsequent administration of the known drug with respect to the administration of a synthetic peptide of the present invention.
  • a person of skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and the synthetic peptides of the present invention.
  • the synthetic peptides of this invention will be used, either alone or in combination with each other or in combination with one or more other therapeutic medications as described herein, or their salts or esters, for manufacturing a medicament for the purpose of providing treatment for an infection, disease or condition to a patient or subject in need thereof.
  • compositions named in this invention can be present in unsalified form, or in salified form, and the naming o such compounds is intended to include both the original (unsalified) compound and its pharmaceutically acceptable salt.
  • the present invention includes pharmaceutically acceptable salt forms of a synthetic peptides of Formula I to IV (and/or having the amino acid sequence of any of SEQ ID NOs:l-I9, 23, 24).
  • the compositions may include pharmaceutically acceptable adjuvants, stabilizers or carriers.
  • the synthetic peptides may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl ester, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, gelatin, acacia, sodium alginate, polyvinyl-pyrrolidone and/or polyvinyl alcohol, and thus tableted or encapsulated for convenient administration.
  • the synthetic peptides may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cotton seed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride and/or various buffers.
  • ointments, cremes or gels are, for example, paraffin, vaseline, natural waxes, starch cellulose, or polyethyleneglycol.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art and can be implemented here as appropriate.
  • a pharmaceutical composition of the invention can optionally contain, in addition to at least one synthetic peptide, at least one other therapeutic agent useful in the treatment or prevention of an infection, disease and/or condition.
  • synthetic peptides of Formulas I to IV can be combined physically with other compounds in fixed dose
  • the synthetic peptides can be provided in combination with other therapeutics that are provided concurrently, prior to or after the antimicrobial peptides but in separate dosages from the synthetic peptides of this invention.
  • antibiotic compositions are encompassed here, which might include one or more of the synthetic peptides described herein (e.g., as defined by the Formulas I to IV and/or the amino acid sequence of any one of SEQ ID NOs: 1-19, 23, 24), plus combinations of the antimicrobial peptides of this invention and/or other known antimicrobial agents including but not limited to 2-p-sulfanilyanilinoethanol, 4,4'-sulfmyldianiline, 4- sulfanilamidosalicylic acid, acediasulfone, acetosulfone, amikacin, amoxicillin, amphotericin B, ampicillin, apalcillin, apicycline, apramycin, arbekacin, aspoxicillin, azidamfenicol, azithromycin, aztreonam, bacitracin, bambermycin(s), biapenem, brodimoprim, butirosin, capreomycin, carbenicillin, carbenicillin
  • sulfamidochrysoidine sulfanilic acid, sulfoxone, teicoplanin, temafloxacin, temocillin, tetracycline, tetroxoprim, thiamphenicol, thiazolsulfone, thiostrepton, ticarcillin, tigemonam, tobramycin, tosufloxacin, trimethoprim, trospectomycin, trovafloxacin, tuberactinomycin, vancomycin and the like.
  • antimicrobial agents may also include, but are not limited to, anti-fungals, such as amphotericin B, azaserine, candicidin(s), chlorphenesin, dermostatin(s), filipin, fungichromin, mepartricin, nystatin, oligomycin(s), perimycin A, tubercidin, imidazoles, triazoles, griesofulvin and the like.
  • anti-fungals such as amphotericin B, azaserine, candicidin(s), chlorphenesin, dermostatin(s), filipin, fungichromin, mepartricin, nystatin, oligomycin(s), perimycin A, tubercidin, imidazoles, triazoles, griesofulvin and the like.
  • anti-fungals such as amphotericin B, azaserine, candicidin(s), chlorphenesin, dermostatin(s), filipin, fungich
  • compositions of this invention can comprise other antimicrobial peptides known and/or later developed.
  • exemplary AMPs include Magainin peptides, pexiganan acetate, omiganan, Novexatin, and Arenicin.
  • the synthetic peptides and compositions thereof may be administered to a subject by any conventional route of administration, including, but not limited to, oral, buccal, topical, systemic (e.g., transdermal, intranasal, or by suppository), parenteral (e.g., intramuscular, subcutaneous, or intravenous injection) and/or by inhalation. Inhalation can include, for example, nasal or oral inhalation or both.
  • Administration of the compounds directly to the nervous system can include, for example, administration to intracerebral, intraventricular, intracerebralventricular, intrathecal, intracisternal, intraspinal or peri-spinal routes of administration by delivery via intracranial or intravertebral needles or catheters with or without pump devices.
  • the synthetic peptides, and/or compositions thereof described herein may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (9 th Ed. 1995).
  • the active compound(s) (including the physiologically acceptable salts thereof) is typically admixed with, for example, an acceptable carrier.
  • the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient.
  • the carrier may be a solid or a liquid, or both, and is preferably formulated with the compound(s) as a unit-dose formulation, for example, a tablet, which may contain from 0.01 or 0.5% to 95%, or 99%, or any range or amount therein, by weight of the active compound.
  • One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well-known techniques of pharmacy comprising admixing the components, optionally including one or more accessory ingredients.
  • the formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular peptide which is being used.
  • forms suitable for oral administration include solid forms, such as pills, gel caps, tablets, caplets, capsules, granules, and powders (each including immediate release, timed release and sustained release formulations).
  • forms suitable for oral administration also include liquid forms, such as solutions, syrups, elixirs, emulsions, and suspensions.
  • forms useful for parenteral administration include sterile solutions, emulsions and suspensions.
  • compositions of this invention comprise one or more antimicrobial synthetic peptides of this invention (e.g., peptides of Formulas I-IV and/or SEQ ID NOs:l-19, 23, 24) or a salt thereof without any pharmaceutical carriers or excipients.
  • pharmaceutical compositions of this invention comprise one or more synthetic peptides or a salt thereof intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • Carriers are inert pharmaceutical excipients, including, but not limited to, binders, suspending agents, lubricants, flavorings, sweeteners, preservatives, dyes, and coatings.
  • compositions in oral dosage form any of the usual pharmaceutical carriers may be employed.
  • suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like;
  • suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
  • aqueous suspensions comprising the synthetic peptides of the invention in an admixture with excipients suitable for the manufacture of aqueous suspensions can include, for example, a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g. , polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g.
  • a suspending agent such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia
  • dispersing or wetting agents such as a naturally occurring phosphatide (
  • heptadecaethylene oxycetanol a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g. , polyoxyethylene sorbitan mono-oleate).
  • the aqueous suspension can also contain one or more preservatives including but not limited to benzalonium chloride, chlorbutanol, ethyl or n-propyl p-hydroxybenzoate, and/or antioxidants such as sulfite, ascorbic acid, citric acid and/or its salts and/or sodium EDTA.
  • the aqueous suspension can also further contain one or more coloring agents, one or more flavoring agents, and/or one or more sweetening agents, such as sucrose, aspartame or saccharin may also be included in a formulation. Formulations can be adjusted for osmolarity.
  • Oil suspensions for use in the present methods can be formulated by suspending a synthetic peptide compound of this invention in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin, or a mixture of these.
  • the oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cety alcohol.
  • Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose.
  • These formulations can be preserved by the addition of an antioxidant such as ascorbic acid.
  • an injectable oil vehicle see Minto, J. Pharmacol. Exp. Ther. 281 :93 (1997).
  • the pharmaceutical formulations of the invention can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil, as described above, or a mixture of these.
  • Suitable emulsifying agents include naturally occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as
  • the emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.
  • the synthetic peptide compounds of the invention alone or in combination with other suitable components can be made into aerosol formulations (/ ' . e. , they can be
  • Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Formulations of the present invention suitable for parenteral administration can include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • aqueous and non-aqueous sterile suspensions can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter.
  • the compounds are sufficiently soluble they can be dissolved directly in normal saline with or without the use of suitable organic solvents, such as propylene glycol or polyethylene glycol. Dispersions of the finely divided compounds can be made-up in aqueous starch or sodium carboxymethyl cellulose solution, or in suitable oil, such as arachis oil. These formulations can be sterilized by conventional, well-known sterilization techniques.
  • the formulations can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • the concentration of a synthetic peptide in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs.
  • the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, such as a solution of 1 ,3-butanediol.
  • the formulations of commends can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • a synthetic peptide composition suitable for use in the practice of this invention can be administered orally.
  • composition can vary widely depending on the type of composition, size of a unit dosage, kind of excipients, and other factors well known to those of skill in the art.
  • the final composition can comprise, for example, from 0.01 percent by weight (% w) to 100% w of the synthetic peptide, e.g., 0.5% w to 50% w, with the remainder being the excipient or excipients.
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical formulations to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc. suitable for ingestion by the patient. In other embodiments, pharmaceutical formulations for oral administration can be formulated without using any pharmaceutically acceptable carriers.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the pharmaceutical formulation suspended in a diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • a diluents such as water, saline or PEG 400
  • capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin
  • suspensions in an appropriate liquid such as water, saline or PEG 400
  • compositions for oral use can be obtained through combination of one or more synthetic peptides of the present invention with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, i desired, to obtain tablets or dragee cores.
  • suitable solid excipients are carbohydrate or protein fillers and include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxymethyl cellulose, hydroxypropylmethyl-ceUulose or sodium carboxymethylcellulose; and gums including arable and tragacanth; as well as proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, com starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • a flavor e.g., sucrose
  • an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • the synthetic peptides of the present invention can also be administered in the form of suppositories for rectal administration of the drug.
  • These formulations can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperatures and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperatures and will therefore melt in the rectum to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • the synthetic peptides of the present invention can also be administered by intranasal, intraocular, intravaginal, and intrarectal routes including suppositories,
  • the synthetic peptides of the present invention can be delivered transdermally by a topical route formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • Encapsulating materials can also be employed with the synthetic peptides of the present invention and the term "composition" can include the active ingredient in
  • microspheres can be administered via intradermal injection of drug (e.g., mifepristone)-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater. Sci. Polym. Ed. 7:623 (1995); as biodegradable and injectable gel formulations (see, e.g., Gao, Pharm. Res. 12:857 (1995)); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669 (1997)). Both transdermal and intradermal routes afford constant delivery for weeks or months. Cachets can also be used in the delivery of the compounds of the present invention.
  • drug e.g., mifepristone
  • a synthetic peptide may also be delivered by the use of monoclonal antibodies as individual carriers to which the synthetic peptide can be coupled.
  • Synthetic peptides may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxy-ethyl-aspart amide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues.
  • a synthetic peptide can be coupled to a class of
  • biodegradable polymers useful in achieving controlled release of a drug for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels.
  • compositions of the present invention may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydropropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes and/or microspheres.
  • a controlled- release preparation is a pharmaceutical composition capable of releasing the active ingredient at the required rate to maintain constant pharmacological activity for a desirable period of time.
  • dosage forms provide a supply of a drug to the body during a predetermined period of time and thus maintain drug levels in the therapeutic range for longer periods of time than conventional non-controlled formulations.
  • a controlled release composition of the invention provides continuous release of an active agent over, for example, a three day, seven day, ten day and/or fourteen day period of time.
  • the synthetic peptides can be in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, foams, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories, for oral parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation.
  • compositions of the invention may be presented in a form suitable for once-weekly or once-monthly administration; for example, an insoluble salt of the active compound, such as the decanoate salt, may be adapted to provide a depot preparation for intramuscular injection.
  • an insoluble salt of the active compound such as the decanoate salt
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • products comprising at least one synthetic antimicrobial peptide of the present invention, and/or salt thereof, or a pharmaceutical composition thereof, are provided.
  • Non-limiting examples of such products include a bandage, a plaster, a suture, a soap, a tampon, a diaper, a shampoo, a tooth paste, an anti-acne compound, a suncream, a textile, an adhesive, a cleaning solution, a contact lens, and/or an implant.
  • the product can be coated or impregnated or both coated and impregnated with one or more synthetic antimicrobial peptide of the present invention.
  • the product can be a medical device that is coated and/or impregnated with one or more synthetic antimicrobial peptide of the present invention.
  • a shunt designed to drain fluids from the brain following surgery could be coated and/or impregnated with at least one synthetic peptide of this invention to prevent infection.
  • an artificial joint, a dental implant and the like can be coated and/or impregnated with at least one synthetic peptide of this invention to prevent infection following implantation.
  • the pharmaceutical compositions herein will contain, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful, suppository and the like, an amount of the active ingredient necessary to deliver an effective dose as described above.
  • the pharmaceutical compositions herein can contain, per dosage unit, from about 10 to about 2000 mg of the active ingredient, e.g., from aboutlOO mg to about 1000 mg of the active ingredient, e.g., from about 25 to about 600 mg of the active ingredient, e.g., from about 75 to about 400 mg of the active ingredient, e.g., about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200,
  • synthetic antimicrobial peptides suitable for use in the practice of this invention will be administered either singly or concomitantly with at least one or more other compounds or therapeutic agents, e.g., with other agents that treat or prevent an infection, disease and/or condition.
  • the synthetic peptides of this invention can take the form of any known method of administration, depending on the age, weight, health and condition of a patient, and the type of infection, disease and/or condition sought to be treated and stage of infection, disease and/or condition.
  • the synthetic antimicrobial peptides of Formulas I to IV may be useful for treatment of an ulcer (e.g., diabetic foot ulcer infections), a wound (e.g., traumatic blast wound, surgical wound, and the like), a burn, a skin infection, and/or acne.
  • the antimicrobial peptides may be useful to treat or prevent bloodstream infections (e.g., catheter-related bloodstream infections), invasive fungal infections, lung infections in cystic fibrosis patients, systemic multi-resistant Gram positive bacterial infections, and other infections including but not limited to sepsis and/or septic shock.
  • the present invention provides methods of treating an infection, disease, and/or condition in a subject comprising, administering to a subject in need thereof an effective amount of at least one synthetic antimicrobial peptide of the invention, or salt thereof, a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention, or salt thereof, and/or a product comprising one or more synthetic antimicrobial peptides of the invention, or salt thereof, wherein the infection, disease, and/or condition in said subject is treated (e.g., the infection, disease, and/or condition is ameliorated, alleviated, severity reduced, symptoms reduced and the like as compared to a similar subject not treated with the synthetic peptides of this invention), thereby treating the infection, disease and/or condition.
  • the infection, disease, and/or condition is ameliorated, alleviated, severity reduced, symptoms reduced and the like as compared to a similar subject not treated with the synthetic peptides of this invention
  • a still further aspect of the invention is directed to a method of preventing an infection, disease, and/or condition in a subject comprising, administering to a subject in need thereof (e.g., identified as having an increased risk of developing said infection, disease and/or condition) an effective amount of at least one synthetic antimicrobial peptide of the invention, or salt thereof, a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention, or salt thereof, and/or a product comprising one or more synthetic antimicrobial peptides of the invention, or salt thereof, wherein said infection, disease, and/or condition is prevented from developing in said subject or severity of onset of said infection, disease, and/or condition is reduced (as compared to the development or severity of onset of the infection, disease, and/or condition in a similar subject not administered the synthetic antimicrobial peptide of the invention), thereby preventing the infection, disease and/or condition.
  • a subject in need thereof e.g., identified as having an increased risk of developing said infection, disease
  • Another aspect of the invention is an antimicrobial peptide of the invention for use in the treatment of an infection, disease, and/or condition.
  • An additional aspect is an antimicrobial peptide of the invention for use in the treatment of an infection, disease, and/or condition.
  • antimicrobial peptide of the invention for use in the prevention of an infection, disease, and/or condition.
  • a still further aspect of the invention is the use of an antimicrobial peptide of the invention for the manufacture of a medicament for treatment of an infection, disease, and/or condition.
  • An additional aspect is the use of an antimicrobial peptide of the invention for the manufacture of a medicament for prevention of an infection, disease, and/or condition.
  • the infection, disease or condition can be an ulcer (e.g., a diabetic foot ulcer), a wound (e.g., a traumatic blast wound), a burn, and/or a skin infection.
  • a subject may receive a wound or a burn and to prevent an infection from occurring in the wound or burn area(s), the subject can be administered the synthetic antimicrobial peptides of the invention.
  • the wound or burn area may already be infected or diseased and be in need of treatment to reduce, alleviate, ameliorate the infection or disease condition.
  • an infection, disease or condition can be a lung infection in a subject having cystic fibrosis, and/or it can be sepsis and/or septic shock.
  • the infection, disease or condition can be caused by a microorganism, e.g., a bacterium, fungus, virus or combination thereof.
  • the disease or condition to be treated or prevented can be cancer (e.g., breast cancer, ovarian cancer, bladder cancer, stomach cancer, lung cancer or leukemia, or any combination thereof).
  • AMPs Antimicrobial peptides
  • AMPs interact differently with different bacterial species and strains depending on the chemical composition of the bacteria's cell membranes.
  • AMPs interact differently with gram positive bacteria, gram negative bacteria, Mycobacteria.
  • AMPs also interact differently with the membranes of other microorganisms as well as the membranes of malignant cells.
  • the potency and selectivity of an AMP of this invention is believed to be controlled by the complementarity of the physicochemical properties of the AMP and the membrane of the target microorganism or malignant cell.
  • physiochemical properties include but are not limited to amino acid sequence, net charge, amphipathicity,
  • the infection, disease or condition in a subject can be a bacterial or fungal infection or disease
  • the bacterium can be a gram positive bacterium or a gram negative bacterium.
  • the bacterium can be a Mycobacterium spp., which have cell wails that are neither truly gram negative or gram positive.
  • the infection to be treated or prevented is caused by a bacterium that is resistant to traditional antibiotics (e.g., Methicillin-resistant Staphylococcus aureus (MRSA)).
  • MRSA Methicillin-resistant Staphylococcus aureus
  • a method of treating or preventing a infection, disease, or condition in a subject comprising administering to said subject at least one synthetic peptide of this invention, wherein said disease, infection, and/or conditions is caused by a bacteria and/or a fungus.
  • the infection, disease, or condition to be treated or prevented by at least one synthetic peptide of this invention is caused by a Gram-positive and/or a Gram-negative bacterium.
  • an infection, disease or condition in a subject that can be treated or prevented using at least one synthetic peptide of the invention can include but is not limited to an Enterococcus spp. infection, Staphylococcus spp. infection. Klebsiella spp. infection, Acinetobacter spp. infection, Pseudomonas spp infection, Enterobacter spp infection, Yersinia spp. infection, Mycobacterium spp. infection, Brucella spp infection, Bacillus spp. infection, Francisella spp. infection, Salmonella spp. infection, Burkholderia spp. infection, Escherichia spp. infection, Campylobacter spp. infection, Gonorrhea spp. infection, Streptococcus spp. infection, Neisseria spp. infection, Shigella spp. infection, or any combination thereof.
  • an infection, disease or condition in a subject that can be treated or prevented using the synthetic peptides of the invention can include but is not limited to an Enterococcus faecium infection, Staphylococcus aureus infection, Klebsiella pnemoniae infection, Acinetobacter baumannii infection, Pseudomonas aeruginosa infection, Enterobacter aerogenes infection, Yersinia pestis infection, Mycobacterium ranae infection, Mycobacterium avium complex (MAC) infection, Brucella suis infection, Bacillus anthracis infection, Francisella tularensis infection, Salmonella typhimurium infection, Burkholderia pseudomallei infection, Escherichia coli infection, or any combination thereof.
  • an Enterococcus faecium infection Staphylococcus aureus infection, Klebsiella pnemoniae infection, Acinetobacter baumannii infection, Pseudomonas aer
  • the infection, disease or condition in a subject that can be treated or prevented using the synthetic peptides of the invention can be caused by a drug resistant microorganism (e.g., a drug resistant bacterium).
  • a drug resistant microorganism e.g., a drug resistant bacterium
  • the drug resistant microorganism can include but is not limited to Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant and vancomysin intermediate Staphylococcus aureus, vancomycin-resistant Enterococci, carbapenem-resistant
  • the infection, disease, and/or condition in a subject to be treated or prevented using the synthetic peptides of the invention can be caused by, for example, Candida spp., Fusarium spp., Aspergillus spp., Cryptococcus spp., Coccidioides spp., Tinea spp., Sporothrix spp., Blastomyces spp., Histoplasma spp.,
  • an infection, disease, and/or condition to be treated or prevented using the synthetic peptides of the invention can be caused by a fungus including but not limited to Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Fusarium solani, Fusarium culmorum, Tinea unguium, Tinea corporis, Tinea cruris, Sporothrix schenckii, Blastomyces dermatitidis, Histoplasma capsulatum, Pneumocystis carinii, Histoplasma duboisii and/or any combination thereof.
  • a fungus including but not limited to Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus
  • a method of treating or preventing a Staphylococcus aureus infection, a MRSA infection, an Enterococcus faecium infection, a Klebsiella pnemoniae infection, an Acinetobacter baumannii infection, a Pseudomonas aeruginosa infection and/or an Enterobacter aerogenes infection in a subject comprising administering to said subject at least one synthetic peptide of the invention, wherein the at least one synthetic peptide can be Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c) K(A6c)KKKK-amide (SEQ ID NO:l).
  • a method of treating or preventing a lung infection in a subject having cystic fibrosis comprising administering to said subject at least one synthetic peptide of the invention, wherein the at least one synthetic peptide can be Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c) K(A6c)KKK -amide (SEQ ID NO:l).
  • the lung infection can be caused by, for example, a bacterium, including but not limited to, Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, and/or Mycobacterium avium complex (MAC).
  • a bacterium including but not limited to, Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, and/or Mycobacterium avium complex (MAC).
  • a bacterium including but not limited to, Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, and/or Mycobacterium avium complex (MAC).
  • MAC Mycobacterium avium complex
  • the present invention provides a method of preventing sepsis and/or septic shock in a subject caused by a bacterium comprising administering to said subject at least one synthetic antimicrobial peptide of the invention.
  • the at least one synthetic peptide can be Ac-
  • methods of treating and/or preventing cancer comprising administering to a subject in need thereof an effective amount of at least one synthetic antimicrobial peptide of the invention, a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention, and/or a product comprising one or more synthetic antimicrobial peptides of the invention, wherein the cancer in said subject is treated or prevented (as compared to a subject that has not been administered the synthetic peptides of this invention).
  • the types of cancer that may be treated or prevented using the synthetic peptides of the invention include to breast cancer, ovarian cancer, bladder cancer, stomach cancer, lung cancer, leukemia, or any combination thereof.
  • the amount of a synthetic peptide necessary to provide treatment or prevention of an infection, disease, or condition is defined as a therapeutically or a pharmaceutically effective dose.
  • the dosage schedule and amounts effective for this use, i.e., the dosing or dosage regimen will depend on a variety of factors including the stage of the disease, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration is also taken into account.
  • a therapeutically effective dose is also one in which any toxic or detrimental side effects of the active agent is outweighed in clinical terms by therapeutically beneficial effects. It is to be further noted that for each particular subject, specific dosage regimens should be evaluated and adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the compounds.
  • compositions or synthetic peptides disclosed herein can be administered to the subject in a single bolus delivery, via continuous delivery over an extended time period, or in a repeated administration protocol (e.g., by an hourly, daily, weekly, repeated administration protocol).
  • the pharmaceutical formulations of the present invention can be administered, for example, one or more times daily, 3 times per week, weekly and the like. In one embodiment of the present invention, the pharmaceutical formulations of the present invention are orally administered once or twice daily.
  • a therapeutically effective dosage of the biologically active agent(s) can include repeated doses within a prolonged treatment regimen that will yield clinically significant results to provide treatment for an identified infection, disease or condition.
  • Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of targeted exposure symptoms or conditions in the subject.
  • Suitable models in this regard include, for example, murine, rat, porcine, feline, non-human primate, and other accepted animal model subjects known in the art.
  • effective dosages can be determined using in vitro models (e.g., immunologic and histopathologic assays).
  • a therapeutically effective amount of the biologically active agent(s) e.g., amounts that are orally effective, intranasally effective, transdermally effective, intravenously effective, or intramuscularly effective to elicit a desired response.
  • the effective amount may be varied depending upon the particular compound used, the mode of administration, the strength of the preparation, the mode of administration, and the advancement of the disease condition.
  • factors associated with the particular patient being treated including patient age, weight, diet and time of administration, will result in the need to adjust dosages.
  • unit dosage forms of the compounds are prepared for standard administration regimens.
  • the composition can be subdivided readily into smaller doses at the physician's direction.
  • unit dosages can be made up in packeted powders, vials or ampoules and preferably in capsule or tablet form.
  • an effective administration of the synthetic antimicrobial peptide compounds of this invention can be a daily dosage from about 0.02 to about 100 mg kg/day may be utilized, more preferably from about 0.1 to about 75 mg/kg/day, more preferably from about 1 to about 50 mg/kg/day.
  • Suitable dosage ranges for intravenous administration are generally about 10-500 micrograms of active compound per kilogram body weight.
  • the daily dosage may be divided, such as being divided equally into two to four times per day daily dosing.
  • the dosage regime, including the number of doses provided daily can be dependent on the route of
  • kits for use in providing treatment or prevention of an infection, disease or condition After a pharmaceutical composition comprising one or more synthetic antimicrobial peptide compounds of this invention, with the possible addition of one or more other compounds of therapeutic benefit, has been formulated in a suitable carrier, it can be placed in an appropriate container and labeled for providing treatment or prevention of an infection, disease or condition. Additionally, another pharmaceutical comprising at least one other therapeutic agent can be placed in the container as well and labeled for treatment of the indicated infection, disease or condition. Such labeling can include, for example, instructions concerning the amount, frequency and method of administration of each pharmaceutical.
  • Antibacterial screening The peptides that exhibited activity in the calcein leakage assay are selected for screening for antibacterial activity. In vitro antibacterial assays are conducted to determine the antibiotic activity of these synthetic AMPs against the following (ESKAPE) pathogens, which are known to commonly infect chronic wounds. Enterococcus faecium
  • CD spectroscopy is used to determine if the synthesized peptides interact differently with zwitterionic (models for human cell membranes) and anionic (models for bacteria cell membranes) micelles and liposomes.
  • CD spectroscopy is very sensitive and its use to monitor conformational changes in peptides and proteins is well documented (Russell et al. Chem. Phys. Lipids 163:488 (2010); Correa et al. African Journal of Biochemistry Research, 3:164 (2009)). Changes in the intensity or shape of the CD spectrum of a peptide in the presence of a micelle or liposome indicates that the peptide is adopting different
  • LUVs Large unilamellar vesicles (LUVs) comprised of only l-Palmitoyl-2-01eoyl-i7?-Glycero-3- Phosphocholine (POPC) were selected as a simple zwitterionic bilayer whereas LUVs consisting of (4:1 mole ratio) l-Palmitoyl-2-01eoyl-in-Glycero-3-Phosphocholine / 1- Palmitoyl-2-01coyl->s" «-Glycero-3-[Phospho-ra -(l -glycerol)] (Sodium Salt) (POPG)) were selected as a simple anionic bilayer models for human and bacteria membranes respectiviely (Bringezu et al.
  • One member of this new series of synthesized AMPs comprises the amino acid sequence of Ac-GF(A6c)G(A6c) (A6c)G(A6c)F(A6c)G(A6c)GK(A6c)KKKK-amide (SEQ ID NO:l) referred to as "TSP-1" (an AMP of Formula I; see, Figure 2).
  • A6c represents the unnatural amino acid 1 -aminocycohexane carboxylic acid. As shown below, this peptide exhibits broad spectrum in vitro antibacterial activity against at least five clinically relevant drug resistant bacteria strains.
  • TSP-1 was synthesized by the Commercial Research Organization New England Peptide and shipped to ECU on February 29, 2012. The molecular weight of TSP-1 was determined to be 2285 using mass spectral analysis, which is within 0.1% of its calculated average molecular weight. HPLC analysis showed by the percent area method that the purity of the peptide is greater than 95%. Additional Biological Evaluation
  • MIC Minimum Inhibitory Concentrations
  • MIC Minimum inhibitory concentration
  • MMC minimum bactericidal concentrations
  • MBC Minimum bactericidal concentration
  • Meuller Hinton VWR International, Radnor, PA
  • OD optical density
  • Aqueous peptide solution 100 ⁇ at two times of the highest test concentration was added to each well of a sterile 96-well, flat bottomed plate (TPP,
  • the antimicrobial peptide solution was 2-fold serially diluted with sterile distilled water in the wells, with the final peptide concentrations ranging from 0.78 to 100 ng/mL. After adding 100 ⁇ , aliquots of the suspension of the target organisms to the wells, the plates were incubated at 37° C for 24 hours. The MIC was determined by recording the lowest concentration of the peptide that prevented visible turbidity of the target organism, as measured at 600 nm by using an ELISA reader (Biotek synergy HT). Visible turbidity was determined by the OD readings of the tested samples that were significantly greater than that of the medium or background.
  • the MBC was determined by plating 100 ⁇ , from each clear well (>MIC) onto 5% sheep blood agar plates. After incubation for 24 hours, the MBC was determined as the lowest concentration of the test peptide that did not permit visible growth on the surface of the agar (See, Table 1).
  • TSP-1 Sodium dodecyl sulfate (SDS) micelles which are anionic were used as a simple model for the membranes of bacteria cells, while dodecylphosphocholine (DPC) micelles which are zwitterionic were used as a simple model for mammalian cells.
  • FIG. 3 As seen in Figure 3, the CD spectrum of TSP-1 in aqueous buffer exhibits characteristics of a random coil or disordered conformation. On binding to the surface of SDS and DPC micelles, TSP-1 clearly adopts an a-helical like structure. The CD spectra suggest these structures are somewhat different indicating that the helical structures are somewhat different. This data suggest there may be some difference in the selectivity of TSP-1 for bacteria verses mammalian cells. Two dimensional NMR studies are used to confirm this observation.
  • the amide to alkyl region of the TOCSY spectrum ( Figure 5) of TSP-1 in the presence of SDS micelles is very different from the spectrum observed in buffer only.
  • the TOCSY spectrum shows only one amide to alkyl connectivity for each residue type, one Gly, one Phe and one Lys instead o the expected five, two and six connectivities respectively.
  • This data suggest that the peptide is highly ordered and of the residues are in similar environments. This would be observed if the peptide adopted a helical structure. This observation is consistent with the CD spectrum of TSP-1 in the presence of SDS micelles which also indicates that TSP-1 adopts a helical structure in the presence of SDS micelles.
  • the peptide encoded by SEQ ID NO:l is at least as active if not more active than compound 23 against all six bacteria.
  • Compound 53 where the Lys residue has been replaced with 1 ,4-diaminobutanoic acid and compound 56 where the Lys was replaced with Arg exhibited increased activity compared to compound 23. This is because of the derealization of the side chain positive charge associated with the Arg and 1 ,4-diaminobutanoic acid residues compared to Lys residues. A similar or greater increase in activity is expected with the incorporation of the Arg and 1 ,4-diaminobutanoic acid residues into SEQ ID NO:l .
  • Table 2 Comparison of biological activity of TSP-1 to other AMPs against (ESKAPE) atho ens
  • the peptides were tested for activity against 18 pathogenic bacteria: Enterobacter aerogenes, Acinetobacter baumannii, Salmonella enteric, Psuedomonas aeruginosa, Klebsiella pneumonia, Eenterococcus faecalis, MRSA NRS 384, Clostridium difficile, Staphylococcus aureus, Steptococcus pneumonia, Neisseria gonorrhoeae, Bacillus anhtracis (A mes), Francisella tularensis, Yersinia pes I is, Burkholderia pseudomallei, Mycobacterium 5 tuberculosis, MDR-TB, XDR-TB.
  • the peptides were tested at the following concentrations: 100 50 ⁇ g/ml, 25 ⁇ g/ml, 12.5 ⁇ g/ml, 6.25 g ml, 3.13 ⁇ / ⁇ 1, 1.6 ⁇ , 0.8 ⁇ g ml, 0.4 ⁇ g ml, 0.2 ⁇ ⁇ , and 0.1 ⁇ g/ml.
  • Table S Minimum inhibitory concentration of the peptides for eighteen bacterial pathogens and six novel peptides. Results are in pg/ml and ⁇ .
  • AMP 1 (SEQ ID NO:2) incorporates two internal back-to-back Lys substitutions resulting in a very high density of positive charge in these two regions of the peptide.
  • This AMP is the most potent analog against the three strains; Mycobacterium tuberculosis, MDR- TB and XDR-TB exhibiting a MIC value of 4.9 ⁇ .
  • AMP 3 (SEQ ID NO:4), incorporating two single internal Orn substitutions, is the next potent against the three strains exhibiting a MIC value of 1 1.4 ⁇ .
  • AMP 6 (SEQ I NO:23), incorporating two single internal Lys substitutions, exhibits interesting activity with MC valves of 11.4 ⁇ against Mycobacterium tuberculosis, and XDR-TB while the MIC value increases to 22.9 ⁇ against MDR-TB.
  • AMP 4 (SEQ ID NO:5), incorporating two single internal Dab substitutions, and AMP 5 (SEQ ID NO: 6), incorporating two single internal Dpr substitutions, exhibited MIC values of 11.8 and 24.6 ⁇ respectively against Mycobacterium tuberculosis, and increased MIC values of 23.7 and 49.3 ⁇ respectively against MDR-TB and XDR-TB.
  • AMP 2 (SEQ ID NO:3), incorporating two single internal Arg substitutions, exhibited MIC values of 40.8 ⁇ against Mycobacterium tuberculosis, and MDR-TB however the potency against XDR-TB increased to 10.2 mM.
  • the variables include the type, number and placement of the basic amino acids within the AMP's primary sequence. Since the mechanism of action of these AMPs is assumed to involve membrane disruption of some type, this data implies that the chemical compositions of the membranes of the three strains of Mycobacterium tuberculosis vary. Specifically there appears to be a variation in the physicochemical properties of the membranes that interact with the positively charged side chains of the AMPs. In addition to electrostatic interactions, hydrophobic interaction may also play a role in the interactions with XDR-TB.
  • AMP 2 (SEQ ID NO:3) which incorporates the C" tetra substitute amino acid 1- aminocyclohexane carboxylic acid exhibits a MC value of 10.2 ⁇ against XDR-TB.
  • AMP 7 (SEQ ID NO: 10) replaces the C" tetra substitute amino acid with 1 -aminocyclopentane carboxylic acid which is slightly less hydrophobic and exhibits an increased MIC value of >43 ⁇ against XDR-TB.
  • Enterobacter aerogenes is most often associated with opportunistic infections.
  • the activity profile of the AMPs of the invention against Enterobacter aerogenes is very interesting.
  • AMP 1 (SEQ ID NO:2), which incorporates two internal back-to-back Lys substitutions, exhibits a very poor MIC value of >39 ⁇ against this bacteria.
  • AMP 2 (SEQ ID NO:3), which incorporates Arg residues, exhibits a MIC value of 2.6 ⁇ .
  • AMP 3 (SEQ ID NO:4), which incorporates Orn residues, exhibits an increased MIC value of 22.8 ⁇ .
  • AMP 4 (SEQ ID NO:5), which incorporates Dab residues, exhibits a MIC value of 3 ⁇ .
  • AMP 5 (SEQ ID NO: 6), which incorporates Dpr residues, exhibits a decreased MIC value of 1.5 ⁇ .
  • This data suggest that the derealization of the side chain positive charge density plays a major role in defining the activity of these AMPs against Enterobacter aerogenes. In addition to electrostatic interactions, hydrophobic interaction may also play role in the interactions with Enterobacter aerogenes.
  • AMPs 2, 3, 4 and 5 (SEQ ID NOs: 3, 4, 5, and 6, respectively) all incorporate the C tetra substitute amino acid, 1 -aminocyclohexane carboxylic acid.
  • AMP 7 replaces the C a tetra substitute amino acid with 1- aminocyclopentane carboxylic acid which is slightly more hydrophobic and exhibits an increased MIC value of >42.4 ⁇ against Enterobacter aerogenes.
  • the remaining AMPs 6, 8,9,10 and 11 (SEQ ID NOs: 23, 17, 18, 19 and 24, respectively) all incorporate the amino acid Lys and exhibit increased MIC values of > 40 ⁇ , confirming the importance of positive charge derealization.
  • AMPs 1 , 2, 3, 4, 6, 7 and 1 1 (SEQ ID NOs: 2, 3, 4, 5, 23, and 10, respectively) all exhibit low MIC values ( 1 .5 to 3 ⁇ ) against Acinetobacter baumannii.
  • AMP 5 (SEQ ID NO:6), which incorporates the amino acid Dpr, exhibited an increased MIC value of 49.3 ⁇ .
  • AMPs 1 , 2, 3, 4, 6, 7 and 1 1 (SEQ ID NOs: 2, 3, 4, 5, 23, and 10, respectively) all exhibit low MIC values (1.5 to 5.7 ⁇ ) against Salmonella enterica.
  • AMPs 5 and 10 (SEQ ID NO:6 and SEQ ID NO:19, respectively) exhibit moderate MIC values (24.6 and 11.2 ⁇ , respectively). It is interesting to point out that replacing three of the seven the C" tetra substitute amino acid, 1 -aminocyclohexane carboxylic acid with the un-natural amino acids Tic or Oic, dramatically alters the MIC values against Salmonella enterica.
  • AMP 8 (SEQ ID NO:17) incorporates three TIC residues and
  • AMP 9 (SEQ ID NO:18) incorporates three Oic residues.
  • AMP 9(SEQ ID NO: 18) exhibited a MIC value of 41.9 ⁇ , while AMP 8 (SEQ ID NO: 17) exhibited a MIC value of 5.2 ⁇ .
  • This observation suggests that the phenyl ring associated with the Tic residues may increase binding of AMP 8 to the surface of the membrane in a conformation which favors membrane disruption more than the cyclohexane ring associated with the Oic residue.
  • AMPs 1 , 2, 3, 4, 5, 7, 10 and 1 1 (SEQ ID NOs: 2, 3, 4, 5, 6, 10, 19, and 23, respectively) all exhibit low MIC values (1.4 to 5.6 ⁇ ) against Psuedomonas aeruginosa.
  • AMPs 6, 8 and 9 (SEQ ID NOs: 23, 17 and 18, respectively) exhibit moderate MIC values (10-23 ⁇ ).
  • Overall Psuedomonas aeruginosa appears to be relatively insensitive to changes in the structure and physicochemical changes within this series of AMPs.
  • AMPs 4, 5 and 7 (SEQ ID NOs: 5, 6 and 10, respectively) exhibited low MIC values of 2.97, 3.08 and 5.31 ⁇ against Klebsiella pneumonia.
  • AMP 4 (SEQ ID NO:5) contains Dab residues
  • AMP 5 (SEQ ID NO:6) contains Dpr residues
  • AMP 7 (SEQ ID NO: 10) contains Arg residues.
  • AMP 2 (SEQ ID NO: 3) also contains Arg residues but exhibits an increased MIC values of 20.4 ⁇ . The only difference between the two AMPs is that AMP 2 contains 1 -aminocyclohexane carboxylic acid residues and AMP 7 contains 1 -aminocylopentane carboxylic acid residues.
  • AMPs 1 , 2, 3, 4, 5, 7, and 1 1 (SEQ ID NOs: 2, 3, 4, 5, 6, 10, and 24, respectively) exhibited MIC values in the range 0.62 to 6.25 ⁇ against Neisseria gonorrhoeae.
  • AMP 1 (SEQ ID NO:2) is the most active of the AMPs exhibiting a MIC value of 0.61 ⁇ .
  • AMP 1 contains double internal Lys substitutions.
  • AMP 6 (SEQ ID NO: 2) incorporates single internal Lys and replaces 1-aminocyclhexance carboxylic acid residues with 1- aminocylcopentane carboxylic acid residues and further exhibits an increased MIC value of 22.9 ⁇ .
  • AMP 10 (SEQ ID NO: 19), which incorporates alternating 1 -aminocyclohexane carboxylic acid residues with 1 -aminocylcopentane carboxylic acid residues, exhibits a decreased MIC value of 1 1.2 ⁇ . This suggest that the increased hydrophobicity or steric bulk, of the cyclohexane ring favors membrane binding and disruption of the membrane compared to the cyclopentane ring.
  • AMPs 2, 4, 5, and 7 (SEQ ID NOs:3, 5, 6, and 10, respectively) exhibited MIC values in the range of 1.3 to 5.3 ⁇ against Eenterococcus faecalis. These four AMPs containing the four basic amino acids with the highest degree of delocalization of positive charge, Arg, Dab, Dpr and Arg respectively.
  • AMP 3 incorporates Orn residues exhibits a MIC value of 1 1.4 ⁇ , while all Lys containing AMPs except AMP 1 (SEQ ID NO:2), with double internal Lys substitutions (19.7 ⁇ ) and AMP 11 (SEQ ID NO.-24) (Lys cluster relocated from the C-terminus to the N-terminus) (SEQ ID NO: 18; >42 ⁇ ) exhibited MIC values of 21.9 ⁇ .
  • AMPs 2, 4, 5 and 7 (SEQ ID NOs:3, 5, 6, and 10, respectively) exhibited MIC values in the range of 5.1 to 6.1 ⁇ against MRSA NRS 384. These four AMPs contain the four basic amino acids with the highest degree of delocalization of positive charge, Arg, Dab, Dpr and Arg, respectively.
  • AMP 3 (SEQ I NO:4) incorporates Orn residues exhibits a MIC value of 22.8 ⁇ , while all Lys containing AMPs exhibited MIC values of >39.35 ⁇ .
  • the increased activity of AMPs with a degree of positive charge derealization is consist with the fact that the lipid composition of some strains of drug resistant strains of S.
  • aureus contain as much as 38% lysylphosphatidylglycerol, which is a cationic lipid that is known to repel antimicrobial peptides.
  • lysylphosphatidylglycerol which is a cationic lipid that is known to repel antimicrobial peptides.
  • AMPs 2, 3, 4, 5, and 7 (SEQ ID Os:3, 4, 5, 6, and 10, respectively) exhibited MIC values in the range of 1.3 to 6.2 ⁇ against Staphylococcus aureus. Derealization of the side chain positive charge seems to play a role in defining antibacterial activity, however no direct correlation is observed. All of the Lys containing AMPs except AMP 1 (SEQ ID NO:1) (SEQ ID NO:2).
  • AMPs 2, 5 and 1 1 (SEQ ID NOs:3, 6 and 24, respectively), exhibited moderate MIC values of 10.2, 24.6 and 21.9 ⁇ against Steptococcus pneumonia. The remaining AMPs all exhibited MIC values of greater than 39.4 ⁇ .
  • AMPs 1 , 2, 3, 4, 5, 7 and 1 1 (SEQ ID NOs: 2, 3, 4, 5, 6, 10, and 24, respectively) exhibited MIC values in the range 1.4 to 6.2 ⁇ against Bacillus anhtracis (Ames).
  • AMPs 8, 9 and 10 (SEQ ID NOs: 17, 18 and 19, respectively) exhibited increased MIC values of 10.3, 20.9 and 22.4 ⁇ , respectively.
  • AMP 6 (SEQ ID NO:23) exhibited an increased MIC value of >46 ⁇ .
  • Tables 5 and 6 show that the novel peptides have antibacterial activity that is the equivalent of or better than that provided by the comparator art known antibiotic in the case of one or more of the pathogenic bacterial species tested, thereby showing that these peptides can be useful in treating bacterial infections.
  • Microtiter plates (96 well) will be loaded with 200 of medium and 10 4 spores of the fungi under study.
  • a serial dilution of AMP 100 g/mL to 0.01 ng/mL will be prepared and added to different wells of the microtiter plates.
  • 7-hydroxy-3H-phenoxazin-3-one-10-oxide will be used as the redox indicator at a
  • the synthetic peptides of the invention will be screened for inhibitory activity against at least the following fungi: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Candida albicans, Coccidioides immitis,
  • Cryptococcus neoformans Fusarium solani, Fusarium culmorum, Tinea unguium, Tinea corporis, Tinea cruris, Sporothrix schenckii, Blastomyces dermatitidis, Histoplasma capsulatum, Pneumocystis carinii, and Histoplasma duboisii.
  • Cells were grown in RPMI-1640 Supplemented with L-Glutamine, sodium pyruvate and 10% FBS.
  • the cell lines used were CFPAC-1 (Pancreatic cancer), MCF-7 (Breast cancer), IGROV- 1 (Ovarian cancer); Colo205 (colon cancer), DLD-1 (colon cancer), UACC- 62 (Melanoma), MiaPaca-2 (Pancreatic cancer), A498 (Kidney cancer), PC-3 (Prostate cancer), A549 (Lung cancer), NCI-H69 (Small Cell Lung cancer) and RL (Lymphoma).
  • ND denotes insufficient peptide available to conduct assay; CFPAC-1 (Pancreatic cancer), MCF-7 (Breast cancer), IGROV-1 (Ovarian cancer); Colo205 (colon cancer), DL - (colon cancer), UACC-62 (Melanoma), MiaPaca-2 (Pancreatic cancer), A498 (Kidney cancer), PC-3 (Prostate cancer), A549 (Lung cancer), NCI-H69 (Small Cell Lung cancer and RL (Lymphoma)
  • One or more of the 1 -peptides tested exhibited !C 5 o values of less than 7.5 ⁇ against all 12 cancer cell lines. Also, not all 11 -peptides exhibited equal activity against the 12 cancer cell lines indicating a level of selecti vity for the various cancer cell lines that can be used to improve the potency and selectivity of these peptides.

Abstract

The invention relates to synthetic antimicrobial peptides that comprise unnatural amino acids including cyclic Cα tetra substituted amino acids, and methods of use of these synthetic antimicrobial peptides for preventing and/or treating an infection and/or disease.

Description

ANTI MICROBIAL PEPTIDES INCORPORATING CYCLIC ALPHA TETRA- SUBSTITUTED UNNATURAL AMINO ACIDS
STATEMENT OF PRIORITY
This application claims the benefit, under 35 U.S.C. § 1 19 (e), of U.S.
Provisional Application No. 61/897,892, filed October 31 , 2013, the entire contents of which are incorporated by reference herein.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821 , entitled 5218-222WO_ST25.txt, 27,240 bytes in size, generated on October 27, 2013 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.
FIELD OF THE INVENTION
The invention relates to synthetic antimicrobial peptides that include unnatural amino acids and their methods of use for preventing and treating an infection and/or disease.
BACKGROUND OF THE INVENTION
Over the past two decades there has been a dramatic increase in the emergence and spread of multiple drug resistant pathogenic microorganisms (MDROs) (Cookson, B. (2005) JAppl Microbiol 99, 989-996; Davies, J. (1994) Science 264, 375-382; Schutze et al.. (1994) Infection 22, 233-237; Lohner, K. (2009) Gen. Physiol. Biophys. 28, 105-1 16). The development of drug resistant strains of Gram-negative bacteria has become a major worldwide health concern ( Prevention, C. D. C. (2013) Antibiotic Resistance threats in the United States, 2013). One of these strains is Pseudomonas aeruginosa which is the leading cause of severe infections in cystic fibrosis patients ( Rowe et al. (2005) N. Engl. J. Med. 352, 1992- 2001). Pseudomonas aeruginosa is also the causative agent of over 200,000 cases o severe hospital-acquired infections each year in the United States (Marr et al. (2006) Curr. Opin. Pharmacol 6, 468-472; Gooderhamet al. (2008) J. Bacterial 190, 5624-5634; Lyczak et al. (2000) Microbes Infect. 2, 1051 -1060; Kapoor et al. (2011) Antimicrob Agents Chemother 55, 3054-3057). Pseudomonas aeruginosa infections associated with chronic and traumatic wounds, burns and medical implants are a critical concern due to their difficulty to treat. (Overhage et al. (2008) Infection and Immunity 76, 4176-4182; Costerton et al. (1999) Science 284, 1318-1322; Dieter, R. S. (2004) Cardiovasc. Interv. 62, 281 ). The evolution of drug resistant strains of P. aeruginosa have been reported over the past decade (Hancock, R. E. W. and Speert, D. P. (2000) Drug Resist. Updat. 3, 247-255; Chan, C, L., B., and Deber, C. (2004) J Biological Chemistry 279, 38749-38754; Chopra, L.E.A. (2008) Lancet Infect. Dis. 8, 133-139), which has only intensified the health care crisis associated with P.
aeruginosa infections. Particularly problematic has been the development of carbapenem- resistant Enterobacteriaceae (CRE) (including E. coli and Klebsiella pneumonia) due to their difficulty to treat coupled with their high mortality rates as much as 50% (Prevention, C. D. C. .(2013) Vital Signs: Carbapenem-Resistant Enterobacteriaceae, In Morbidity and Mortality Weekly Report). The Enterobacteriaceae are a family of Gram-negative bacteria that inhabit the gastrointestinal tract of humans as well as some animals. (Altieri et al. (1995) J. Am. Chem. Soc. 1 17, 7566-7567). They are associated with infections in hospitals and other health-care facilities. (Prevention, C. D. C.(2013) Vital Signs: Carbapenem-Resistant Enterobacteriaceae, In Morbidity and Mortality Weekly Report. Carbapenem-resistant Enterobacteriacease were rarely observed in the United States before 2000. (Gaynes, R. P., and Culver, D. I I. (1992) Infect. Control I osp. Epidemiol. 13, 10-14). The dramatic and continued evolution of MDROs has resulted in an international health care crisis (Hicks et al. (2007), J Med. Chem. 50, 3026-3036; Bush, K. (2004), ASM News 70, 282-287; Klevens et al. (2007) 2002, Public Health Reports 122, 160-166), stimulating an intensive research effort to develop new classes of antibiotics that exhibit novel mechanisms of activity. (Shlaeset al. (2004) ASM News 70, 275-281 ; Huang et al. (2010) Protein Cell 1, 143-152; Godballe et al. (2011) Chem Biol Drug Des 77, 107-116; Song, J. (2008) Int. J. Antimicrob. Agents 32, S207-S213; Findlay et al. (2010) Antimicrobial Agents and Chemotherapy 54, 4049-4058; Hartl, G. (2000) World Health Organization).
Antimicrobial peptides (AMPs) were first discovered in the 1990's and thus, are a relatively new class of compounds showing promise as potential antibiotic drugs. Naturally occurring AMPs are produced by almost every class of living organism as a defense against invading microorganisms (e.g., bacteria, fungi, protozoa, viruses and the like). They are generally small (10-50 amino acid residues), highly positively charged (+2 to +9)
amphipathic molecules with well defined hydrophobic and hydrophilic regions. For most AMPs, it is their amphipathicity that allows them to interact with and disrupt bacterial membranes. (See, U.S. patent No: 8,1 88,033; Hoskin et al. Biochim Biophys Acta
1778(2):357-375 (2008)).
AMPs are divided into two major classes, those that are membrane disruptive and those that are nonmembrane disruptive. The membrane disrupting AMPs are further divided into those that are cell selective and those that are non-selective. A cell selective AMP is active against bacterial cells, while being inactive against mammalian cells and non-selective membrane disrupting AMPs are active against both mammalian and bacterial cells. The selectivity is believed to be based on the differences in chemical composition between mammalian cells and bacterial cells. (Id.)
The selectivity of AMP's for prokaryotic versus eukaryotic cells is believed to be derived from the differences in the chemical compositions of their respective membranes. Dennison et al. (2005) Protein and peptide Letters 12, 31-39; Fernandez et al. (2009) Biochemica et Biophysica Acta 1788, 1630-1638) Bacterial cells contain a high percentage of negatively charged phospholipids while mammalian cells contain a much higher concentration of zwitterionic phospholipids in addition to cholesterol (Papo, N., and Shai, Y. (2003) Biochemistry 42, 9346-9354; Findlay et al. (2010) Antimicrobial Agents and
Chemotherapy 54, 4049-4058; Godballe et al. (2011) Chem Biol Drug Des 77, 107-116; Huang et al. (2010) Protein Cell I, 143-152; Yeaman, M. R., and Yount, N. Y. (2003) Pharmacological Reviews 55, 27-55; Leontiadou et al. (2006) J. Am. Chem. Soc. 128, 12156- 12161 ; Hancock, R. E. W., and Lehrer, R. (1998) Trends Biotechnol 16, 82-88). The electrostatic interactions that occur between the AMP(s) and the surface of the cell membrane are believed to be determinant for cell selectivity (Bechinger, B. (201 1) J. Pept, Set. 17, 306- 314; Lohner, K. (2009) Gen. Physiol. Biophys. 28, 105-1 16; 12; Wenk, M. R., and Seelig, j. (1998) Biochemistry 37, 3909-3916). Hancock and co-workers (Powers, J.-P. S., and
Hancock, R. E. W. (2003) Peptides 24, 1681-1691) have extended this hypothesis to propose the differences in membrane composition between different strains of bacteria are responsible for the diversity in the potency and selectivity exhibited by a particular AMP against different strains of bacteria.
Generally, AMPs have a reduced tendency to induce resistance in the target organisms or target cells as compared to traditional antibiotics and therefore present promise for assisting in overcoming the loss of traditional antibiotics.
Accordingly, the present invention overcomes the deficiencies in the art by providing new synthetic antimicrobial peptides and methods for their use in treating recalcitrant infections and other diseases, disorders and conditions.
SUMMARY OF THE INVENTION
One aspect of the invention is a synthetic antimicrobial peptide comprising the formula of: Acetyl-Gly-Phe-A-B-A-C-A-B-E-D-NH2 (Formula I);
Acetyl-D-Gly-Phe-A-B-A-C-A-B-B-NH2 (Formula II);
Acetyl -Gly-Phe-A-B-A-C-A-B-A-C-A-B-E-D-NH2 (Formula III); or Acetyl -D-Gly-Phe-A-B-A-C-A-B-A-C-A-B-E-NH2 (Formula IV);
wherein
a) A is a three amino acid sequence of Y-Z-Y, and
i) each Y is a cyclic C" tetra substituted amino acid, each Y can be the same or a different Cu tetra substituted amino acid and
ii) Z is Glycine, β-alanine, GABA or 6-aminohexanoic acid;
b) each B is independently X, U-X, X-U, U-X-U, or X-X and
i) each X is independently lysine, arginine, histidine, ornithine, 2,3- diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dab), 4-aminopiperidine-4- carboxylic acid (Apc4), or 3-aminopiperidine-3-carboxylic acid (Apc3); and
ii) each U is independently glycine, alanine, β-alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12- Adda), or 16-aminopalmitic acid (16-Apa);
c) each C is independently S, R-S, S-T or R-S-T, and
i) each S is independently phenylalanine, tyrosine, tryptophan, 4- flurophenylalanine (Fpa), 4-clorophenylalanine (Cph), 4-nitrophenylalanine (Nph), phenyl glycine (Phg), valine or isoleucine,
ii) each R is independently alanine, β-alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa), and
iii) each T is independently alanine, β-alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa);
d) each D is independently three to six of any combination of lysine, arginine, histidine, ornithine, 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dab), 4- aminopiperidine-4-carboxylic acid (Apc4), or 3-aminopiperidine-3-carboxylic acid (Apc3), and
e) E is a cyclic C" tetra substituted amino acid.
Figure imgf000007_0001
In another aspect of the invention, the synthetic antimicrobial peptide comprises the acid sequence of:
a. Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c) K(A6c) K K-amide (SEQ ID NO:l);
b. Ac-GF(A6c)G(A6c)KK(A6c)G(A6c)F(A6c)G(A6c)G K(A6c)KKKK- amide (SEQ ID NO:2);
c. Ac-GF(A6c)G(A6c)R(A6c)G(A6c)F(A6c)G(A6c)GR(A6c)RRRR-amide (SEQ ID NO:3);
d. Ac-GF(A6c)G(A6c)Orn(A6c)G(A6c)F(A6c)G(A6c)GOm(A6c)Orn-Orn- Orn-Orn-amide (SEQ ID NO:4);
e. Ac-GF(A6c)G(A6c)Dab(A6c)G(A6c)F(A6c)G(A6c)GDab(A6c)Dab-Dab- Dab-Dab-amide (SEQ ID NO:5);
f. Ac-GF(A6c)G(A6c)Dpr(A6c)G(A6c)F(A6c)G(A6c)GDpr(A6c)Dpr-Dpr- Dpr-Dpr-amide (SEQ ID NO:6);
g. Ac-GF(A6c)P-Ala(A6c)K(A6c)p-Ala (A6c)F(A6c) -Ala(A6c)p- AlaK(A6c) KKK-amide (SEQ ID NO:7); h. Ac-GF(A6c)GABA(A6c)K(A6c)GABA(A6c)F(A6c)GABA(A6c)GABA (A6c)KKKK-amide (SEQ ID NO:8);
i. Ac-GF(A5c)G(A5c)KK(A5c)G(A5c)F(A5c)G(A5c)GKK(A5c)KKKK- amide (SEQ ID NO:9);
j . Ac-GF(A5c)G(A5c)R(A5c)G(A5c)F(A5c)G(A5c)GR(A5c)RRRR-amide (SEQ ID NO.-10);
k. Ac-GF(A5c)G(A5c)Om(A5c)G(A5c)F(A5c)G(A5c)GOrn(A5c)Orn-Orn- Orn- Orn-amide (SEQ ID NO: 11):
1. Ac-GF(A5c)G(A5c)Dab(A5c)G(A5c)F(A5c)G(A5c)GDab(A5c)Dab-Dab- Dab-Dab-amide (SEQ ID NO: 12);
m. Ac-GF(A5c)G(A5c)Dpr(A5c)G(A5c)F(A5c)G(A5c)GDpr(A5c)Dpr-Dpr- Dpr-Dpr-amide (SEQ ID NO: 13);
n. Ac-GF(A5c)p-Ala(A5c) (A5c)p-Ala(A5c)F(A5c)P-Ala(A5c)p- AlaK(A5c)KKKK-amide (SEQ ID NO:14);
o. Ac-GF(A5c)GABA(A5c)K(A5c)GABA(A5c)F(A5e)GABA(A5c)GABAK (A5c) KKKK-amide (SEQ ID NO: 15);
p. Ac-GF(A6c)G(A5c)K(A6c)G(A5c)F(A6c)G(A5c)GK(A6c)KKKK-aniide (SEQ ID NO:16);
q. Ac-GF(A6c)G(Tic) (A6c)G(Tic)F(A6c)G(Tic)GK(Tic)KKKK-amide (SEQ ID NO: 17);
r. Ac-GF(A6c)G(Oic)K(A6c)G(Oic)F(A6c)G(Oic)GK(Oic)KKKK-amide (SEQ ID NO: 18);
s. Ac-GF(A5c)G(A6c)K(A5c)G(A6c)F(A5c)G(A6c)G (A5c) KK -amide (SEQ ID NO: 19);
t. Ac-GF(A5c)G(A5c) (A5c)G(A5c)F(A5c)G(A5c)GK(A5c)KKKK-amide (SEQ ID NO:23);
u. Ac-KKKKGF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c)GK(A6c)-amide (SEQ ID NO:24); or any combination thereof, wherein A6c is 1 -aminocycohexane carboxylic acid, A5c is 1 -aminocyclopentane carboxylic acid, GABA is Gamma aminobutyric acid, Oic is Octahydroindolecarboxylic acid, Tic is
Tetrahydroisoquinolinecarboxylic acid, Dab is diaminobutionic acid, Dpr is diaminopropionic acid, G is glycine, F is phenyalanine, K is lysine, and Ac is acetyl. Another aspect of the invention provides a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention and/or salt thereof, and a pharmaceutically acceptable carrier.
Further aspects of the invention provide methods of treating or preventing an infection, disease, and/or condition in a subject comprising, administering to a subject in need thereof an effective amount of at least one synthetic antimicrobial peptide of the invention, a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention, and/or a product comprising one or more synthetic antimicrobial peptides of the invention.
Embodiments of the invention include the use, for the preparation of a medicament for the treatment or prevention of an infection, disease, and/or condition in a subject, of a synthetic antimicrobial peptide as described herein.
Additional aspects of the invention provide kits comprising one or more synthetic antimicrobial peptides of the invention, and/or a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention, and/or a product comprising one or more synthetic antimicrobial peptides of the invention.
These and other aspects of the invention are set forth in more detail in the description of the invention below. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows representative amino acids useful with this invention.
Figure 2 shows the amino acid sequence of TSP-1 (SEQ ID NO: I). Figure 3 shows the CD spectra of TSP-1 in the presence of (A) 150 mM sodium acetate buffer (pH= 4.4) (— ) (B) 100 mM SDS micelles in buffer (-·) and (C) 100 mM DPC micelles in buffer (— ). The buffer only spectrum (— ) exhibits characteristics of a random coil or a disordered conformation. The micelle spectra (("") and (— )) exhibit characteristics of -helical conlbrmers.
Figure 4 shows the amide to alkyl region of the TOCSY spectrum of TSP-1 in buffer. Phe connectivities, Gly connectivities and Lys connectivities are shown. Figure 5 shows the amide to alkyl region of the TOCSY spectrum of TSP-1 in the presence of SDS micelles. Phe connectivities, Gly connectivities and Lys connectivities are shown. Figure 6 shows the amide to alkyl region of the TOCSY spectrum of TSP-1 in the presence of DPC micelles. Phe connectivities, Gly connectivities and Lys connectivities are shown.
DETAILED DESCRIPTION OF THE INVENTION
The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.
Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
As used in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of
combinations when interpreted in the alternative ("or").
The term "about," as used herein when referring to a measurable value such as a dosage or time period and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.
As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y."
The term "comprise," "comprises" and "comprising" as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
As used herein, the transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term "consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising."
As used herein, the terms "increase," "increases," "increased," "increasing," and similar terms indicate an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.
As used herein, the terms "reduce," "reduces," "reduced," "reduction," and similar terms mean a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more. In particular embodiments, the reduction results in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.
"Effective amount" as used herein refers to an amount of a compound, composition and/or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an "effective amount" in any individual case can be determined by one of skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.
By the term "treat," "treating," or "treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation, or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or condition, and/or delay o the onset of a disease or illness. With respect to an infection, a disease or a condition, the term refers to, e.g., a decrease in the symptoms or other manifestations of the infection, disease or condition. In some embodiments, treatment provides a reduction in symptoms or other manifestations of the infection, disease or condition by at least about 5%, e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.
Further, with respect to an infection, a disease or a condition, the terms "treat," "treating," or "treatment of and the like refer to, e.g., elimination of or a decrease in the presence or amount of a microorganism (e.g., bacteria, fungi, virus) in said subject. Thus, by treating the infection, disease, and/or condition in said subject, the infection, disease, and/or condition is ameliorated, alleviated, severity reduced, symptoms reduced and the like as compared to a similar subject not treated with the synthetic peptides of this invention, thereby treating the infection, disease and/or condition. Thus, in some embodiments, the treatment of an infection by a bacteria or fungus as described herein can be, for example, bactericidal and/or bacteriostatic and/or fungicidal and/or fungistatic.
The terms "bactericidal," "fungicidal," and the like, refer to killing the bacteria or fungi, respectively), and the terms "bacteriostatic" or "fungistatic," and the like, refer to inhibiting or retarding the growth of the bacteria or the fungi, respectively, without killing the microorganism.
As used herein, the terms "eliminate," "eliminated," and/or "eliminating" refer to complete cessation of the specified acti vity. As used herein, the terms "retarding the growth" or "retardation of growth" refers to reducing, delaying, inhibiting, and/or hindering the activity contributing to the growth and multiplication of a microorganism.
In some embodiments, a subject in need of treatment may be identified by, for example, well-established hallmarks of an infection, such as fever, puis, culture of organisms, and the like, or a subject may be treated prior to infection to prevent or reduce the likelihood of infection in said subject.
A "therapeutically effective" amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
The terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to prevention and/or delay of the onset of an infection, disease, condition and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the infection, disease, condition and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. The prevention can be complete, e.g., the total absence of the infection, disease, condition and/or clinical symptom(s). The prevention can also be partial, such that the occurrence of the infection, disease, condition and/or clinical symptom(s) in the subject and/or the severity of onset is less than what would occur in the absence of the present invention. Thus, the terms "prevent," "preventing," and "prevention" and like terms are used herein to include imparting any level of prevention or protection which is of some benefit to a subject, such that there is a reduction in the incidence and/or the severity of the disease in a treated subject, regardless of whether the protection or reduction in incidence and/or severity is partial or complete. With respect to an infection, a disease, and/or a condition in a subject, the term refers to, e.g., preventing the infection, disease, and/or condition from occurring if the treatment is administered prior to the onset of the infection, disease, or condition. In some embodiments, prevention refers to a decrease in the symptoms or other manifestations of the infection, disease or condition compared to the symptoms or other manifestations of the infection, disease or condition in the absence of administration of the synthetic peptides of the invention.
A "prevention effective" amount as used herein is an amount of a synthetic peptide of the invention that is sufficient to prevent and/or delay the onset of an infection, disease, condition and/or associated clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of an infection, disease, condition and/or associated clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.
A "subject" of the invention includes any animal that has or is susceptible to an infection, disease or condition involving bacteria and/or fungi, or a disease such as cancer. Thus, such subject can be mammals, avians, reptiles, amphibians, or fish. Mammalian subjects include but are not limited to humans, non-human primates (e.g., gorilla, monkey, baboon, and chimpanzee, etc.), dogs, cats, goats, horses, pigs, cattle, sheep, and the like, and laboratory animals (e.g., rats, guinea pigs, mice, gerbils, hamsters, and the like). Avian subjects include but are not limited to chickens, ducks, turkeys, geese, quail, pheasants, and birds kept as pets (e.g., parakeets, parrots, macaws, cockatoos, canaries, and the like).
Suitable subjects include both males and females and subjects of any age, including embryonic (e.g., i utero or in ovo), infant, juvenile, adolescent, adult and geriatric subjects A "subject in need" of the methods of the invention can be a subject known to have, suspected of having, or having an increased risk of developing an infection, disease, or condition, including secondary infections, caused by, for example, bacteria or fungi, or a disease or condition such as cancer.
The research focus of the present inventor is to develop novel AMPs which incorporate unnatural amino acids at specific locations in the primary sequence as effective antibacterial therapeutic agents ( Bhonsle et al.. (2007) J. Med. Chem. 50, 6545-6553).
Incorporation of unnatural amino acids into a peptide sequence provides a "toolbox" of different physicochemical properties that are not available in peptides incorporating only the 20 RNA encoded amino acids.(Goodman, M., and Shao, H. (1996) Pure and Appl. CHem. 66, 1303-1308; Hendrickson et al. (2004) Annu. Rev. Biochem. 73, 147-176; Ma, J. S. (2003) CHIMICA OGGI Chemistry today, 65-68; Oh, J. E., and Lee, K. H. ( 1999) Bioorganic & Medicinal Chemistry 7, 2985-2990; Vasudev et al. (2010) Chemical Reviews
10.1021/crlOOlOOx published online). These physicochemical properties include molecular flexibility, the ability to adopt novel helical conformations, modification of the electronic surface properties and hydrophobicity among others.
It is well accepted that the non-covalent interactions that occur between two molecules is controlled by the complementary nature of the surface physicochemical properties of the two (Hartell et al. J Pharm Sci, 93:2076 (2004)). It is also well known that the chemical composition of the membranes of mammalian and bacteria cells are different, while the chemical composition of different bacteria strains also varies (Hancock, R. E. W. Exp. Opin. Invest. Drugs 7:167 (1998)). Therefore the physicochemical surface properties of the membranes will vary (Bhonsle et al. J. Med. Chem. 50:6545 (2007)). The conformation of antimicrobial peptides change on binding with lipid membranes and the final conformation adopted is a result of maximizing attractive interactions and minimizing the repulsive interactions occurring between the surfaces of the two. By careful placement of unnatural amino acid residues with well defined hydrophobic and electrostatic properties, AMPs with increased potency and selectivity against specific bacteria strains may be developed (Bhonsle et al. J. Med. Chem. 50:6545 (2007); (I licks et al. J. Med. Chem. 50:3026 (2007); (Venugopal et al. Bioorganic & Medicinal Chemistry 18:5137 (2010)). Using protocols developed in the inventor's laboratory (Hicks, R. P.; Russell, A. L. In Unnatural Amino Acids: Methods and Protocols, Methods in Molecular Biology; Pollegioni, L., Servi, S., Eds.; Springer, Science 2012; Vol. 794, pp. 135) a new series of antimicrobial peptides are designed having greater potency and selectivity for drag restraint strains of bacteria.
Thus, this invention is directed in part to the discovery that the novel synthetic antimicrobial peptides (AMPs) described herein, which incorporate unnatural amino acids, have physicochemical properties distinct from previously known AMPs. These properties allow the novel synthetic peptides of this invention to be designed to be selective for specific membranes such as those of bacteria, fungi and/or malignant cells. The unnatural amino acids induce semi-rigid conformations onto the peptide backbone, as well as specific regions of hydrophobicity and charge, thus controlling the three-dimensional physicochemical properties of the resulting peptide. The incorporation of unnatural amino acids into the AMPs of this invention offers several advantages over antimicrobial peptides consisting of solely natural amino acids. These advantages include greater metabolic stability, greater control over local and global molecular structural and physicochemical properties. Thus, the synthetic peptides of this invention can be tailored to kill not only bacteria, fungi, and/or malignant cells but can be tailored to the membranes of specific bacterial and/or fungal species and strains or specific cancer cell types by modulating the charge density and location, hydroplasticity, and molecular flexibility of the peptides.
Accordingly, one aspect of the invention is a synthetic antimicrobial peptide comprising, consisting essentially of, consisting of the formula of:
Acetyl-Gly-Phe-A-B-A-C-A-B-E-D-NH2 (Formula I);
Acetyl-D-Gly-Phe-A-B-A-C-A-B-E-NH2 (Formula H);
Acetyl -Gly-Phe-A-B-A-C-A-B-A-C-A-B-E-D-NH2 (Formula III); or Acetyl -D-Gly-Phe-A-B-A-C-A-B-A-C-A-B-E-NH2 (Formula IV); wherein
a) A is a three amino acid sequence of Y-Z-Y, and
i) each Y is a cyclic C" tetra substituted amino acid, each Y can be the same or a different Cu tetra substituted amino acid and
ii) Z is Glycine, β-alanine, GABA or 6-aminohexanoic acid;
b) each B is independently X, U-X, X-V, U-X-V, or X-X and
i) each X is independently lysine, arginine, histidine, ornithine, 2,3- diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dab), 4-aminopiperidine-4- carboxylic acid (Apc4), or 3-aminopiperidine-3-carboxylic acid (Apc3);
ii) each U is independently glycine, alanine, β-alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa); and
iii) each V is independently glycine, alanine, β-alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid ( 16-Apa);
c) each C is independently S, R-S, S-T or R-S-T, and
i) each S is independently phenylalanine, tyrosine, tryptophan, 4- flurophenylalanine (Fpa), 4-clorophenylalanine (Cph), 4-nitrophenylalanine (Nph), phenyl glycine (Phg), valine or isoleucine,
ii) each R is independently alanine, β-alanine, gamma-am i nobutyri c acid (GABA), c-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (l OAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid ( 16-Apa), and
iii) each T is independently alanine, β-alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa);
d) each D is independently three to six of any combination of lysine, arginine, histidine, ornithine, 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dab), 4- aminopiperidine-4-carboxylic acid (Apc4), or 3-aminopiperidine-3-carboxylic acid (Apc3), and
e) E i a cyclic Ca tetra substituted amino acid. The amino acids described above and their structures are known in the art. See
Figure 1 for structures of many of these amino acids.
In some aspects of the invention, the cyclic Ca tetra substituted amino acid of the synthetic antimicrobial peptide can comprise, consisting essentially of, or consisting of one more of the followin :
Figure imgf000017_0001
Based on Formulas I to IV, a number of new synthetic peptides have been synthesized having the amino acid sequence of SEQ ID NOs:l to 19. Thus, in representative embodiments of the invention, a synthetic antimicrobial peptide comprises, consists essentially of, or consists of the amino acid sequence of:
a. Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c) K(A6c)KKKK-amide (SEQ ID
NO: l); b. Ac-GF(A6c)G(A6c)K (A6c)G(A6c)F(A6c)G(A6c)GK (A6c) K K-amide (SEQ ID NO:2);
c. Ac-GF(A6c)G(A6c)R(A6c)G(A6c)F(A6c)G(A6c)GR(A6c)RRRR-amide (SEQ ID NO:3);
d. Ac-GF(A6c)G(A6c)0rn(A6c)G(A6c)F(A6c)G(A6c)G0rn(A6c)0rn-0rn-0m- Orn-amide (SEQ ID NO: 4);
e. Ac-GF(A6c)G(A6c)Dab(A6c)G(A6c)F(A6c)G(A6c)GDab(A6c)Dab-Dab-Dab- Dab-amide (SEQ ID NO:5);
f. Ac-GF(A6c)G(A6c)Dpr(A6c)G(A6c)F(A6c)G(A6c)GDpr(A6c)Dpr-Dpr-Dpr-Dpr- amide (SEQ ID NO:6);
g. Ac-GF(A6c) -Ala(A6c) (A6c)(3-Ala (A6c)F(A6c)P-Ala(A6c)p- AlaK(A6c)KKKK-amidc (SEQ ID NO:7);
h. Ac-GF(A6c)GABA(A6c)K(A6c)GABA(A6c)F(A6c)GABA(A6c)GABAK (A6c)KKK -amide (SEQ ID NO:8);
i. Ac-GF(A5c)G(A5c)KK(A5c)G(A5c)F(A5c)G(A5c)GKK(A5c)KKKK-amide (SEQ ID NO:9);
j. Ac-GF(A5c)G(A5c)R(A5c)G(A5c)F(A5c)G(A5c)GR(A5c)RRRR-amide (SEQ ID NO: 10);
k. Ac-GF(A5c)G(A5c)Orn(A5c)G(A5c)F(A5c)G(A5c)GOrn(A5c)Orn-Orn-Om-
Orn-amide (SEQ ID NO: 11);
1. Ac-GF(A5c)G(A5c)Dab(A5c)G(A5c)F(A5c)G(A5c)GDab(A5c)Dab-Dab-Dab- Dab-amide (SEQ ID NO: 12);
m. Ac-GF(A5c)G(A5c)Dpr(A5c)G(A5c)F(A5c)G(A5c)GDpr(A5c)Dpr-Dpr-Dpr- Dpr-amide (SEQ ID NO: 13);
n. Ac-GF(A5c) -Ala(A5c)K(A5c) -Ala(A5c)F(A5c)p-Ala(A5c)P- AlaK(A5c)KKKK-amide (SEQ ID NO:14);
o. Ac-GF(A5c)GABA(A5c)K(A5c)GABA(A5c)F(A5c)GABA(A5c)GABAK (A5c) KKKK-amide (SEQ ID NO: 15);
p. Ac-GF(A6c)G(A5c)K(A6c)G(A5c)F(A6c)G(A5c)GK(A6c)KKKK-aniide (SEQ
ID NO: 16);
q. Ac-GF(A6c)G(Tic)K(A6c)G(Tic)F(A6c)G(Tic)GK(Tic)KKKK-amide (SEQ ID NO:17); r. Ac-GF(A6c)G(Oic)K(A6c)G(Oic)F(A6c)G(Oic)G (Oic) KK-amide (SEQ ID NO:18);
s. Ac-GF(A5c)G(A6c)K(A5c)G(A6c)F(A5c)G(A6c)GK(A5c)KKKK-amide (SEQ ID NO:19);
t. Ac-GF(A5c)G(A5c)K(A5c)G(A5c)F(A5c)G(A5c)GK(A5c)KKKK-amide (SEQ
ID NO:23);
u. Ac-K K GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c)GK(A6c)-amide (SEQ ID NO:24); or any combination thereof, wherein A6c is 1 -aminocycohexane carboxylic acid, A5c is 1 -aminocyclopentane carboxylic acid, GABA is Gamma aminobutyric acid, Oic is Octahydroindolecarboxylic acid, Tic is Tetrahydroisoquinolinecarboxylic acid, Dab is diaminobutionic acid, Dpr is diaminopropionic acid, G is glycine, F is phenyalanine, K is lysine, and Ac is acetyl.
Peptides of the present invention may be synthesized using any well-known technique in the art, including solid phase synthesis. Means for synthesizing peptide libraries on a solid phase support are well known in the art. (See U.S. Patent No. 5,834,318; U.S. Patent No. 6,207,807; U.S. Patent No. 6,670,142; U.S. Patent No. 6,599,875; R. Merrifield, J. Am. Chem. Soc, 85:2149-2154 (1963); G. Baray and R. Merrifield, Solid phase peptide synthesis, In The Peptides, E. Gross and J. Meinhofer, eds., Academic Press, New York, l-284p (1980); G. Fields, Solid-Phase Peptide Synthesis, Academic Press, San Diego (1997), all of which are incorporated herein by reference).
Another aspect of the present invention provides pharmaceutical compositions comprising, consisting essentially of, or consisting of one or more synthetic antimicrobial peptides of the invention (e.g., as defined by the Formulas I to IV and/or the amino acid sequence of any one of SEQ ID NOs:l-19, 23, 24) and/or a salt thereof, and a
pharmaceutically acceptable carrier. The active ingredient in such formulations can comprise from 0.01 to 99.9 weight percent. "Pharmaceutically acceptable carrier" as used herein means any carrier, diluents or excipient which is compatible with the other ingredients of the formulation and not deleterious to the recipient.
"Pharmaceutically acceptable" as used herein means that the compound or
composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
The term "pharmaceutically acceptable salts" shall mean non-toxic salts of the compounds employed in this invention which are generally prepared by reacting the free acid with a suitable organic or inorganic base or the free base with a suitable organic or inorganic acid. Examples of such salts include, but are not limited to, acetate, aluminum,
benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, diethanolamine, dihydrochloride, edetate, edisylate, estolate, esylate, iumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, , hydrobromide, hydrochloride, hydroxynapthoatc, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine, oleate, oxalate, pamoate, palmitate, panthothenate, phosphate/diphosphate, polygalacturonate, potassium, salicylate, , stcarate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, tromethamine, or valerate, and the like.
As used herein the term "concomitant administration" or "combination
administration" of a compound, therapeutic agent or known drug with a synthetic peptide of the present invention means administration of a known medication or drug and, in addition, the one or more synthetic peptides of the invention at such time that both the known drug and the one or more synthetic peptides will have a therapeutic effect. In some cases this therapeutic effect will be synergistic. Such concomitant administration can involve concurrent (i.e., at the same time), prior, or subsequent administration of the known drug with respect to the administration of a synthetic peptide of the present invention. A person of skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and the synthetic peptides of the present invention.
In addition, in some embodiments, the synthetic peptides of this invention will be used, either alone or in combination with each other or in combination with one or more other therapeutic medications as described herein, or their salts or esters, for manufacturing a medicament for the purpose of providing treatment for an infection, disease or condition to a patient or subject in need thereof.
Compounds named in this invention can be present in unsalified form, or in salified form, and the naming o such compounds is intended to include both the original (unsalified) compound and its pharmaceutically acceptable salt. The present invention includes pharmaceutically acceptable salt forms of a synthetic peptides of Formula I to IV (and/or having the amino acid sequence of any of SEQ ID NOs:l-I9, 23, 24). The compositions may include pharmaceutically acceptable adjuvants, stabilizers or carriers. For instance, the synthetic peptides may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl ester, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, gelatin, acacia, sodium alginate, polyvinyl-pyrrolidone and/or polyvinyl alcohol, and thus tableted or encapsulated for convenient administration. Alternatively, the synthetic peptides may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cotton seed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride and/or various buffers. Appropriate additives for the use as ointments, cremes or gels are, for example, paraffin, vaseline, natural waxes, starch cellulose, or polyethyleneglycol. Other adjuvants and modes of administration are well known in the pharmaceutical art and can be implemented here as appropriate.
A pharmaceutical composition of the invention can optionally contain, in addition to at least one synthetic peptide, at least one other therapeutic agent useful in the treatment or prevention of an infection, disease and/or condition. For example the synthetic peptides of Formulas I to IV can be combined physically with other compounds in fixed dose
combinations to simplify their administration. Alternatively, the synthetic peptides can be provided in combination with other therapeutics that are provided concurrently, prior to or after the antimicrobial peptides but in separate dosages from the synthetic peptides of this invention.
For instance, antibiotic compositions are encompassed here, which might include one or more of the synthetic peptides described herein (e.g., as defined by the Formulas I to IV and/or the amino acid sequence of any one of SEQ ID NOs: 1-19, 23, 24), plus combinations of the antimicrobial peptides of this invention and/or other known antimicrobial agents including but not limited to 2-p-sulfanilyanilinoethanol, 4,4'-sulfmyldianiline, 4- sulfanilamidosalicylic acid, acediasulfone, acetosulfone, amikacin, amoxicillin, amphotericin B, ampicillin, apalcillin, apicycline, apramycin, arbekacin, aspoxicillin, azidamfenicol, azithromycin, aztreonam, bacitracin, bambermycin(s), biapenem, brodimoprim, butirosin, capreomycin, carbenicillin, carbomycin, carumonam, cefadroxil, cefamandole, cefatrizine, cefbuperazone, cefclidin, cefdinir, cefditoren, cefepime, cefetamet, cefixime, cefmenoxime, cefininox, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefozopran, cefpimizole, eefpiramide, cefpirome, cefprozil, cefroxadine, ceftazidime, cefteram, ceftibuten, ceftriaxone, cefuzonam, cephalexin, cephaloglycin, cephalosporin C, cephradine, chloramphenicol, chlortetracycline, ciprofloxacin, clarithromycin, clinafloxacin, clindamycin, clindamycin phosphate, clomocycline, colistin, cyclacillin, dapsone, demeclocycline, diathymosulfone, dibekacin, dihydrostreptomycin, dirithromycin, doxycycline, enoxacin, enviomycin, epicillin, erythromycin, flomoxef, fortimicin(s), gentamicin(s), glucosulfone solasulfone, gramicidin S, gramicidin(s), grepafloxacin, guamecycline, hetacillin, imipenem, isepamicin, josamycin, kanamycin(s), leucomycin(s), lincomycin, lomefloxacin, lucensomycin, lymecycline, meclocycline, meropenem, methacycline, micronomicin, midecamycin(s), minocycline, moxalactam, mupirocin, nadifloxacin, natamycin, neomycin, netilmicin, norfloxacin, oleandomycin, oxytetracycline, p-sulfanilylbenzylamine, panipenem, paromomycin, pazufloxacin, penicillin N, pipacycline, pipemidic acid, polymyxin, primycin, quinacillin, ribostamycin, rifamide, rifampin, rifamycin SV, rifapentine, rifaximin, ristocetin, ritipenem, rokitamycin, rolitetracycline, rosaramycin, roxithromycin, salazosulfadimidine, sancycline, sisomicin, sparfloxacin, spectinomycin, spiramycin, streptomycin, succisulfone, sulfachrysoidine, sulfaloxic acid,
sulfamidochrysoidine, sulfanilic acid, sulfoxone, teicoplanin, temafloxacin, temocillin, tetracycline, tetroxoprim, thiamphenicol, thiazolsulfone, thiostrepton, ticarcillin, tigemonam, tobramycin, tosufloxacin, trimethoprim, trospectomycin, trovafloxacin, tuberactinomycin, vancomycin and the like. Exemplary antimicrobial agents may also include, but are not limited to, anti-fungals, such as amphotericin B, azaserine, candicidin(s), chlorphenesin, dermostatin(s), filipin, fungichromin, mepartricin, nystatin, oligomycin(s), perimycin A, tubercidin, imidazoles, triazoles, griesofulvin and the like. Further exemplary antimicrobial agents can include, but are not limited to anti-virals, such as acyclovir, valacyclovir, famcyclovir, gancyclovir, amantadine and others known in the art or later developed. In addition, the compositions of this invention can comprise other antimicrobial peptides known and/or later developed. Exemplary AMPs include Magainin peptides, pexiganan acetate, omiganan, Novexatin, and Arenicin.
The synthetic peptides and compositions thereof may be administered to a subject by any conventional route of administration, including, but not limited to, oral, buccal, topical, systemic (e.g., transdermal, intranasal, or by suppository), parenteral (e.g., intramuscular, subcutaneous, or intravenous injection) and/or by inhalation. Inhalation can include, for example, nasal or oral inhalation or both. Administration of the compounds directly to the nervous system can include, for example, administration to intracerebral, intraventricular, intracerebralventricular, intrathecal, intracisternal, intraspinal or peri-spinal routes of administration by delivery via intracranial or intravertebral needles or catheters with or without pump devices. Depending on the route of administration, the synthetic peptides, and/or compositions thereof described herein may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (9th Ed. 1995). In the manufacture of a pharmaceutical formulation according to the invention, the active compound(s) (including the physiologically acceptable salts thereof) is typically admixed with, for example, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound(s) as a unit-dose formulation, for example, a tablet, which may contain from 0.01 or 0.5% to 95%, or 99%, or any range or amount therein, by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well-known techniques of pharmacy comprising admixing the components, optionally including one or more accessory ingredients.
The formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular peptide which is being used. For example, forms suitable for oral administration include solid forms, such as pills, gel caps, tablets, caplets, capsules, granules, and powders (each including immediate release, timed release and sustained release formulations). Forms suitable for oral administration also include liquid forms, such as solutions, syrups, elixirs, emulsions, and suspensions. In addition, forms useful for parenteral administration include sterile solutions, emulsions and suspensions.
In certain embodiments, pharmaceutical compositions of this invention comprise one or more antimicrobial synthetic peptides of this invention (e.g., peptides of Formulas I-IV and/or SEQ ID NOs:l-19, 23, 24) or a salt thereof without any pharmaceutical carriers or excipients. In other embodiments, pharmaceutical compositions of this invention comprise one or more synthetic peptides or a salt thereof intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. Carriers are inert pharmaceutical excipients, including, but not limited to, binders, suspending agents, lubricants, flavorings, sweeteners, preservatives, dyes, and coatings. In preparing
compositions in oral dosage form, any of the usual pharmaceutical carriers may be employed. For example, fo liquid oral preparations, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like; for solid oral preparations, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
Thus, aqueous suspensions comprising the synthetic peptides of the invention in an admixture with excipients suitable for the manufacture of aqueous suspensions can include, for example, a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g. , polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g. , heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g. , polyoxyethylene sorbitan mono-oleate).
The aqueous suspension can also contain one or more preservatives including but not limited to benzalonium chloride, chlorbutanol, ethyl or n-propyl p-hydroxybenzoate, and/or antioxidants such as sulfite, ascorbic acid, citric acid and/or its salts and/or sodium EDTA. The aqueous suspension can also further contain one or more coloring agents, one or more flavoring agents, and/or one or more sweetening agents, such as sucrose, aspartame or saccharin may also be included in a formulation. Formulations can be adjusted for osmolarity.
Oil suspensions for use in the present methods can be formulated by suspending a synthetic peptide compound of this invention in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin, or a mixture of these. The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cety alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther. 281 :93 (1997). The pharmaceutical formulations of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, as described above, or a mixture of these.
Suitable emulsifying agents include naturally occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as
polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. The synthetic peptide compounds of the invention, alone or in combination with other suitable components can be made into aerosol formulations (/'. e. , they can be
"nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations of the present invention suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, can include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Among the acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter.
Where the compounds are sufficiently soluble they can be dissolved directly in normal saline with or without the use of suitable organic solvents, such as propylene glycol or polyethylene glycol. Dispersions of the finely divided compounds can be made-up in aqueous starch or sodium carboxymethyl cellulose solution, or in suitable oil, such as arachis oil. These formulations can be sterilized by conventional, well-known sterilization techniques. The formulations can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
The concentration of a synthetic peptide in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For intravenous administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, such as a solution of 1 ,3-butanediol. The formulations of commends can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
A synthetic peptide composition suitable for use in the practice of this invention can be administered orally. The amount of a compound of the present invention in the
composition can vary widely depending on the type of composition, size of a unit dosage, kind of excipients, and other factors well known to those of skill in the art. In general, the final composition can comprise, for example, from 0.01 percent by weight (% w) to 100% w of the synthetic peptide, e.g., 0.5% w to 50% w, with the remainder being the excipient or excipients.
Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical formulations to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc. suitable for ingestion by the patient. In other embodiments, pharmaceutical formulations for oral administration can be formulated without using any pharmaceutically acceptable carriers.
Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the pharmaceutical formulation suspended in a diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
Pharmaceutical preparations for oral use can be obtained through combination of one or more synthetic peptides of the present invention with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, i desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers and include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxymethyl cellulose, hydroxypropylmethyl-ceUulose or sodium carboxymethylcellulose; and gums including arable and tragacanth; as well as proteins such as gelatin and collagen.
If desired, disintegrating or solubilizing agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, com starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
The synthetic peptides of the present invention can also be administered in the form of suppositories for rectal administration of the drug. These formulations can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperatures and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.
The synthetic peptides of the present invention can also be administered by intranasal, intraocular, intravaginal, and intrarectal routes including suppositories,
insufflation, powders and aerosol formulations (for examples of steroid inhalants, see
Rohatagi, J. Clin. Pharmacol. 35:1 187 (1995); Tjwa, Ann. Allergy Asthma Immunol. 75:107 (1995)).
The synthetic peptides of the present invention can be delivered transdermally by a topical route formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
Encapsulating materials can also be employed with the synthetic peptides of the present invention and the term "composition" can include the active ingredient in
combination with an encapsulating material as a formulation, with or without other carriers. For example, the synthetic peptides of the present invention can also be delivered as microspheres for slow release in the body. In one embodiment, microspheres can be administered via intradermal injection of drug (e.g., mifepristone)-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater. Sci. Polym. Ed. 7:623 (1995); as biodegradable and injectable gel formulations (see, e.g., Gao, Pharm. Res. 12:857 (1995)); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669 (1997)). Both transdermal and intradermal routes afford constant delivery for weeks or months. Cachets can also be used in the delivery of the compounds of the present invention.
A synthetic peptide may also be delivered by the use of monoclonal antibodies as individual carriers to which the synthetic peptide can be coupled. Synthetic peptides may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxy-ethyl-aspart amide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, a synthetic peptide can be coupled to a class of
biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels.
Thus, the pharmaceutical compositions of the present invention may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydropropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes and/or microspheres. In general, a controlled- release preparation is a pharmaceutical composition capable of releasing the active ingredient at the required rate to maintain constant pharmacological activity for a desirable period of time. Such dosage forms provide a supply of a drug to the body during a predetermined period of time and thus maintain drug levels in the therapeutic range for longer periods of time than conventional non-controlled formulations. In some embodiments of the invention, a controlled release composition of the invention provides continuous release of an active agent over, for example, a three day, seven day, ten day and/or fourteen day period of time.
In other embodiments, the synthetic peptides can be in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, foams, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories, for oral parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation.
Alternatively, the compositions of the invention may be presented in a form suitable for once-weekly or once-monthly administration; for example, an insoluble salt of the active compound, such as the decanoate salt, may be adapted to provide a depot preparation for intramuscular injection.
Methods of formulating pharmaceutical compositions have been described in numerous publications such as Pharmaceutical Dosage Forms: Tablets. Second Edition. Revised and Expanded. Volumes 1-3, edited by Lieberman et al. Pharmaceutical Dosage Forms: Parenteral Medications. Volumes 1-2, edited by Avis et al. and Pharmaceutical Dosage Forms: Disperse Systems. Volumes 1-2, edited by Lieberman et al. ; published by Marcel Dekker, Inc, the disclosure of each of which are herein incorporated by reference in their entireties and for all purposes.
The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
In other embodiments, products comprising at least one synthetic antimicrobial peptide of the present invention, and/or salt thereof, or a pharmaceutical composition thereof, are provided. Non-limiting examples of such products include a bandage, a plaster, a suture, a soap, a tampon, a diaper, a shampoo, a tooth paste, an anti-acne compound, a suncream, a textile, an adhesive, a cleaning solution, a contact lens, and/or an implant. The product can be coated or impregnated or both coated and impregnated with one or more synthetic antimicrobial peptide of the present invention.
In some embodiments, the product can be a medical device that is coated and/or impregnated with one or more synthetic antimicrobial peptide of the present invention. In a representative embodiment, a shunt designed to drain fluids from the brain following surgery could be coated and/or impregnated with at least one synthetic peptide of this invention to prevent infection. In a further example of the usefulness of said synthetic peptides, an artificial joint, a dental implant and the like can be coated and/or impregnated with at least one synthetic peptide of this invention to prevent infection following implantation.
The pharmaceutical compositions herein will contain, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful, suppository and the like, an amount of the active ingredient necessary to deliver an effective dose as described above. For example, the pharmaceutical compositions herein can contain, per dosage unit, from about 10 to about 2000 mg of the active ingredient, e.g., from aboutlOO mg to about 1000 mg of the active ingredient, e.g., from about 25 to about 600 mg of the active ingredient, e.g., from about 75 to about 400 mg of the active ingredient, e.g., about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 mg or more or any range or amount therein of the active ingredient.
In some embodiments of the present invention, synthetic antimicrobial peptides suitable for use in the practice of this invention will be administered either singly or concomitantly with at least one or more other compounds or therapeutic agents, e.g., with other agents that treat or prevent an infection, disease and/or condition. As described herein, the synthetic peptides of this invention can take the form of any known method of administration, depending on the age, weight, health and condition of a patient, and the type of infection, disease and/or condition sought to be treated and stage of infection, disease and/or condition. For example, as a topical application the synthetic antimicrobial peptides of Formulas I to IV may be useful for treatment of an ulcer (e.g., diabetic foot ulcer infections), a wound (e.g., traumatic blast wound, surgical wound, and the like), a burn, a skin infection, and/or acne. As examples of parenteral use, the antimicrobial peptides may be useful to treat or prevent bloodstream infections (e.g., catheter-related bloodstream infections), invasive fungal infections, lung infections in cystic fibrosis patients, systemic multi-resistant Gram positive bacterial infections, and other infections including but not limited to sepsis and/or septic shock. These are examples of the types o infections, diseases and/or conditions that can be treated or prevented using the synthetic peptides of the invention and are not meant to be limiting of the wide range of uses, forms and methods of application of the synthetic AMPs of this invention. Someone having ordinary skill in the area of antimicrobial peptides and antibiotics in general would be able to readily adapt the synthetic AMPs to the particular mode of administration and dosage that is appropriate to treat and/or prevent an infection, disease and/or condition in a subject in need thereof.
Thus, the present invention provides methods of treating an infection, disease, and/or condition in a subject comprising, administering to a subject in need thereof an effective amount of at least one synthetic antimicrobial peptide of the invention, or salt thereof, a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention, or salt thereof, and/or a product comprising one or more synthetic antimicrobial peptides of the invention, or salt thereof, wherein the infection, disease, and/or condition in said subject is treated (e.g., the infection, disease, and/or condition is ameliorated, alleviated, severity reduced, symptoms reduced and the like as compared to a similar subject not treated with the synthetic peptides of this invention), thereby treating the infection, disease and/or condition.
A still further aspect of the invention is directed to a method of preventing an infection, disease, and/or condition in a subject comprising, administering to a subject in need thereof (e.g., identified as having an increased risk of developing said infection, disease and/or condition) an effective amount of at least one synthetic antimicrobial peptide of the invention, or salt thereof, a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention, or salt thereof, and/or a product comprising one or more synthetic antimicrobial peptides of the invention, or salt thereof, wherein said infection, disease, and/or condition is prevented from developing in said subject or severity of onset of said infection, disease, and/or condition is reduced (as compared to the development or severity of onset of the infection, disease, and/or condition in a similar subject not administered the synthetic antimicrobial peptide of the invention), thereby preventing the infection, disease and/or condition.
Another aspect of the invention is an antimicrobial peptide of the invention for use in the treatment of an infection, disease, and/or condition. An additional aspect is an
antimicrobial peptide of the invention for use in the prevention of an infection, disease, and/or condition.
A still further aspect of the invention is the use of an antimicrobial peptide of the invention for the manufacture of a medicament for treatment of an infection, disease, and/or condition. An additional aspect is the use of an antimicrobial peptide of the invention for the manufacture of a medicament for prevention of an infection, disease, and/or condition.
In representative embodiments, the infection, disease or condition can be an ulcer (e.g., a diabetic foot ulcer), a wound (e.g., a traumatic blast wound), a burn, and/or a skin infection. Thus, for example, a subject may receive a wound or a burn and to prevent an infection from occurring in the wound or burn area(s), the subject can be administered the synthetic antimicrobial peptides of the invention. In other embodiments, the wound or burn area may already be infected or diseased and be in need of treatment to reduce, alleviate, ameliorate the infection or disease condition. As a further example, an infection, disease or condition can be a lung infection in a subject having cystic fibrosis, and/or it can be sepsis and/or septic shock. In some embodiments, the infection, disease or condition can be caused by a microorganism, e.g., a bacterium, fungus, virus or combination thereof. In still further embodiments, the disease or condition to be treated or prevented can be cancer (e.g., breast cancer, ovarian cancer, bladder cancer, stomach cancer, lung cancer or leukemia, or any combination thereof). These are examples of the types of infections, diseases and/or conditions that can be treated or prevented using the synthetic peptides of the invention and are not meant to be limiting of the wide range of uses, forms and methods of application of the synthetic AMPs of this invention.
Antimicrobial peptides (AMPs) interact differently with different bacterial species and strains depending on the chemical composition of the bacteria's cell membranes. Thus, AMPs interact differently with gram positive bacteria, gram negative bacteria, Mycobacteria. It is further believed that AMPs also interact differently with the membranes of other microorganisms as well as the membranes of malignant cells. Without being bound by any particular theory, the potency and selectivity of an AMP of this invention is believed to be controlled by the complementarity of the physicochemical properties of the AMP and the membrane of the target microorganism or malignant cell. Such physiochemical properties include but are not limited to amino acid sequence, net charge, amphipathicity,
hydrophobicity, electrostatic potential surface charge density, and structural folding
(including secondary structure, dynamics and orientation) in the membrane. Therefore, by varying the physicochemical properties of the AMP, it is possible to "fine tune" the activity of the AMP.
In particular embodiments, the infection, disease or condition in a subject can be a bacterial or fungal infection or disease, in some embodiments, the bacterium can be a gram positive bacterium or a gram negative bacterium. In other embodiment, the bacterium can be a Mycobacterium spp., which have cell wails that are neither truly gram negative or gram positive. In still further embodiments, the infection to be treated or prevented is caused by a bacterium that is resistant to traditional antibiotics (e.g., Methicillin-resistant Staphylococcus aureus (MRSA)).
Therefore, in some aspects of the invention, a method of treating or preventing a infection, disease, or condition in a subject is provided, comprising administering to said subject at least one synthetic peptide of this invention, wherein said disease, infection, and/or conditions is caused by a bacteria and/or a fungus. In representative embodiments, the infection, disease, or condition to be treated or prevented by at least one synthetic peptide of this invention is caused by a Gram-positive and/or a Gram-negative bacterium.
In other embodiments, an infection, disease or condition in a subject that can be treated or prevented using at least one synthetic peptide of the invention can include but is not limited to an Enterococcus spp. infection, Staphylococcus spp. infection. Klebsiella spp. infection, Acinetobacter spp. infection, Pseudomonas spp infection, Enterobacter spp infection, Yersinia spp. infection, Mycobacterium spp. infection, Brucella spp infection, Bacillus spp. infection, Francisella spp. infection, Salmonella spp. infection, Burkholderia spp. infection, Escherichia spp. infection, Campylobacter spp. infection, Gonorrhea spp. infection, Streptococcus spp. infection, Neisseria spp. infection, Shigella spp. infection, or any combination thereof.
In particular embodiments, an infection, disease or condition in a subject that can be treated or prevented using the synthetic peptides of the invention can include but is not limited to an Enterococcus faecium infection, Staphylococcus aureus infection, Klebsiella pnemoniae infection, Acinetobacter baumannii infection, Pseudomonas aeruginosa infection, Enterobacter aerogenes infection, Yersinia pestis infection, Mycobacterium ranae infection, Mycobacterium avium complex (MAC) infection, Brucella suis infection, Bacillus anthracis infection, Francisella tularensis infection, Salmonella typhimurium infection, Burkholderia pseudomallei infection, Escherichia coli infection, or any combination thereof.
In some embodiments, the infection, disease or condition in a subject that can be treated or prevented using the synthetic peptides of the invention can be caused by a drug resistant microorganism (e.g., a drug resistant bacterium). In representative embodiments, the drug resistant microorganism can include but is not limited to Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant and vancomysin intermediate Staphylococcus aureus, vancomycin-resistant Enterococci, carbapenem-resistant
Enterobacteriaceae Clostridium difficile and Acinetobacter.
In other embodiments of the invention, the infection, disease, and/or condition in a subject to be treated or prevented using the synthetic peptides of the invention can be caused by, for example, Candida spp., Fusarium spp., Aspergillus spp., Cryptococcus spp., Coccidioides spp., Tinea spp., Sporothrix spp., Blastomyces spp., Histoplasma spp.,
Pneumocystis spp., or a combination thereof. In representative embodiments, an infection, disease, and/or condition to be treated or prevented using the synthetic peptides of the invention can be caused by a fungus including but not limited to Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Fusarium solani, Fusarium culmorum, Tinea unguium, Tinea corporis, Tinea cruris, Sporothrix schenckii, Blastomyces dermatitidis, Histoplasma capsulatum, Pneumocystis carinii, Histoplasma duboisii and/or any combination thereof.
In representative embodiments, a method of treating or preventing a Staphylococcus aureus infection, a MRSA infection, an Enterococcus faecium infection, a Klebsiella pnemoniae infection, an Acinetobacter baumannii infection, a Pseudomonas aeruginosa infection and/or an Enterobacter aerogenes infection in a subject is provided, comprising administering to said subject at least one synthetic peptide of the invention, wherein the at least one synthetic peptide can be Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c) K(A6c)KKKK-amide (SEQ ID NO:l). In a further embodiment, a method of treating or preventing a lung infection in a subject having cystic fibrosis is provided, comprising administering to said subject at least one synthetic peptide of the invention, wherein the at least one synthetic peptide can be Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c) K(A6c)KKK -amide (SEQ ID NO:l). In some embodiments, the lung infection can be caused by, for example, a bacterium, including but not limited to, Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, and/or Mycobacterium avium complex (MAC).
In a further embodiment, the present invention provides a method of preventing sepsis and/or septic shock in a subject caused by a bacterium comprising administering to said subject at least one synthetic antimicrobial peptide of the invention. In particular
embodiments, the at least one synthetic peptide can be Ac-
GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c) K(A6c)KKKK-amide (SEQ ID NO:l). With regard to treatment and prevention of sepsis, without wishing to be bound by any particular theory, it is hypothesize that the helical conformation of the synthetic peptides of this invention allow them to interact with the bacterial lipopolysaccharide (LPS) thereby binding the LPS and in doing so inhibiting the release of cytokines generally produced in response to the presence of LPS. The synthetic peptides of this invention are believed to prevent or reduce endotoxic or septic shock by this binding of the LPS.
Additionally provided herein, are methods of treating and/or preventing cancer, comprising administering to a subject in need thereof an effective amount of at least one synthetic antimicrobial peptide of the invention, a pharmaceutical composition comprising one or more synthetic antimicrobial peptides of the invention, and/or a product comprising one or more synthetic antimicrobial peptides of the invention, wherein the cancer in said subject is treated or prevented (as compared to a subject that has not been administered the synthetic peptides of this invention). Non-limiting examples of the types of cancer that may be treated or prevented using the synthetic peptides of the invention include to breast cancer, ovarian cancer, bladder cancer, stomach cancer, lung cancer, leukemia, or any combination thereof. While not wishing to be limited to any particular theory of the invention, it is likely that the differences between the membranes of malignant and non-malignant cells account for the ability of some AMPs (or anticancer peptides (ACPs)) to selectively kill cancer cells as opposed to normal cells (Hoskin et al. Biochim Biophys Acta 1778(2):357-375 (2008)).
The amount of a synthetic peptide necessary to provide treatment or prevention of an infection, disease, or condition is defined as a therapeutically or a pharmaceutically effective dose. The dosage schedule and amounts effective for this use, i.e., the dosing or dosage regimen will depend on a variety of factors including the stage of the disease, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration is also taken into account.
A person of skill in the art having regard to that skill and this disclosure will be able to determine without undue experimentation a therapeutically effective amount of a particular synthetic antimicrobial peptide for practice of this invention (see, e.g.. Lieberman,
Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd, 1999, The Art, Science and
Technology of Pharmaceutical Compounding; and Pickar, 1999, Dosage Calculations). A therapeutically effective dose is also one in which any toxic or detrimental side effects of the active agent is outweighed in clinical terms by therapeutically beneficial effects. It is to be further noted that for each particular subject, specific dosage regimens should be evaluated and adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the compounds.
For treatment purposes, the compositions or synthetic peptides disclosed herein can be administered to the subject in a single bolus delivery, via continuous delivery over an extended time period, or in a repeated administration protocol (e.g., by an hourly, daily, weekly, repeated administration protocol). The pharmaceutical formulations of the present invention can be administered, for example, one or more times daily, 3 times per week, weekly and the like. In one embodiment of the present invention, the pharmaceutical formulations of the present invention are orally administered once or twice daily.
In this context, a therapeutically effective dosage of the biologically active agent(s) can include repeated doses within a prolonged treatment regimen that will yield clinically significant results to provide treatment for an identified infection, disease or condition.
Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of targeted exposure symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (e.g., immunologic and histopathologic assays).
Using such models, only ordinary calculations and adjustments are typically required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the biologically active agent(s) (e.g., amounts that are orally effective, intranasally effective, transdermally effective, intravenously effective, or intramuscularly effective to elicit a desired response). The effective amount, however, may be varied depending upon the particular compound used, the mode of administration, the strength of the preparation, the mode of administration, and the advancement of the disease condition. In addition, factors associated with the particular patient being treated, including patient age, weight, diet and time of administration, will result in the need to adjust dosages.
In an exemplary embodiment of the present invention, unit dosage forms of the compounds are prepared for standard administration regimens. In this way, the composition can be subdivided readily into smaller doses at the physician's direction. For example, unit dosages can be made up in packeted powders, vials or ampoules and preferably in capsule or tablet form.
The specific dose of a compound according to the invention to obtain therapeutic benefit for treatment of an infection, disease or condition will, of course, be determined by the particular circumstances of the individual patient including the weight, age and sex of the patient, the nature and stage of the disease, the aggressiveness of the disease, and the route of administration of the compound. For example, an effective administration of the synthetic antimicrobial peptide compounds of this invention can be a daily dosage from about 0.02 to about 100 mg kg/day may be utilized, more preferably from about 0.1 to about 75 mg/kg/day, more preferably from about 1 to about 50 mg/kg/day. Suitable dosage ranges for intravenous administration are generally about 10-500 micrograms of active compound per kilogram body weight. Higher or lower doses are also contemplated as it may be necessary to use dosages outside these ranges in some cases. The daily dosage may be divided, such as being divided equally into two to four times per day daily dosing. However, the dosage regime, including the number of doses provided daily can be dependent on the route of
administration, as well as other patient specific factors.
The methods of this invention also provide for kits for use in providing treatment or prevention of an infection, disease or condition. After a pharmaceutical composition comprising one or more synthetic antimicrobial peptide compounds of this invention, with the possible addition of one or more other compounds of therapeutic benefit, has been formulated in a suitable carrier, it can be placed in an appropriate container and labeled for providing treatment or prevention of an infection, disease or condition. Additionally, another pharmaceutical comprising at least one other therapeutic agent can be placed in the container as well and labeled for treatment of the indicated infection, disease or condition. Such labeling can include, for example, instructions concerning the amount, frequency and method of administration of each pharmaceutical.
The invention will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention.
EXAMPLES Example 1. Synthetic Peptide characterization
To identify useful synthetic AMPs, various cyclic a-tetrasubstituted amino acids (Grauer, et al. Beilstein Journal of Organic Chemistry 5 (2009) (doi: 10.3762/bjoc.5.5) as well as other unnatural and natural amino acids (Hicks et al J. Med. Chem. 50:3026 (2007);
No wick. Acc. Chem. Res. 41 :1319 (2008)) were incorporated at specific positions within the primary sequence of a peptide having the amino acid sequence of any of Formulas I-IV (e.g., SEQ ID NOs: 1 to4) as described herein. The peptides are first tested for their activity against bacteria and other organisms (or cells (e.g., cancer cells)) then tested using membrane model systems for the specific organism to determine the for well defined physicochemical and structural properties that will increase potency and selectivity for microbial membranes or membranes of malignant cells over normal mammalian membranes.
Peptide analysis: For each AMP synthesized the following analyses is carried out to determine whether the peptide understudy study interacts selectively with bacteria cell membranes and also kills bacterial cells in vitro. The interaction of an AMP with a cell membrane is generally divided into two separate but related bound states (Brogden, Nature Reviews Microbiology 3:238 (2005)). In the first state the AMP is localized near the polar head groups of the lipids, their hydrophobic side chains may insert into the hydrophobic core of the lipid bilayer. This state is commonly referred to as the S- state (surface bound state) (Id.). As the local surface bound concentration of the AMP increases, a critical concentration is reached where the peptides self-assemble to form complexes of 4-6AMP (Id.). These oligomeric complexes are thought to insert into the hydrophobic core so that the long axis of the peptide is now oriented perpendicular to the membrane surface and the peptide is now in the I-state (inserted state).
Example 2. Antibacterial peptide analysis
1. Antibacterial screening. The peptides that exhibited activity in the calcein leakage assay are selected for screening for antibacterial activity. In vitro antibacterial assays are conducted to determine the antibiotic activity of these synthetic AMPs against the following (ESKAPE) pathogens, which are known to commonly infect chronic wounds. Enterococcus faecium
Staphylococcus aureus
Klebsiella pnemoniae
Acinetobacter baumannii
Pseudomonas aeruginosa
Enterobacter species
2. Calcein fluorescence leakage experiments are conducted to evaluate the potential for the synthetic peptides of this invention to induce leakage and/or pore formation of POPC and 4:1 POPC/POPG liposomes. Peptide induced calcein leakage monitored through
fluorescence is well documented as a technique for probing AMP activity (Andrushchenkoet al. Biochimica et Biophysica Acta 1778:1004 (2008)). Calcein is a relatively large water soluble molecule and its release from the liposome is assumed to involve the formation of some type of pore in the liposome (Russellet al. Chem. Phys. Lipids 163:488 (2010). The observation of a lower percentage of calcein leakage from POPC liposomes as compared to 4:1 POPC/POPG mixed liposomes in the presence of the synthetic peptides of the invention will confirm the selectivity of these peptides (selectivity of these peptides also shown by the observation of the different CD spectra in the presence of POPC and 4:1 POPC/POPG liposomes as described above).
3. CD spectroscopy is used to determine if the synthesized peptides interact differently with zwitterionic (models for human cell membranes) and anionic (models for bacteria cell membranes) micelles and liposomes. CD spectroscopy is very sensitive and its use to monitor conformational changes in peptides and proteins is well documented (Russell et al. Chem. Phys. Lipids 163:488 (2010); Correa et al. African Journal of Biochemistry Research, 3:164 (2009)). Changes in the intensity or shape of the CD spectrum of a peptide in the presence of a micelle or liposome indicates that the peptide is adopting different
conformations when interacting with that particular micelle or liposome as compared to another environment such as a buffer (Glattli, et al. J. Am. Chem Soc. 124: 12972 (2002)). In order to investigate the S -state in isolation, DPC micelles (Jinget al. J. Pept. Res. 61 :219 (2003)) were selected as a simple zwitterionic model and SDS micelles (Watson et al.
Biochemistry 40: 14037 (2001)) were selected as a simple anionic model. Following these initial studies the I-state may be probed using liposomes as simple bilayer membrane models. Large unilamellar vesicles (LUVs) comprised of only l-Palmitoyl-2-01eoyl-i7?-Glycero-3- Phosphocholine (POPC) were selected as a simple zwitterionic bilayer whereas LUVs consisting of (4:1 mole ratio) l-Palmitoyl-2-01eoyl-in-Glycero-3-Phosphocholine / 1- Palmitoyl-2-01coyl->s"«-Glycero-3-[Phospho-ra -(l -glycerol)] (Sodium Salt) (POPG)) were selected as a simple anionic bilayer models for human and bacteria membranes respectiviely (Bringezu et al. Biochemistry 46:5678 (2007)). Different CD spectra will be exhibited for the peptides of interest when interacting with anionic and zwitterionic micelles and lipids. Further, synthetic peptides selective for bacteria membranes versus human membranes will also provide different CD spectra in the presence of POPC and 4: 1 POPC/POPG liposomes. Example 3. Synthetic antimicrobial peptide TSP-1
One member of this new series of synthesized AMPs comprises the amino acid sequence of Ac-GF(A6c)G(A6c) (A6c)G(A6c)F(A6c)G(A6c)GK(A6c)KKKK-amide (SEQ ID NO:l) referred to as "TSP-1" (an AMP of Formula I; see, Figure 2). A6c represents the unnatural amino acid 1 -aminocycohexane carboxylic acid. As shown below, this peptide exhibits broad spectrum in vitro antibacterial activity against at least five clinically relevant drug resistant bacteria strains.
Synthesis
TSP-1 was synthesized by the Commercial Research Organization New England Peptide and shipped to ECU on February 29, 2012. The molecular weight of TSP-1 was determined to be 2285 using mass spectral analysis, which is within 0.1% of its calculated average molecular weight. HPLC analysis showed by the percent area method that the purity of the peptide is greater than 95%. Additional Biological Evaluation
Minimum Inhibitory Concentrations (MIC) values to inhibit growth of the following bacteria in vitro were determined.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) were determined by a procedure based on the National Committee for Clinical Laboratory Standards broth microdilution method. MIC is defined as the lowest
concentration of the test antimicrobial that would inhibit the growth of the target organism as detected spectrophotometrically. Minimum bactericidal concentration (MBC) is defined as the lowest concentration of the test antimicrobial required to kill the target organism as determined by subculturing of the treated organism to agar media. The susceptibility study was performed as previously described. (Concannon et al. (2003) Journal of Medical
Microbiology 52, 1083-1093) Briefly, Meuller Hinton (VWR International, Radnor, PA) was used as the assay medium for the test organisms. Freshly grown bacterial cultures at exponential phase were adjusted to an optical density (OD) at 600 nm of 0.05 (equivalent to 2 107 CFU/mL) and further diluted to obtain a concentration of 2 χ 106 CFU/mL to be used as the inoculum. Aqueous peptide solution (100 μ at two times of the highest test concentration was added to each well of a sterile 96-well, flat bottomed plate (TPP,
Switzerland). The antimicrobial peptide solution was 2-fold serially diluted with sterile distilled water in the wells, with the final peptide concentrations ranging from 0.78 to 100 ng/mL. After adding 100 μΐ, aliquots of the suspension of the target organisms to the wells, the plates were incubated at 37° C for 24 hours. The MIC was determined by recording the lowest concentration of the peptide that prevented visible turbidity of the target organism, as measured at 600 nm by using an ELISA reader (Biotek synergy HT). Visible turbidity was determined by the OD readings of the tested samples that were significantly greater than that of the medium or background. The MBC was determined by plating 100 μΐ, from each clear well (>MIC) onto 5% sheep blood agar plates. After incubation for 24 hours, the MBC was determined as the lowest concentration of the test peptide that did not permit visible growth on the surface of the agar (See, Table 1).
Table 1. Results o antibacterial activity testing.
Bacterium MBC*
S. aureus 25 μβΛηΙ. (10.9 μΜ)
A. baumonnii 25 ng/mL (10.9 μΜ)
K. pneumonia 100 g/mL (43 μΜ)
P. aeruginosa 50 y%J L (21.9 μΜ)
E. aerogenes 50 ^ig/mL (21.9 μΜ)
E. faecium 25 με ιη1 Π0.9 μ )
MBOMinimum bactericidal concentration
Structural and functional characterization
Sodium dodecyl sulfate (SDS) micelles which are anionic were used as a simple model for the membranes of bacteria cells, while dodecylphosphocholine (DPC) micelles which are zwitterionic were used as a simple model for mammalian cells. The CD spectra of TSP-1 is the presence of (A) 150 mM sodium acetate buffer (pl l= 4.4) (— ); (B) 100 mM SDS micelles in buffer ("""); and (C) 100 mM DPC micelles in buffer (— ) are shown in
Figure 3. As seen in Figure 3, the CD spectrum of TSP-1 in aqueous buffer exhibits characteristics of a random coil or disordered conformation. On binding to the surface of SDS and DPC micelles, TSP-1 clearly adopts an a-helical like structure. The CD spectra suggest these structures are somewhat different indicating that the helical structures are somewhat different. This data suggest there may be some difference in the selectivity of TSP-1 for bacteria verses mammalian cells. Two dimensional NMR studies are used to confirm this observation.
Two dimensional Ή TOCSY NMR data was collected on TSP-1 in predeuteriated
150 mM sodium acetate buffer pH=4.4 (Figure 4), in the presence of 100 mM SDS micelles in the same buffer (Figure 5) and in the presence of 100 mM DPC micelles in the same buffer (Figure 6). The amide to alkyl region of the TOCSY spectrum in buffer is shown in Figure 4. This spectrum clearly shows six separate amide proton connectivities for the six Lys residues, five separate connectivitives for the five Gly residues and two separate connectivities for the two Phe residues in the peptide. The observation of separate amide connectivities for these residues is consistent with the observed CD spectrum in the presence of buffer indicating a random coil or unordered conformation in this environment.
The amide to alkyl region of the TOCSY spectrum (Figure 5) of TSP-1 in the presence of SDS micelles is very different from the spectrum observed in buffer only. The TOCSY spectrum shows only one amide to alkyl connectivity for each residue type, one Gly, one Phe and one Lys instead o the expected five, two and six connectivities respectively. This data suggest that the peptide is highly ordered and of the residues are in similar environments. This would be observed if the peptide adopted a helical structure. This observation is consistent with the CD spectrum of TSP-1 in the presence of SDS micelles which also indicates that TSP-1 adopts a helical structure in the presence of SDS micelles.
The amide to alkyl region of the TOCSY spectrum of TSP-1 in the presence of DPC micelles is given in Figure 6. This spectrum is again different from the spectra obtained in buffer and in the presence of SDS micelles. Again, only one amide to alkyl connectivity is observed for the Phe and Gly residues. However three Lys amide to alkyl connectivities are observed. This suggests that the C-terminal Lys residues are in somewhat different environments or adopt a different structure compared to the two internal Lys residues. This data suggest that the peptide adopts a helical conformation from the N-terminus to the C- terminus, with the helical structure being disrupted somewhere in the last four C-terminal Lys residues. The difference in the helical structures adopted by TSP-1 on binding to SDS and DPC micelles are confirmed by the difference in the CD spectra of TSP-1 in both
environments.
The observed differences in the CD and TOCSY spectra of TSP-1 in buffer and in the presence of SDS and DPC micelles confirms that this peptide adopts different conformations on binding to the surface of anionic and zwitterionic micelles suggesting that TSP-1 may exhibit selectivity for bacteria verses mammalian cells. Comparison of biological activity of TSP-1 peptide to other AMPs
As a point of reference for the activity of compound Ac- GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c)GK(A6c)KKKK-amide (SEQ ID NO:l) against the bacteria listed in Table 1 we have compared the in vitro MIC values for three other AMPs containing unnatural amino acids developed by the Hicks group. (United States Patent No. 8,188,033, issued 05/29/12 to Hicks et al.); Hicks et al. (2013) Bioorg. Med.
Chem. 21, 205-214; Hicks et al. (2007) J. Med. Chem. 50, 3026-3036). These AMPs are based on the incorporation of the peptide skeleton of the unnatural AMPs designed in our laboratory is based on the placement of three L-Tic(Tetrahydroiso-quinoline-carboxylic acid) -L-Oic (Octahydroindolecarboxylic acid) dipeptide units into the polypeptide backbone to reduce the overall molecular flexibility of the peptide. The basic skeleton of the AMPs based on the incorporation of three Tic-Oic dipeptide units as shown Table 3. Compound 23 contains Lys residues as does SEQ ID NO:l. As shown in Table 2, the peptide encoded by SEQ ID NO:l is at least as active if not more active than compound 23 against all six bacteria. Compound 53 where the Lys residue has been replaced with 1 ,4-diaminobutanoic acid and compound 56 where the Lys was replaced with Arg exhibited increased activity compared to compound 23. This is because of the derealization of the side chain positive charge associated with the Arg and 1 ,4-diaminobutanoic acid residues compared to Lys residues. A similar or greater increase in activity is expected with the incorporation of the Arg and 1 ,4-diaminobutanoic acid residues into SEQ ID NO:l . Table 2. Comparison of biological activity of TSP-1 to other AMPs against (ESKAPE) atho ens
Figure imgf000043_0002
MIC=minimum inhibitory concentration
Table 3. Amino Acid Se uences of the AMPs listed in Table 2.
Figure imgf000043_0003
Example 3. Antibacterial activity of the synthetic peptides.
Additional testing of the peptides was carried out using methods as set forth in Example 2, above.
The peptides were tested for activity against 18 pathogenic bacteria: Enterobacter aerogenes, Acinetobacter baumannii, Salmonella enteric, Psuedomonas aeruginosa, Klebsiella pneumonia, Eenterococcus faecalis, MRSA NRS 384, Clostridium difficile, Staphylococcus aureus, Steptococcus pneumonia, Neisseria gonorrhoeae, Bacillus anhtracis (A mes), Francisella tularensis, Yersinia pes I is, Burkholderia pseudomallei, Mycobacterium 5 tuberculosis, MDR-TB, XDR-TB.
The peptides were tested at the following concentrations: 100
Figure imgf000043_0001
50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 g ml, 3.13 μ /ηι1, 1.6 μ^ιηΐ, 0.8 μg ml, 0.4 μg ml, 0.2 μ^η ΐ, and 0.1 μg/ml.
Art known antibiotics (gentamicin, chloramphenicol, vancomycin, tetracycline, and doxycycline) were tested simultaneously as comparators.
The peptides tested are provided in Table 4 and the results of the testing are provided in Table 5 and Table 6. Table 4. Peptide sequences.
Figure imgf000044_0001
Table S. Minimum inhibitory concentration of the peptides for eighteen bacterial pathogens and six novel peptides. Results are in pg/ml and μΜ.
Figure imgf000045_0001
Table 6. Minimum inhibitory concentration of the peptides for eighteen bacterial pathogens and five novel peptides. Results are in μα/ml and μΜ.
Figure imgf000046_0001
A discussion of the in vitro antibacterial inhibitory activity of the antimicrobial peptides incorporating cyclic tetra-substituted Ca amino acids and other unnatural amino acids as presented in Tables 4, 5, and 6, is provided below.
Mycobacterium tuberculosis
AMP 1 (SEQ ID NO:2) incorporates two internal back-to-back Lys substitutions resulting in a very high density of positive charge in these two regions of the peptide. This AMP is the most potent analog against the three strains; Mycobacterium tuberculosis, MDR- TB and XDR-TB exhibiting a MIC value of 4.9 μΜ. AMP 3 (SEQ ID NO:4), incorporating two single internal Orn substitutions, is the next potent against the three strains exhibiting a MIC value of 1 1.4 μΜ. AMP 6 (SEQ I NO:23), incorporating two single internal Lys substitutions, exhibits interesting activity with MC valves of 11.4 μ against Mycobacterium tuberculosis, and XDR-TB while the MIC value increases to 22.9 μΜ against MDR-TB.
AMP 4 (SEQ ID NO:5), incorporating two single internal Dab substitutions, and AMP 5 (SEQ ID NO: 6), incorporating two single internal Dpr substitutions, exhibited MIC values of 11.8 and 24.6 μΜ respectively against Mycobacterium tuberculosis, and increased MIC values of 23.7 and 49.3 μΜ respectively against MDR-TB and XDR-TB. AMP 2 (SEQ ID NO:3), incorporating two single internal Arg substitutions, exhibited MIC values of 40.8 μΜ against Mycobacterium tuberculosis, and MDR-TB however the potency against XDR-TB increased to 10.2 mM. Relocating the C-terminal Arg cluster of four residues found in AMP 2 (SEQ ID NO:3) to the N-terminus of AMP 1 1 (SEQ ID NO:24) dramatically alters the activity against Mycobacterium tuberculosis (21.9 μΜ), MDR-TB (5.5 μΜ) and XDR-TB (10.9 μΜ). This data indicates that the following factors effect activity against these three strains of Mycobacterium tuberculosis: 1) the position and number of positively charged amino acids, 2) the derealization of positive charge density of the basic amino acids, 3) the hydrophobicity of the side chains of the basic amino acids and 4) the molecular flexibility of the side chains of the basic amino acids.
The electrostatic surface potential maps for the tripeptides Ac-Ala-X-Ala-NHMe where X = Lys, Orn, Dab, Dpr and Arg have been calculated. It was determined as the length of the side chain decreases the electropositive region of the protonated terminal amine approaches the electronegative region of the backbone amide carbonyl oxygen, thus in effect delocalizing the positive charge. By varying the number of -CH2- groups in side chain, from 1 to 4, the distance (the calculated distance is given in Table 7, below) between the positive charge and the peptide backbone will decrease resulting in a) less side chain flexibility during binding, b) the positive charge density will reside closer to the peptide backbone, and c) the overall hydrophobicity of the residue will vary. ( Bhonsle, J. B., et. al (2013) Current Topics in Medicinal Chemistry, Ώ, 3205-3224). These basic residues exhibit negative
hydrophobicity and by shortening the side chain the protonated nitrogen is moved closer to the peptide backbone. This results in reducing the solvent accessibility of the protonated nitrogen and thus increasing the hydrophobicity. The combined Consensus Scale (CCS) developed by Tossi and co-workers (Tossiet al. In Proceeding of the 27th European Peptide Symposium Sorrento; Benedetti, E., Pedone, C, Eds.; Edizioni Ziino: Napoli, Italy, 2002) (hydrophobicity) for each residue used is given in Table X. Table 7: Physicochemical Properties of the Basic Amino Acids used in this invention.
Amino Acid Residue Distance a Hydrophobicity value b # of carbons in side chain
Dpr 2.6 A -9.3 1
Dab 3.9 A -9.5 2
Orn 5.0 A -9.0 3
Lys 6.4 A -9.9 4
Arg 7.0 A -10.0 3 a. distance in angstroms from CG carbon of the peptide backbone to the positively charged nitrogen atom(s); b. combined consensus scale hydrophobicity for each charge residue
Thus, the variables include the type, number and placement of the basic amino acids within the AMP's primary sequence. Since the mechanism of action of these AMPs is assumed to involve membrane disruption of some type, this data implies that the chemical compositions of the membranes of the three strains of Mycobacterium tuberculosis vary. Specifically there appears to be a variation in the physicochemical properties of the membranes that interact with the positively charged side chains of the AMPs. In addition to electrostatic interactions, hydrophobic interaction may also play a role in the interactions with XDR-TB. AMP 2 (SEQ ID NO:3) which incorporates the C" tetra substitute amino acid 1- aminocyclohexane carboxylic acid exhibits a MC value of 10.2 μΜ against XDR-TB. AMP 7 (SEQ ID NO: 10) replaces the C" tetra substitute amino acid with 1 -aminocyclopentane carboxylic acid which is slightly less hydrophobic and exhibits an increased MIC value of >43 μΜ against XDR-TB. Gram Negative Bacteria
Enterobacter aerogenes
Enterobacter aerogenes is most often associated with opportunistic infections. The activity profile of the AMPs of the invention against Enterobacter aerogenes is very interesting. AMP 1 (SEQ ID NO:2), which incorporates two internal back-to-back Lys substitutions, exhibits a very poor MIC value of >39 μΜ against this bacteria. AMP 2 (SEQ ID NO:3), which incorporates Arg residues, exhibits a MIC value of 2.6 μΜ. AMP 3 (SEQ ID NO:4), which incorporates Orn residues, exhibits an increased MIC value of 22.8 μΜ. AMP 4 (SEQ ID NO:5), which incorporates Dab residues, exhibits a MIC value of 3 μΜ. AMP 5 (SEQ ID NO: 6), which incorporates Dpr residues, exhibits a decreased MIC value of 1.5 μΜ. This data suggest that the derealization of the side chain positive charge density plays a major role in defining the activity of these AMPs against Enterobacter aerogenes. In addition to electrostatic interactions, hydrophobic interaction may also play role in the interactions with Enterobacter aerogenes. AMPs 2, 3, 4 and 5 (SEQ ID NOs: 3, 4, 5, and 6, respectively) all incorporate the C tetra substitute amino acid, 1 -aminocyclohexane carboxylic acid. AMP 7(SEQ ID NO: 10) replaces the Ca tetra substitute amino acid with 1- aminocyclopentane carboxylic acid which is slightly more hydrophobic and exhibits an increased MIC value of >42.4 μΜ against Enterobacter aerogenes. The remaining AMPs 6, 8,9,10 and 11 (SEQ ID NOs: 23, 17, 18, 19 and 24, respectively) all incorporate the amino acid Lys and exhibit increased MIC values of > 40 μΜ, confirming the importance of positive charge derealization.
Acinetobacter baumannii
AMPs 1 , 2, 3, 4, 6, 7 and 1 1 (SEQ ID NOs: 2, 3, 4, 5, 23, and 10, respectively) all exhibit low MIC values ( 1 .5 to 3 μΜ) against Acinetobacter baumannii. AMP 5 (SEQ ID NO:6), which incorporates the amino acid Dpr, exhibited an increased MIC value of 49.3 μΜ. It is interesting to point out that replacing three of the seven the C" tetra substitute amino acid, 1 -aminocyclohexane carboxylic acid with the un-natural amino acids Tic or Oic, or alternating the 1 -aminocyclohexane carboxylic acid residues with 1 -aminocyclopentane carboxylic acid residues result in analogs with increased MIC values of > 42μΜ. These observations suggest that the increased hydrophobicity of the Tic and Oic residues or their allowed binding conformational space may not be favorable for membrane disruption. Also the alternating of 1 -aminocyclohexane carboxylic acid residues with 1 -aminocyclopentane carboxylic acid residues may somehow prevents the AMP from adopting a binding conformation that induces membrane disruption. Salmonella enterica
AMPs 1 , 2, 3, 4, 6, 7 and 1 1 (SEQ ID NOs: 2, 3, 4, 5, 23, and 10, respectively) all exhibit low MIC values (1.5 to 5.7 μΜ) against Salmonella enterica. AMPs 5 and 10 (SEQ ID NO:6 and SEQ ID NO:19, respectively) exhibit moderate MIC values (24.6 and 11.2 μΜ, respectively). It is interesting to point out that replacing three of the seven the C" tetra substitute amino acid, 1 -aminocyclohexane carboxylic acid with the un-natural amino acids Tic or Oic, dramatically alters the MIC values against Salmonella enterica. AMP 8 (SEQ ID NO:17) incorporates three TIC residues and AMP 9 (SEQ ID NO:18) incorporates three Oic residues. AMP 9(SEQ ID NO: 18) exhibited a MIC value of 41.9 μΜ, while AMP 8 (SEQ ID NO: 17) exhibited a MIC value of 5.2 μΜ. This observation suggests that the phenyl ring associated with the Tic residues may increase binding of AMP 8 to the surface of the membrane in a conformation which favors membrane disruption more than the cyclohexane ring associated with the Oic residue.
Psuedomonas aeruginosa
AMPs 1 , 2, 3, 4, 5, 7, 10 and 1 1 (SEQ ID NOs: 2, 3, 4, 5, 6, 10, 19, and 23, respectively) all exhibit low MIC values (1.4 to 5.6 μΜ) against Psuedomonas aeruginosa. AMPs 6, 8 and 9 (SEQ ID NOs: 23, 17 and 18, respectively) exhibit moderate MIC values (10-23 μΜ). Overall Psuedomonas aeruginosa appears to be relatively insensitive to changes in the structure and physicochemical changes within this series of AMPs.
Klebsiella pneumoniae
AMPs 4, 5 and 7 (SEQ ID NOs: 5, 6 and 10, respectively) exhibited low MIC values of 2.97, 3.08 and 5.31 μΜ against Klebsiella pneumonia. AMP 4 (SEQ ID NO:5) contains Dab residues, AMP 5 (SEQ ID NO:6) contains Dpr residues, and AMP 7 (SEQ ID NO: 10) contains Arg residues. It is interesting to note that AMP 2 (SEQ ID NO: 3) also contains Arg residues but exhibits an increased MIC values of 20.4 μΜ. The only difference between the two AMPs is that AMP 2 contains 1 -aminocyclohexane carboxylic acid residues and AMP 7 contains 1 -aminocylopentane carboxylic acid residues. This observation suggest either 1) the 1 -aminocyclohexane carboxylic acid residues adopt a different binding conformation from the 1 -aminocyclopentane carboxylic acid which reduces membrane disruption, or 2) the increased hydrophobicity or steric bulk of the cyclohexane ring disfavors membrane binding and disruption. AMP 1 (SEQ ID NO:2) and AMP 3 (SEQ ID NO:4) (Orn) exhibited MIC values of 19.7 and 22.8 μΜ respectfully. The remaining AMPs all exhibit MIC values >43 μΜ.
Neisseria gonorrhoeae
AMPs 1 , 2, 3, 4, 5, 7, and 1 1 (SEQ ID NOs: 2, 3, 4, 5, 6, 10, and 24, respectively) exhibited MIC values in the range 0.62 to 6.25 μΜ against Neisseria gonorrhoeae. AMP 1 (SEQ ID NO:2) is the most active of the AMPs exhibiting a MIC value of 0.61 μΜ. AMP 1 contains double internal Lys substitutions. AMP 6 (SEQ ID NO: 2) incorporates single internal Lys and replaces 1-aminocyclhexance carboxylic acid residues with 1- aminocylcopentane carboxylic acid residues and further exhibits an increased MIC value of 22.9 μΜ. AMP 10 (SEQ ID NO: 19), which incorporates alternating 1 -aminocyclohexane carboxylic acid residues with 1 -aminocylcopentane carboxylic acid residues, exhibits a decreased MIC value of 1 1.2 μΜ. This suggest that the increased hydrophobicity or steric bulk, of the cyclohexane ring favors membrane binding and disruption of the membrane compared to the cyclopentane ring.
Francisella tularensis, Yersinia pestis and Burkholderia pseudomalle
All of the AMPs exhibited relatively poor MIC values against these three strains of bacteria.
Gram Positive bacteria
Enterococcus faecalis
AMPs 2, 4, 5, and 7 (SEQ ID NOs:3, 5, 6, and 10, respectively) exhibited MIC values in the range of 1.3 to 5.3 μΜ against Eenterococcus faecalis. These four AMPs containing the four basic amino acids with the highest degree of delocalization of positive charge, Arg, Dab, Dpr and Arg respectively. AMP 3 (SEQ ID NO:4) incorporates Orn residues exhibits a MIC value of 1 1.4 μΜ, while all Lys containing AMPs except AMP 1 (SEQ ID NO:2), with double internal Lys substitutions (19.7 μΜ) and AMP 11 (SEQ ID NO.-24) (Lys cluster relocated from the C-terminus to the N-terminus) (SEQ ID NO: 18; >42 μΜ) exhibited MIC values of 21.9 μΜ.
MRSA NRS 384
AMPs 2, 4, 5 and 7 (SEQ ID NOs:3, 5, 6, and 10, respectively) exhibited MIC values in the range of 5.1 to 6.1 μΜ against MRSA NRS 384. These four AMPs contain the four basic amino acids with the highest degree of delocalization of positive charge, Arg, Dab, Dpr and Arg, respectively. AMP 3 (SEQ I NO:4) incorporates Orn residues exhibits a MIC value of 22.8 μΜ, while all Lys containing AMPs exhibited MIC values of >39.35 μΜ. The increased activity of AMPs with a degree of positive charge derealization is consist with the fact that the lipid composition of some strains of drug resistant strains of S. aureus contain as much as 38% lysylphosphatidylglycerol, which is a cationic lipid that is known to repel antimicrobial peptides. (Teixeira, V. (2012) Prog. Lipid Res. 51, 149) Andra, J. (201 1) J. Biol. Chem 286, 18692) (Kilelee, E.(2011) Antimicrobial Agents and Chemotherapy, 54, 4476).
Clostridium difficile
All of the AMPs exhibited MIC values in the range of 1.37 to 5.69 μΜ against Clostridium difficile.
Staphylococcus aureus
AMPs 2, 3, 4, 5, and 7 (SEQ ID Os:3, 4, 5, 6, and 10, respectively) exhibited MIC values in the range of 1.3 to 6.2 μΜ against Staphylococcus aureus. Derealization of the side chain positive charge seems to play a role in defining antibacterial activity, however no direct correlation is observed. All of the Lys containing AMPs except AMP 1 (SEQ ID
NO:2) with double internal Lys substitutions ( 19.7 μΜ), and AMP 1 1 (SEQ ID NO:24) (Lys cluster relocated from the C-terminus to the N-terminus ) (SEQ ID NO: 18, >42 μΜ) exhibited MIC values of 10.9 μΜ.
Steptococcus pneumoniae
AMPs 2, 5 and 1 1 (SEQ ID NOs:3, 6 and 24, respectively), exhibited moderate MIC values of 10.2, 24.6 and 21.9 μΜ against Steptococcus pneumonia. The remaining AMPs all exhibited MIC values of greater than 39.4 μΜ.
Bacillus anthracis (Ames)
AMPs 1 , 2, 3, 4, 5, 7 and 1 1 (SEQ ID NOs: 2, 3, 4, 5, 6, 10, and 24, respectively) exhibited MIC values in the range 1.4 to 6.2 μΜ against Bacillus anhtracis (Ames). AMPs 8, 9 and 10 (SEQ ID NOs: 17, 18 and 19, respectively) exhibited increased MIC values of 10.3, 20.9 and 22.4 μΜ, respectively. AMP 6 (SEQ ID NO:23) exhibited an increased MIC value of >46 μΜ.
Thus, Tables 5 and 6 show that the novel peptides have antibacterial activity that is the equivalent of or better than that provided by the comparator art known antibiotic in the case of one or more of the pathogenic bacterial species tested, thereby showing that these peptides can be useful in treating bacterial infections.
Example 4. Fungicidal activity of the synthetic peptides
Fungal strains will be cultured in supplemented minimal medium (Hill, T. W. Kafer,
E., 2001 Fungal Genet. News 48, 20-21 ). Microtiter plates (96 well) will be loaded with 200 of medium and 104 spores of the fungi under study. A serial dilution of AMP ( 100 g/mL to 0.01 ng/mL) will be prepared and added to different wells of the microtiter plates. 7-hydroxy-3H-phenoxazin-3-one-10-oxide will be used as the redox indicator at a
concentration of 100 μΜ from a stock solution of 100 mM in H20 (double distilled). Methyl - l(butylcarbamoyl)-2-benzimidazol carbamate, a microtubule-destablizing drug, (1 mg/mL dimethyl sulfoxide stock solution) will be added to the medium in a concentration range of 0.1 to 10.0 μg/ml. The color change of the 7-hydroxy-3H-phenoxazin-3-one- 10-oxide in the microliter plates at 570 nm will be determined using a microtiter plate reader. (Mania et al., Applied and Environmental Microbiology, 2010, 7102-7108)
The synthetic peptides of the invention will be screened for inhibitory activity against at least the following fungi: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Candida albicans, Coccidioides immitis,
Cryptococcus neoformans, Fusarium solani, Fusarium culmorum, Tinea unguium, Tinea corporis, Tinea cruris, Sporothrix schenckii, Blastomyces dermatitidis, Histoplasma capsulatum, Pneumocystis carinii, and Histoplasma duboisii.
Example 5. Anticancer activity of the synthetic peptides
Cells were tested for their ability to kill cancer cells from a variety of cancer cell lines.
Cells were grown in RPMI-1640 Supplemented with L-Glutamine, sodium pyruvate and 10% FBS. The cell lines used were CFPAC-1 (Pancreatic cancer), MCF-7 (Breast cancer), IGROV- 1 (Ovarian cancer); Colo205 (colon cancer), DLD-1 (colon cancer), UACC- 62 (Melanoma), MiaPaca-2 (Pancreatic cancer), A498 (Kidney cancer), PC-3 (Prostate cancer), A549 (Lung cancer), NCI-H69 (Small Cell Lung cancer) and RL (Lymphoma).
Five 96 well plates of each cell line were seeded with the optimized number of cells per well in a total volume of 50 xL per well. The plates were left overnight. A media control was included. The following day, the cells were expose to the peptides as described below. At same time as drug exposure, carry out a CellTiter-Glo® luminescent cell viability(CTG) assay on 5th plate (a full plate is not necessary) for 0 hr count.
2X stocks of all peptides in media were prepared for dosing as follows :- 15 μΜ: 40 of 1.5 raM peptide DMSO stock in 4 mL medium
4 μΜ: 1.333 mL 15 μΜ + 3.667 mL mix
0.8 μΜ: 1 mL 4 μΜ + 4 mL mix
0.16 μΜ: 1 mL 0.8 μΜ + 4 mL mix
0.032 μΜ: 1 mL 0.16 μΜ + 4 mL mix
0.0064 μΜ: 1 mL 0.032 μΜ + 4 mL mi
0.00128 μΜ: 1 mL 0.0064 μΜ + 4 mL mix
0.000256 μΜ: 1 mL 0.00128 μΜ + 4 mL mix
DMSO Mix: 5 mL DMSO + 495 mL medium
Fifty ί, of above 2X stocks were be added to the cells and medium already on plate to give the final concentrations outlined in Table 8. 50 μΐ. media were added to cell and media wells and 50 iL DMSO mix added to vehicle control wells. 10 μΜ Dox was also be added to appropriate wells. The cells were incubated at 37°C with 5% C02 for 72 hr followed by the CTG luminescent cell viability assay assay.
Table 8. Example Plate Layout for 3 Peptides in 1 Cell Line (Repeated for remaining 11 Cell Lines )
Figure imgf000055_0001
CTG Assay
At the end of the 72 hr exposure period, plates were removed from 37°C, 5% C0 incubator and place on the bench at room temperature for 30 mins. For the CTG assay 100 iL CellTiter-Glo® reagent was added and mixed for 2 mins, followed by a further 10 min incubation at room temperature at which point the luminescence was recorded and the concentration of each of the peptides tested at which 50 percent of the cancer cells die was determined and is shown in Table 9.
Table 9. The concentration of each of the e tides tested at which 50 ercent of the cancer cells die.
Figure imgf000057_0001
ND denotes insufficient peptide available to conduct assay; CFPAC-1 (Pancreatic cancer), MCF-7 (Breast cancer), IGROV-1 (Ovarian cancer); Colo205 (colon cancer), DL - (colon cancer), UACC-62 (Melanoma), MiaPaca-2 (Pancreatic cancer), A498 (Kidney cancer), PC-3 (Prostate cancer), A549 (Lung cancer), NCI-H69 (Small Cell Lung cancer and RL (Lymphoma)
The results show that the peptides were effective in killing the cancer cells at concentrations that are commonly used for other peptide based anti-cancer agents, indicating that these peptides may be useful for treating many types of cancer. ICso's of less than 7.5 μΜ for membrane disrupting peptides are considered to indicate very good anti-cancer activity. (Hansen, T., et. Al. (2012) European J of Med. Chem. 58, 22-29) ( Sinthuvanich C, et. Al. (2013) J Am. Chem. Soc. 134, 6210-6217) (Kang, S.-J. et.al. (2012) Arch Pharm Res 35, 7910799). One or more of the 1 -peptides tested exhibited !C5o values of less than 7.5 μΜ against all 12 cancer cell lines. Also, not all 11 -peptides exhibited equal activity against the 12 cancer cell lines indicating a level of selecti vity for the various cancer cell lines that can be used to improve the potency and selectivity of these peptides.
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:
1. A synthetic antimicrobial peptide comprising the formula of:
Acetyl-Gly-Phe-A-B-A-C-A-B-E-D-NH2 (Formula I);
Acetyl-D-Gly-Phe-A-B-A-C-A-B-E-NHi (Formula II);
Acetyl -Gly-Phe-A-B-A-C-A-B-A-C-A-B-E-D-NH2 (Formula III); or Acetyl -D-Gly-Phe-A-B-A-C-A-B-A-C-A-B-E-NH2 (Formula IV);
wherein
a) A is a three amino acid sequence of Y-Z-Y, and
i) each Y is a cyclic Ca tetra substituted amino acid, each Y can be the same or a different Cu tetra substituted amino acid and
ii) Z is Glycine, β-alanine, GAB A or 6-aminohexanoic acid;
b) each B is independently X, U-X, X-V, U-X-V, or X-X and
i) each X is independently lysine, arginine, histidine, ornithine, 2,3- diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dab), 4-aminopiperidine-4- carboxylic acid (Apc4), or 3-aminopiperidine-3-carboxylic acid (Apc3);
ii) each U is independently glycine, alanine, β-alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (l OAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa); and
iii) each V is independently glycine, alanine, β-alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa);
c) each C is independently S, R-S, S-T or R-S-T, and
i) each S is independently phenylalanine, tyrosine, tryptophan, 4- flurophenylalanine (Fpa), 4-clorophenylalanine (Cph), 4-nitrophenylalanine (Nph), phenyl glycine (Phg), valine or isoleucine,
ii) each R is independently alanine, β-alanine, gamma-aminobutyric acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (l OAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa), and
iii) each T is independently alanine, β-alanine, gamma-aminobutyri c acid (GABA), e-aminohexanoic acid (Ahx), phenylglycine (Phg), 9-aminooctanoic acid (9-Aoa), 10-aminodecanoic acid (lOAda), 12-aminododecanoic acid (12-Adda), or 16-aminopalmitic acid (16-Apa);
d) each D is independently three to six of any combination of lysine, arginine, histidine, ornithine, 2,3-diaminopropionic acid (Dpr),
2,4-diaminobutanoic acid (Dab), 4- aminopiperidine-4-carboxylic acid (Apc4), or 3-aminopiperidine-3-carboxylic acid (Apc3), and
e) E is a cyclic Cu tetra substituted amino acid.
Figure imgf000060_0001
3. The synthetic antimicrobial peptide of claim 1 or claim 2, wherein the peptide comprises the amino acid sequence of
a. Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c) K(A6c)KKKK-amide (SEQ ID
NO:l); b. Ac-GF(A6c)G(A6c) (A6c)G(A6c)F(A6c)G(A6c)GKK(A6c)KKKK-amide (SEQ ID NO:2); c. Ac-GF(A6c)G(A6c)R(A6c)G(A6c)F(A6c)G(A6c)GR(A6c)RRRR-amide (SEQ ID
NO:3);
d. Ac-GF(A6c)G(A6c)Orn(A6c)G(A6c)F(A6c)G(A6c)GOrn(A6c)Orn-Orn-Orn- Orn-amide (SEQ ID NO:4);
e. Ac-GF(A6c)G(A6c)Dab(A6c)G(A6c)F(A6c)G(A6c)GDab(A6c)Dab-Dab-Dab-
Dab-amide (SEQ ID NO: 5);
f. Ac-GF(A6c)G(A6c)Dpr(A6c)G(A6c)F(A6c)G(A6c)GDpr(A6c)Dpr-Dpr-Dpr-Dpr- amide (SEQ ID NO:6);
g. Ac-GF(A6c) -Ala(A6c)K(A6c)p-Ala (A6c)F(A6c)p-Ala(A6c)p- AlaK(A6c)KKKK-amide (SEQ ID NO:7);
h. Ac-GF(A6c)GABA(A6c)K(A6c)GABA(A6c)F(A6c)GABA(A6c)GABAK (A6c) KKK-amide (SEQ ID NO:8);
i. Ac-GF(A5c)G(A5c)KK(A5c)G(A5c)F(A5c)G(A5c)GKK(A5c)KKKK-amide (SEQ ID NO:9);
j. Ac-GF(A5c)G(A5c)R(A5c)G(A5c)F(A5c)G(A5c)GR(A5c)RRRR-amide (SEQ ID
NO: 10);
k. Ac-GF(A5c)G(A5c)Orn(A5c)G(A5c)F(A5c)G(A5c)GOrn(A5c)Orn-Orn-Orn- Orn-amide (SEQ ID NO: 11);
1. Ac-GF(A5c)G(A5c)Dab(A5c)G(A5c)F(A5c)G(A5c)GDab(A5c)Dab-Dab-Dab- Dab-amide (SEQ ID NO: 12);
m. Ac-GF(A5c)G(A5c)Dpr(A5c)G(A5c)F(A5c)G(A5c)GDpr(A5c)Dpr-Dpr-Dpr- Dpr-amide (SEQ ID NO: 13);
n. Ac-GF(A5c) -Ala(A5c)K(A5c) -Ala(A5c)F(A5c)p-Ala(A5c)P- AlaK(A5c)KKKK-amide (SEQ ID NO:14);
o. Ac-GF(A5c)GABA(A5c)K(A5c)GABA(A5c)F(A5c)GABA(A5c)GABAK (A5c)
KKKK-amide (SEQ ID NO: 15);
p. Ac-GF(A6c)G(A5c)K(A6c)G(A5c)F(A6c)G(A5c)GK(A6c)KKKK-amide (SEQ ID NO: 16);
q. Ac-GF(A6c)G(Tic)K(A6c)G(Tic)F(A6c)G(Tic)GK(Tic)KKKK-amide (SEQ ID NO: 17);
r. Ac-GF(A6c)G(Oic)K(A6c)G(Oic)F(A6c)G(Oic)G (Oic)KK K-amide (SEQ ID NO:18);
s. Ac-GF(A5c)G(A6c) (A5c)G(A6c)F(A5c)G(A6c)GK(A5c)KK K-amide (SEQ ID NO: 19); t. Ac-GF(A5c)G(A5c)K(A5c)G(A5c)F(A5c)G(A5c)GK(A5c)KKKK-amide (SEQ ID NO:23);
u. Ac-K KGF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c)GK(A6c)-amide (SEQ ID NO:24)or any combination thereof, wherein A6c is 1 -aminocycohexane carboxylic acid, A5c is 1 -aminocyclopentane carboxylic acid, GABA is Gamma aminobutyric acid, Oic is Octahydroindolecarboxylic acid, Tic is Tetrahydroisoquinolinecarboxylic acid, Dab is diaminobutionic acid, Dpr is diaminopropionic acid, G is glycine, F is phenyalanine, K is lysine, and Ac is acetyl.
4. A pharmaceutical composition comprising the synthetic antimicrobial peptide of any one of the preceding claims and/or salt thereof, and a pharmaceutically acceptable carrier.
5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is in the form of a granule, a powder, a tablet, a capsule, a suppository, a syrup, an emulsion, a gel, an ointment, a dispersion, a suspension, a cream, a foam, an aerosol, a droplet, an injectable form and/or a coating.
6. A product comprising the antimicrobial peptide of anyone of claims 1 to 3, and/or the pharmaceutical composition of claim 4 or claim 5, wherein the product comprises a bandage, plaster, suture, soap, tampon, diaper, shampoo, tooth paste, anti-acne compound, suncream, textile, adhesive, cleaning solution, contact lens, implant or any combination thereof.
7. A kit comprising the antimicrobial peptide of any one of claims 1-3, the pharmaceutical composition of claim 4 or claim 5, and/or the product of claim 6.
8. A method of treating or preventing an infection, disease, and/or condition in a subject in need thereof comprising,
administering to a subject in need thereof an effective amount of at least one synthetic antimicrobial peptide of any one of claims 1 to 3, the pharmaceutical composition of claim 4 or claim 5, and/or the product of claim 6.
9. The method of claim 8, wherein the infection, disease or condition is an ulcer, a wound, a burn, a skin infection, a lung infection in a subject having cystic fibrosis, sepsis, septic shock, or cancer.
10. The method of claim 8 or claim 9, wherein the infection, disease, and/or condition to be treated or prevented is caused by a bacteria and/or a fungus.
1 1. The method of claim 10, wherein the bacteria is a gram negative or a gram positive bacterium.
12. The method of any one of claims 8 to 11, wherein the infection, disease, and/or condition to be treated or prevented is caused by Enterococcus spp., Staphylococcus spp., Klebsiella spp., Acinetobacter spp., Pseudomonas spp, Enterobacter spp, Yersinia spp., Mycobacterium spp., Brucella spp., Bacillus spp., Francisella spp., Salmonella spp.,
Burkholderia spp., Escherichia spp., or any combination thereof.
13. The method of any one of claims 8 to 12, wherein the infection, disease, and/or condition to be treated or prevented is caused by Enterococcus faecium, Staphylococcus aureus, Klebsiella pnemoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,
Enterobacter aerogenes, Yersinia pestis, Mycobacterium ranae, Mycobacterium avium complex (MAC), Brucella suis, Bacillus anthracis, Francisella tularensis, Salmonella typhimurium, Burkholderia pseudomallei, Escherichia coli, or any combination thereof.
14. The method of claim 13, wherein the Staphylococcus aureus is a Methicillin- resistant Staphylococcus aureus (MRS A).
15. The method of claim 8 or claim 9, wherein the infection, disease, and/or condition to be treated or prevented is caused by Candida spp., Fusarium spp., Aspergillus spp., Cryptococcus spp., Coccidioides spp., Tinea spp., Sporothrix spp., Blastomyces spp., Histoplasma spp. and/or Pneumocystis spp.
16. The method of any one of claims 8, 9 or 15, wherein the infection, disease, and/or condition to be treated or prevented is caused by Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Fusarium solani, Tinea unguium, Tinea corporis, Tinea cruris, Sporothrix schenckii, Blastomyces dermatitidis, Histoplasma capsulatum,
Pneumocystis carinii and/or Histoplasma duboisii.
17. The method of claim of any one of claims 8 to 15, wherein the infection, disease, and/or condition to be treated or prevented is caused by Staphylococcus aureus, MRSA, Enterococcus faecium, Klebsiella pnemoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and/or Enterobacter aerogenes and the at least one synthetic antimicrobial peptide is Ac-GF(A6c)G(A6c) (A6c)G(A6c)F(A6c)G(A6c) K(A6c)KKKK-amide.
18. The method of any one of claims 8 to 13, wherein the infection, disease and/or condition to be treated or prevented is a lung infection in a subject having cystic fibrosis and the at least one peptide is Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c) K(A6c)KKKK- amide.
19. The method of claim 18, wherein the lung infection is caused by Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, and/or Mycobacterium avium complex (MAC).
20. The method of claim 9, wherein the infection, condition or disease is sepsis and/or septic shock caused by a gram negative bacteria.
21. The method of claim 9, wherein the cancer is breast cancer, ovarian cancer, bladder cancer, stomach cancer, lung cancer or leukemia, or any combination thereof.
22. The method of any one of claims 8-21, wherein the administering step comprises topical, intravenous, infusion, oral, inhalation, transdermal, parenteral, rectal, intraperitoneal and intravaginal administration
PCT/US2014/062442 2013-10-31 2014-10-27 Anti microbial peptides incorporating cyclic alpha tetra-substituted unnatural amino acids WO2015065916A1 (en)

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EP3612203A4 (en) * 2017-04-19 2021-02-17 The Regents Of The University Of Colorado Antimicrobial peptides and methods of treating gram-negative pathogens: polar and non-polar face analogs
CN107857803A (en) * 2017-12-04 2018-03-30 中国农业科学院饲料研究所 Natural antibacterial peptide and its application
CN111407764A (en) * 2020-03-27 2020-07-14 广州中医药大学(广州中医药研究院) Application of novel skeleton heteroterpene derivative in preparation of medicine for resisting non-alcoholic steatohepatitis and hepatic fibrosis

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