WO2004052915A2 - Novel cyclic peptides comprising alpha-, beta- and gamma- amino acids - Google Patents

Abstract

The present invention provides novel cyclic peptides comprised of α-, β- and Ϝ-amino acids.

Description

NOVEL CYCLIC PEPTIDES COMPRISING o -. β- and v-AMINO ACIDS

This application claims priority from provisional applications U.S.S.N. 60/431,493, filed December 6, 2002, by Juan R. Granja, entitled " Novel Cyclic Peptides Comprising o , β- and γ-Amino Acids", and U.S.S.N. 60/459, 435 filed March 31, 2003, by Granja, the contents of both are hereby incorporated by reference in their entirety.

FD3LD OF THE INVENTION

The present invention relates to homodetic cyclic peptides comprised of α-, β- and γ-amino acids. This invention further concerns compositions comprising such cyclic peptides, in addition to methods of using such cyclic peptides and compositions, for example, as antimicrobial, antifungal, antiviral, and anticancer agents.

BACKGROUND

The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art.

Modern medical science is constantly searching for new and more powerful agents to prevent, treat or retard bacterial and viral infections and cure the diseases they cause. Bacterial and viral infections of humans and domestic animals cost billions of dollars annually. Vast sums of money are spent each year by pharmaceutical companies in an effort to identify, characterize, and produce new antibiotics and antivirals to combat the emerging drug resistant strains which have become a serious problem. Reliable prophylactic treatments for disease prevention are also of major interest. Yet, despite the costs and efforts to identify treatments for viral infections, such as hepatitis and others, for example, fully effective therapies remain elusive. Antibiotic resistance is gaining in importance as a medical problem because more microorganisms are becoming resistant to greater numbers of antibiotics. See, e.g., Antibiotic Resistance, A Growing Threat, website at fda.gov/oc/opacom/hottopics/anti_resist.html; P.J. Koplan et al, Preventing Emerging Infections Diseases. A Strategy for the 21^s' Century, U.S. Department of Health & Human Services, Center for Disease Control & Prevention, Atlanta, GA (1998). Some strains of Staphylococcus aureus, for example, have developed resistance to many distinct antibiotics and are now described as having a "multi-resistant" phenotype. Unfortunately, due to extensive use of antibiotics, such multi-resistant strains of microorganisms are now found in the general population and within hospitals.

"New" antibiotics are often structurally related to, or structurally derived from, a previous generation of antibiotics. For example, cephalosporin is structurally related to penicillin. While these structural analogs of known antibiotics may be successful for a time, increasing levels of resistance are expected to develop as the "new" antibiotic becomes widely used, as illustrated by the case of β-lactam antibiotics. This problem has been alleviated only partially by the production of new generations of antibiotics. For example, valinomycin is a cyclic depsipeptide with an alternating D-D-L-L chiral motif that employs ester linkages within the ring structure. However, the activity, selectivity, in vivo stability, toxicity and bioavailability of valinomycin and other such peptides are not optimal. There is thus an urgent need for new antimicrobial agents, especially with the recent dramatic rise of antibiotic-resistant pathogens and infectious diseases.

There is also currently a significant scarcity of antivirals for treating viral infections, such as adenovirus infections, as well as a shortage of vaccines. In recent years, several novel treatment modalities emerged for a number of virus infections, including lamivudine for hepatitis B virus, abacavir, adefovir dipivoxyl and apropovir disprometil for human immunodeficiency virus, cidofovir for cytomegalovirus, and famciclovir (the oral prodrug of penciclovir) and cidofovir for other herpesviruses (herpes simplex virus and varicella-zoster virus). For all drugs, it has been reported that resistance eventually develops upon prolonged administration to the infected individuals, albeit to a varying extent. In addition, new mutations related to multidrug resistance have recently been identified. Balzarini et al, "New Antivirals - Mechanism of Action and Resistance Development," Current Opinion in Microbiology, 1, 535-546 (1998).

Antibiotics, antivirals and antifungals together comprise about a $32 billion market. Increases in the number of immuno-suppressed patients combined with the emergence of multi-drug resistant infections have created a gap in therapy where existing anti-infectives are increasingly inadequate. As a result, there is a strong demand for new drugs having greater potency, better efficacy against resistant infections, and fewer side effects than existing therapies.

Cancer also continues to be a common cause of death in developed countries. Although advances have been made in detection and therapy, no universally successful method for prevention and/or treatment is currently available. While surgery and non- surgical anticancer therapies such as radiotherapy, chemotherapy, photodynamic therapy, immunotherapy, electric/chemotherapy, hyperthermia therapy, hyperbaric oxygen therapy, ischemia/reperfusion therapy and gene therapy have been found to be effective in the treatment of various types of cancer, all of these treatments are frequently limited by their effectiveness and by tumor recurrence. It remains difficult to evaluate and prevent the metastatic potential of a cancer, and the high mortality observed in cancer patients indicates that improvements are needed in the treatment and/or prevention of the disease. Billions of dollars are spent each year on global efforts to treat and prevent cancer. The cyclic peptides of the invention are designed to meet one or more of these needs. Cyclic peptides of the invention will also serve as valuable tools for unraveling basic cell biological processes.

In addition to valinomycin, various other cyclic peptides have been the subject of previous investigation. See, e.g., U.S. Patent No. 5,916,872, issued June 29, 1999, for "Cyclic peptides having broad spectrum antimicrobial activity," which asserts that cyclic peptides generally comprised of about 10-30 amino acid residues and characterized by a structure containing three main elements or domains (an amphiphilic anti-parallel β-sheet region, a β-turn region and a loop region) have antibiotic activity against Eschericha coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus or vancomycin- resistant Enterococcus faecium and Enterococcus faecalis.

U.S. Patent No. 6,465,427, entitled "Compounds and methods for modulating cell adhesion," describes methods for treating cancer by modulating cell adhesion with cyclic peptides comprising a cadherin ell adhesion recognition sequence. See also U.S. Patent No. 6,433,149, entitled "Compounds and methods for inhibiting cancer metastasis." U.S. Patent No. 6,358,921, entitled "Antimicrobial peptide compositions and method," is said to be directed to cyclized peptides generally greater than 11 amino acids in length that have antimicrobial activity. See also, e.g., U.S. Patent No. 6,337,385, entitled, "Staphylococcus peptides for bacterial interference," which is said to be directed to various cyclic peptides including a cyclic peptide comprising the amino acid sequence of NH₂-X_n -Z-Xy-COOH and a cyclic bond between the Z residue and COOH other than a thioester bond, wherein X is selected from the group consisting of an amino acid, an amino acid analog, a peptidomimetic and a non-amide isostere, Z is selected from the group consisting of a synthetic amino acid and a biosynthetic amino acid, n is 0 to 10 and y is 1 to 10, and U.S. Patent No. 6,307,016, entitled "Parevins and tachytegrins," and which is said to be directed to various straight chain and cyclic antimicrobial peptides effective against a variety of microbes, including bacteria, viruses, retroviruses, fungi, yeast and protozoa.

Others have investigated the use of cyclic D,L-α-peptides, indicating possible applications ranging from the preparation of novel antibacterial, cytotoxic, and drug delivery agents to catalytic and materials science applications. See, e.g., Fernandez-Lopez, et al., Nature 412, 452-455 (2001); Bong, et al, Angew. Chem. Int. Ed. Engl, 40, 988 (2001); Sanchez-Quesada, et al., J Am. Chem. Soc, 122:11757 (2000); Kim, et al., J Am. Chem. Soc, 120(18) 4417-4424 (1998),; Hartgerink, et al., Chem. Eur. J, 4, 1367-1372 (1998); Ghadiri et al., Nature, 366, 324-327 (1993); Khazanovich et al., J Am. Chem. Soc, 116, 6011-6012 (1994); Ghadiri, et al. Nature, 369, 301-304 (1994); Ghadiri, et al, Angew. Chem. Int. Ed. Engl, 34, 93-95 (1995); Granja, et al, J Am. Chem. Soc. , 116, 10785-10786 (1994); Kobayashi, et al, Angew. Chem. Int. Ed. Engl, 34, 95-98 (1995); Ghadiri, Adv. Mater.,1, 615-611 (1995); Engels, et al., J Am. Chem. Soc, 111, 9151-9158 (1995); Clark, et al, J Am. Chem. Soc, 117, 12364-12365 (1995); Hartgerink, et al, J Am. Chem. Soc, 118, 43-50 (1996); Vollmer, et al., Angew. Chem. Int. Ed. , 38, 1598-1601 (1999); Motesharei, et al, J -4w. Chem. Soc , 119, 11306-11312 (1997); Rapaport, et al, J Am. Chem. Soc. , 121, 1186-1191 (1999); Steinem, et al, Langmuir , 15, 3956-3964 (1999); Clark, et al, Chem.Eur. J., 5, 782-792 (1999); Sanchez-Quesada, et al, Angew. Chem. Int. Ed, 40, 2503-2506 (2001); Bong, et aϊ, Angew. Chem. Int. Ed, 40, 2163-2166 (2001); Sanchez-Quesada, et al, J Am. Chem. Soc. , 124, 10004-10005 (2002).

SUMMARY OF THE INVENTION

The present invention provides novel cyclic peptides. Peptides of the invention have desirable therapeutic activities, and may be used as fast-acting, well-tolerated antimicrobial, antiviral, antifungal, and/or anticancer agents. The novel cyclic peptides of the present invention comprise from four to about twenty α-, β- and γ-amino acids.

In one embodiment, the cyclic peptides of the invention include an even number of α-amino acids and at least one β-amino acid. In another embodiment, at least one α-amino acid and at least one γ-amino acid are used to form a cyclic peptide wherein the total number of α- and γ-amino acids combined is an even number. In yet another embodiment, at least one α-amino acid, at least one β-amino acid, and at least one γ-amino acid are used to form a cyclic peptide wherein the total number of the α- and γ-amino acids combined is an even number. In still another embodiment, all γ-amino acids are used to form a cyclic peptide. In a further embodiment β-amino acids and γ-amino acids are used to form a cyclic peptide wherein the total number of the γ-amino acids is an even number.

In each of the embodiments described above, the total number of atoms that form the peptide backbone is an even number and the cyclic peptide is in the flat conformation, where all the amino acid side chains are pointing outwardly in an equatorial position. The cyclic peptides of the present invention are fast-acting, proteolytically stable and easy to synthesize. They are designed to avoid undesired lysis of mammalian cells, for example, as measured by hemo lysis of erythrocytes. This invention further concerns compositions comprising such cyclic peptides, in addition to methods of using such cyclic peptides and compositions, for example, to treat and/or prevent microbial infections, cancer, and viral infections in a subject, for example, a mammal.

In a first aspect, the present invention concerns a cyclic peptide comprising a sequence of from four to about twenty amino acids, wherein the sequence includes an even number of α-amino acids and at least one β-amino acid. In other aspects, cyclic peptides comprising from four to about twelve, from four to about ten, and from six to eight amino acids, wherein the sequence includes an even number of α-amino acids and at least one β- amino acid. In a preferred embodiment, the cyclic peptide has an amino acid sequence represented as: c-{[(α-Aa)₂-]_n(β-Aa)_m-} FORMULA I where n is 2, 3, 4 or 5; and m is 1, 2, 3, 4, 5 or 6. In this and other formulae, "Aa" refers to an amino acid and "c" indicates that the peptide is cyclic. In an additional embodiment, the amino acid sequence of the cyclic peptide may be represented as: c-{[(S-α-Aa)-(R-α-Aa)-]_n(S,S-β-Aa)_m-} FORMULA la where n is 2, 3, 4 or 5; and m is 1, 2, 3, 4, 5 or 6. In this and other formulae, "S" and "R" indicate the chirality of the referenced amino acids. In a related preferred embodiment, an amino acid sequence of Formula I is an enantiomer of a sequence represented by Formula la, i.e., c-{[(R-α-Aa)-(S-α-Aa)-]_n(R,R- β-Aa)_m-}.

In yet another preferred embodiment, the cyclic peptide has an amino acid sequence represented by Formula II: c-[(α-Aa)₂-(β-Aa)_n-]_m FORMULA II where n is 1, 2, 3, 4, 5, or 6; and m is 1, 2, 3, 4, 5, or 6. In an additional embodiment, the amino acid sequence of the cyclic peptide may be represented as: c-[(S-α-Aa)-(R-α-Aa)-(S,S-β-Aa)_n-]_m FORMULA Ila where n is 1, 2, 3, 4, 5, or 6; and m is 1, 2, 3, 4, 5, 6, 7 or 8. In a related preferred embodiment, an amino acid sequence of Formula II is an enantiomer of a sequence represented by Formula Ila, i.e., c-[(R-α-Aa)-(S-α-Aa)-(R,R-β-Aa)_n-]_m.

In yet another preferred embodiment, the cyclic peptide has an amino acid sequence represented by Formula III: c-[(α-Aa)₃-(β-Aa)_n-]_x FORMULA III where n is 1, 2, 3, 4, 5, or 6; and x is 2 or 4. In an additional embodiment, the amino acid sequence of the cyclic peptide may be represented as: c-[(R-α-Aa)-(S-α-Aa)-(R-α-Aa)-( S,S-β-Aa)ni-(S-α-Aa)-(R-α-Aa)-(S-α-Aa)-(R,R

-β-Aa)_n2-]_m FORMULA Ilia where nl is 1, 2, 3, 4, 5, or 6; n2 = 1, 2, 3, 4, 5 or 6; and m is 1 or 2.

In yet another preferred embodiment, the cyclic peptide has an amino acid sequence represented by Formula IV:

c-[(α-Aa)₄-(β-Aa)_n-]m FORMULA IV where n is 1, 2, 3, 4, 5, 6, 1, or 8; and m is 1, 2, 3 or 4. In an additional preferred embodiment, the amino acid sequence of the cyclic peptide may be represented as:

c-[(S-α-Aa)-(R-α-Aa)-(S-α-Aa)-(R-α-Aa)-(S,S-β-Aa)_n-]m FORMULA IVa. where n = 1, 2, 3, 4, 5, 6, 1 or 8; and m is 1, 2, 3 or 4. In a related embodiment, an amino acid sequence of Formula IV is an enantiomer of a sequence represented by Formula IVa, i.e., c-[(R-α-Aa)-(S-α-Aa)-(R-α-Aa)-(S-α-Aa)-(R,R-β-Aa)_n-]_m. In yet another preferred embodiment, the cyclic peptide has an amino acid sequence represented by Formula V: c-[(α-Aa)-(β-Aa)_n-]_x FORMULA V where n is 1, 2, 3, 4, 5, 6, 1, or 8; and x = 2, 4, 6 or 8. In an additional preferred embodiment, the amino acid sequence of the cyclic peptide may be represented as:

c-[(S-α-Aa)-(R,R-β-Aa)ni-(R-α-Aa)-(S,S-β-Aa)_n2-]_m FORMULA Va where nl = 1, 2, 3, 4, 5, 6, 1 or 8; n2 = 1, 2, 3, 4, 5, 6, 7 or 8; and m is 1, 2, 3, or 4. In a related embodiment, an amino acid sequence of Formula V is an enantiomer of a sequence represented by Formula Va, i.e., c-[(R-α-Aa)-(S,S-β-Aa)_nι-(S-α-Aa)-(R,R-β-Aa)_n2-]_m.

In another aspect, the invention concerns cyclic peptides comprising a sequence of from four to about twenty amino acids, wherein the sequence includes at least one α-amino acid and at least one γ-amino acid and wherein the total number of α- and γ-amino acids combined is an even number.

In a preferred embodiment, the cyclic peptide has an amino acid sequence represented as: c-[(α-Aa)_n-(γ-Aa)-]_x FORMULA VI where if n = 1, 3 or 5; then x = 2, 3, 4, 5, 6, 1, or 8; if n = 2, 4, or 6; then x = 2, 4, 6, or 8. In an additional embodiment, the amino acid sequence of the cyclic peptide may be represented as: c-[(R-α-Aa)-(R,R,S-γ-Aa)-]_m FORMULA Via where m = 2, 3, 4, 5, 6, 7 or 8. In a related embodiment, an amino acid sequence of

Formula VI is an enantiomer of a sequence represented by Formula Via, i.e., c-[(S-α-Aa)- (S,S,R-γ-Aa)-]_m.

In an additional preferred embodiment, the amino acid sequence of the cyclic peptide may be represented as: c-{[(S-α-Aa)-(R-α-Aa)-]_n(R,R,S-γ-Aa)-(R-α-Aa)-}_m FORMULA VIb where n = 1, 2, 3, 4 or 5; and m = 1, 2, 3 or 4. In a related embodiment, an amino acid sequence of Formula VI is an enantiomer of a sequence represented by Formula VIb, i.e., c-{[(R-α-Aa)-(S-α-Aa)]_n-(S,S,R-γ-Aa)-(S-α-Aa)-}_m.

In an additional preferred embodiment, the amino acid sequence of the cyclic peptide may be represented as: c-{[(S-α-Aa)-(R-α-Aa)-]ni(R,R,S-γ-Aa)-[(R-α-Aa)-(S-α-Aa)-]_n2(S,S,R-γ-Aa)-}_m

FORMULA Vic where nl is 0, 1, 2, 3, 4 or 5 and n2 are 1, 2, 3, 4 or 5; and m = 1, 2, 3 or 4.

In another preferred embodiment, the cyclic peptide has an amino acid sequence represented by Formula VII: c-[(α-Aa)-(γ-Aa)_n-]_x FORMULA VII where n = 1, 3 or 5; then x = 1, 2, 3 or 4; if n = 2, or 4; then x = 2, 4 or 6.

In an additional preferred embodiment, the amino acid sequence of the cyclic peptide may be represented as: c-{(R-α-Aa)-[(R,R,S-γ-Aa)-(S,S,R-γ-Aa)-]_nι(S-α-Aa)-[(S,S,R-γ-Aa)-(R,R,S-γ-Aa)-

]_n2}_m FORMULA Vila where nl and n2 are both 1 or 2; and m = 1, 2, 3 or 4.

In an additional preferred embodiment, the amino acid sequence of the cyclic peptide may be represented as c-[(S-α-A)-(S,S,R-γ-Aa)-(R,R,S-γ-Aa)-(S,S,R-γ-Aa)-]_m FORMULA Vllb where m = 1, 2, 3, 4 or 5. In a related embodiment, an amino acid sequence of Formula VII is an enantiomer of a sequence represented by Formula Vllb, i.e., c-[(R-α-A)-(R,R,S-γ-

Aa)-(S,S,R-γ-Aa)-(R,R,S-γ-Aa)-]_m.

In a further aspect , the invention concerns a cyclic peptide comprising a sequence of from four to about twenty amino acids, wherein the sequence includes at least one α-

amino acid, at least one β-amino acid, and at least one γ-amino acid, and wherein the total number of α- and γ-amino acids combined is an even number.

In a preferred embodiment, the cyclic peptide has an amino acid sequence represented as: c-[(α-Aa)-(β-Aa)_n-(γ-Aa)-]_m FORMULA VIII where n = 1, 2, 3, or 4; and m = 1, 2, 3, 4, 5 or 6.

In an additional preferred embodiment, the amino acid sequence of the cyclic peptide may be represented as: c-[(R-α-Aa)-(S,S-β-Aa)_n-(R,R,S-γ-Aa)-]_m FORMULA Villa where n = 1, 2, 3 or 4; and m = 1, 2, 3, 4, 5 or 6. In a related embodiment, an amino acid sequence of Formula VIII is an enantiomer of a sequence represented by Formula Villa, le, c-[(S-α-Aa)-(R,R-β-Aa)_n-(S,S,R-γ-Aa)-]_m.

In another preferred embodiment, the cyclic peptide has an amino acid sequence represented by Formula IX: c-[(α-Aa)-(γ-Aa)-(β-Aa)_n-]m FORMULA IX where n = 1, 2, 3 or 4 ; and m = 1, 2, 3, 4, 5 or 6.

In an additional preferred embodiment, the amino acid sequence of the cyclic peptide may be represented as: c-[(S-α-Aa)-(S,S,R-γ-Aa)-(S,S-β-Aa)_n-]_m FORMULA IXa where n = 1, 2, 3 or 4; and m = 1, 2, 3, 4, 5 or 6. In a related embodiment, an amino acid sequence of Formula IX is an enantiomer of a sequence represented by Formula IXa, i.e., c-[(R-α-Aa)-(R,R,S-γ-Aa)-(R.R-β-Aa)_n]m.

In another aspect of the invention, a cyclic peptide of any of the above aspects self- assembles into a supramolecular structure. The supramolecular structure may be a nanotube, a barrel of associated, axially parallel nanotubes, or a carpet of associated nanotubes, or mixtures of one or more of these supramolecular structures. The supramoleculars structure are believed to induce depolarization of membranes of the target microbial organisms selectively over animal cell membrane depolarization or to otherwise induce lysis of a target microbial organism, a target virus, a target fungal organism, or cancer cells, selectively over undesired cells.

The invention further provides a method of identifying cyclic peptides capable of selective association with one or more target biomolecules on a selected cell surface, comprising contacting a solution of the described cyclic peptides with the target biomolecule(s) and determining, for example, whether the peptides selectively associates with the biomolecule(s). Such target biomolecules can include, for example, intracellular, extracellular or membrane-associated proteins, enzymes, nucleic acids, receptors, organelles and the like. This method can involve contacting a solution of cyclic peptides with the target biomolecule under hydrogen bond-promoting conditions and can determine whether the peptides selectively associate with the desired biomolecules and possess biological activity of interest. The target biomolecule can be displayed, for example, on the surface of a living cell, on the surface of a genetically engineered cell or on the surface of a liposome.

In another aspect, the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a cyclic peptide of the invention in an amount effective to treat or prevent an infection caused by a target microbial organism, or a target virus in a subject, for example, a human or other animal.

In a further aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a cyclic peptide of the invention in an amount effective to treat or prevent a cancer in a subject, for example, a human or other animal.

In yet another aspect, the invention concerns a method of treating a microbial infection in a subject comprising administering to the subject a cyclic peptide of the invention in an amount sufficient to induce target cell death without inducing an undesirable amount of non-target cell death.

In a still further aspect, the invention relates to a method of treating a fungal infection in a subject, including but not limited to, those caused by the specific fungi listed herein, which may comprise administering to the subject a cyclic peptide of the invention in an amount sufficient to induce fungi death without inducing an undesirable amount of non-target cell death. In a still further aspect, the invention relates to a method of treating a viral infection in a subject comprising administering to the subject a cyclic peptide of the invention in an amount sufficient to induce virally infected cell death without inducing an undesirable amount of non-target cell death. In a still further aspect, the invention relates to a method of treating a cancer in a subject comprising administering to the subject a cyclic peptide of the invention in an amount sufficient to induce cancer cell death without inducing an undesirable amount of non-target cell death.

The invention also features a method for identifying or evaluating a cyclic peptide for antiviral activity comprising: (a) contacting a target viral organism or organisms with a test cyclic peptide; and (b) determining the antiviral activity of the test cyclic peptide.

In still another aspect, the invention features a method of identifying or evaluating a cyclic peptide selectively for cytotoxicity to a target cancer cell type or types comprising the steps of (1) contacting the selected cancer cell type or types with a test cyclic peptide of the invention; and (2) determining whether said test cyclic peptide induces cell death of said target cancer cell type without inducing substantial or undesired cell death in one or more other cell types.

In still another aspect, the invention features a method of identifying or evaluating a cyclic peptide of the invention for antimicrobial activity comprising the steps of (1) contacting a target microbial organism or organisms with a test cyclic peptide of the invention, and (2) determining whether the test cyclic peptide has antimicrobial activity.

BRIEF DESCRD?TION OF THE FIGURES FIGURES la, lb and lc illustrate examples of cyclic peptides made according to Formula I. FIGURES 2a, 2b, 2c and 2d illustrate examples of cyclic peptides made according to Formula II.

FIGURE 3 provides an example of cyclic peptides made according to Formula III.

FIGURES 4a and 4b illustrate examples of cyclic peptides made according to Formula IV.

FIGURES 5a and 5b illustrate examples of cyclic peptides made according to Formula V. Figure 5c illustrates cyclic peptides in which each group includes two β-amino acids followed by one α-amino acid, the cyclic peptide having the sequence: c-[(S-α-Aa)-(R,R-β-Aa)-(R,R-β-Aa)-(R-α-Aa)-(S,S-β-Aa)-(S,S-β-Aa)-]. FIGURES 6a and 6b illustrate examples of cyclic peptides made according to

Formula VI. Figures 6c, 6d and 6e illustrate other examples of cyclic peptides made according to Formula VIb. Figure 6f illustrates yet another example of cyclic peptides made according to Formula Vic.

FIGURES 7a and 7b illustrate examples of cyclic peptides made according to Formula VII.

FIGURES 8a-8e illustrate examples of cyclic peptides made according to Formula VIII.

FIGURES 9a-9e illustrate examples of cyclic peptides made according to Formula IX.

DETAILED DESCRIPTION

Certain terms used in the context of describing the invention are defined and have the following meanings when used herein and in the appended claims.

The term "amino acid" generally refers to an organic chemical species comprising at least one amino group (i.e., -NH₂) and at least one carboxylic acid group (i.e., -COOH). When incorporated into a peptide, an amino acid is often referred to as an "amino acid residue" due to the fact that an amino acid loses one or more atoms of its amino and carboxylic groups in a dehydration reaction that links one amino acid to another. An amino acid may be derivatized or modified before or after incorporation into a peptide (e.g., by glycosylation, by formation of cystine through the oxidation of the thiol side chains of two non-contiguous cysteine amino acid residues, resulting in a disulfide covalent bond that frequently plays an important role in stabilizing the folded conformation of a protein, etc.).

The term "amino acid" used herein includes alpha(α), beta (β), and gamma (γ) amino acids. An "α-amino acid" is a molecule having the structure wherein the main chain carbon atom (the "alpha (α)-carbon") is linked to a hydrogen atom, a carboxylic acid group, an amino group, and a side chain group, R. A "β-amino acid" is a molecule having the structure wherein the first main chain carbon atom (the "alpha (α)-carbon") is linked to a carboxylic acid group, a side chain group, R_α, and the second main chain carbon atom (the "beta (β)-carbon") which is linked to an amino group and another side chain group, Rβ. A "γ-amino acid" is a molecule having the structure wherein the first main chain carbon atom (the "alpha (α)-carbon") is linked to a carboxylic acid group, a side chain group, R_α, and the second main chain carbon atom (the "beta (β)-carbon") which is linked to another side chain group, R_β, and the third main chain carbon atom (the "gamma (γ)- carbon"). The third main chain carbon atom or the gamma (γ)-carbon is linked to an amino group and yet another side chain group, R_γ.

In the context of this invention, an amino acid may be one that occurs in nature or it may be non-naturally occurring, and produced by synthetic methods such as solution phase, solid state and/or automated synthesis methods. The amino acid notations used herein for the twenty genetically encoded L-α- amino acids, some examples of non-encoded amino acids and β-amino acids are provided in Table 1.

Table 1

Certain commonly encountered amino acids that are not genetically encoded and that can be present in the cyclic peptides of the invention include, but are not limited to, β- alanine (β -Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3- diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; α-aminoisobutyric acid (Aib); methylglycine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t- butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); 2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2- fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); .beta.-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2,3-diaminobutyric acid (Dbu); p- aminophenylalanine (Phe(pNH₂)); N-methyl valine (Me Val); homocysteine (hCys); and homoserine (hSer). Additional amino acid analogs contemplated include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statine, α-methyl-alanine, para-benzoyl- phenylalanine, propargylglycine, and sarcosine. Peptides that are encompassed within the scope of the invention can have any of the foregoing amino acids in the L- or D- configuration, or any other amino acid known to one of skill in the art.

Additional examples of amino acids that can be utilized in the cyclic peptide described herein can be found, for example, in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein. Another source of a wide variety of amino acid residues is provided by the website of RSP Amino Acids Analogues, Inc. (www.amino-acids.com).

The term "chirality" is used to generally describe the stereochemistry of molecules. A chiral molecule has "handedness" and hence is optically active. A carbon atom bonded to four non-identical substituents is termed a "chiral center." The absolute configuration of any chiral center can be unambiguously specified using the R- and S-sequencing rules of Cahn, Ingold and Prelog. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. Conventions for stereochemical nomenclature, methods for the determination of stereochemistry and the separation of stereoisomers are well known in the art (see, e.g., Advanced Organic Chemistry, 3rd edition, March, John Wiley & Sons, New York, 1985). In addition, R and S configurations are herein defined by assigning the following priorities to the groups around the chiral center independent of the side chains: • For alpha carbon (Cα) of the α-amino acid, preference 1 is assigned to the amino group (NH), preference 2 to the carbonyl carbon (C=0) and preference 3 to the side chain group (Rⁿ) attached to the alpha carbon.

• For alpha carbon (Cα) of the β-amino acid, preference 1 is assigned to the carbonyl carbon (C=0), preference 2 to the beta carbon (Cβ) of the main chain and preference 3 to the side chain group (Rⁿα) attached to the alpha carbon.

• For the beta carbon (Cβ) of the β-amino acid, preference 1 is assigned to N, preference 2 to the alpha carbon (Cα) of the main chain and preference 3 to the side chain group (Rⁿβ) attached to the beta carbon. • For the alpha carbon (Cα) of the γ-amino acid, preference 1 is assigned to the carbonyl carbon (C=0), preference 2 to the beta carbon (Cβ) of the main chain and preference 3 to the side chain group (Rⁿ _α) connected to the alpha carbon (Cα). For the beta carbon (Cβ) of the γ-amino acid, preference 1 is assigned to the gamma carbon (Cγ) of the main chain, preference 2 to the alpha carbon (Cα) of the main chain and preference 3 to the side chain group (Rⁿβ) attached to the beta carbon.

• For the gamma carbon (Cγ), preference 1 is assigned to N, preference 2 to the beta carbon (Cβ) of the main chain and preference 3 to the side chain group (Rⁿ _γ) attached to the gamma carbon.

Then, viewing the molecule from the face opposite the group of the lowest priority (H) and drawing an arrow through groups 1, 2 and 3 will determine the configuration of each stereocenter. If the direction of the arrow is clockwise, then the chiral center is R; if anticlockwise, label the center S.

An α-amino acid has one chiral center and has two enantiomeric forms of opposite chirality, R and S. A β-amino acid has two chiral centers and may have the following configurations: RR, SS, RS, and SR. The cyclic peptides of the present invention, however, generally comprise β-amino acids having the SS or RR configuration (configuration of both stereocenters were determined following the rules indicated above). A γ-amino acid has three chiral centers and may have the following configurations: RRR, SSS, RRS, SSR, RSS, SSR, RSR, and SRS. The cyclic peptides of the present invention, however, generally comprise γ-amino acids having the SSR or RRS configuration (as indicated above).

The present invention also encompasses all enantiomers of each of the cyclic peptides described herein. For example, a cyclic peptide of the present invention with α-, β- and γ-amino acids may have a sequence represented by the following formula: c-[(R-α- Aa)-(S,S-β-Aa)-(R,R,S-γ-Aa)-]. It is contemplated that the present invention also includes an enantiomer of such cyclic peptides having a sequence of c-[(S-α-Aa)-(R,R-β-Aa)- (S,S,R-γ-Aa)-].

The term "homodetic" refers to cyclic peptides formed by only normal peptide bonds which join the C-terminus of an amino acid residue to the N-terminus of an adjacent amino acid residue. Homodetic cyclic peptides are contrasted to heterodetic cyclic peptides in which one or more links in the ring are non-peptide bonds, for example, a new covalent bond between amino acid backbone and/or side chain groups located near the N- or C-terminal ends. The term "isomers" refers to compounds having identical molecular formulae but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers." Stereoisomers that are not mirror images of one another are termed "diastereomers", and stereoisomers that are non-superimposable mirror images are termed "enantiomers" or sometimes "optical isomers." As used herein, a "nanotube" or "nanotubule" is a small tubule that may spontaneously form from the cyclic peptides of the present invention. While not wishing or intending to be bound by this mechanism of action, it is believed that the present cyclic peptides stack under appropriate conditions to form nanotubes and/or supramolecular structures composed of nanotubes. Hydrogen bonding between cyclic peptide is believed to help drive the self-assembly of nanotubes and supramolecular nanotube structures from the cyclic peptides of the invention. Each nanotube is believed to have a pore in the center of the tube that is surrounded by a series of peptide backbones of the stacked cyclic peptides that form the nanotubes. The size of the pore will depend upon the number of amino acids in the cyclic peptides that form the nanotube. In general, depending on the ring size of the cyclic peptides employed, water, ions, sugars, and other small molecules can travel through the pores of the nanotubes. Larger molecules can also flow through pores formed from larger cyclic peptides and supramolecular structures formed of aggregates of nanotubes. For example, in some embodiments the supramolecular structure is thought to be a barrel-like structure composed of clusters of nanotubes. In other embodiments, the supramolecular structure is thought to be a "carpet" or "carpet-like" arrangement of nanotubes.

The term "mammal," as used herein, refers to an animal, in general, a warmblooded animal, which is susceptible to or has a microbial infection or other infection or disease state described herein. Mammals include cattle, buffalo, sheep, goats, pigs, horses, dogs, cats, rats, rabbits, mice, and humans. Also included are other livestock, domesticated animals and captive animals. The term "farm animals" includes chickens, turkeys, fish, and other farmed animals.

The term "peptide" as used herein refers to a sequence of amino acids residues in which the α-carboxyl group of one amino acid is joined by an amide bond to the main chain amino group of the adjacent amino acid. The peptides provided herein for use in the described and claimed methods and compositions are cyclic. Peptide sequences specifically recited herein are written with the amino terminus on the left and the carboxyl terminus on the right. However, where the peptides are shown in cyclic form, the first amino acid in the sequence is arbitrarily chosen. Moreover, for formulae of cyclic peptides where the sequence extends onto two lines, the sequence on the second line extends from the N-terminal side on the right to the C-terminal side on the left.

The phrase "pharmaceutically acceptable" refers to a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

According to the present invention, "supramolecular structures" are multi-subunit structures, e.g., nanotubes, barrels and carpets of nanotubules, which are believed to be formed through non-covalent assembly of cyclic peptides. Supramolecular structures may be contrasted with molecular or polymeric systems in which the product of covalent bond formation between reactants or monomers. The proposed peptide supramolecular structures are thermodynamically controlled assemblies that can undergo reversible structural assembly and disassembly. Such assembly/disassembly will depend, for example, on the environment, subunit structure, side chain group selection, side chain group interaction, and the nature and combination of non-covalent forces operating on the system. In contrast, covalent polymeric structures have been used to design thermodynamically and/or kinetically stable structures rather than structures that assemble and dissassemble in response to the environment. Hence, an attractive feature of the present compositions containing peptides that can form supramolecular structures is their ability to select amongst various cell membrane types. Such selection is driven by favorable thermodynamic forces determined by the composition of the cyclic peptide relative to the cell membrane environment and the molecular and/or supramolecular constituents of the cell membrane. Cyclic Peptides

The present invention provides cyclic peptides comprising from about four to about twenty α-, β- and γ-amino acid residues. Cyclic peptides of the present invention are arranged to have certain chirality configurations as illustrated in various embodiments hereinbelow. For example, alpha amino acids that are adjacent to each other are arranged to have alternating S and R chirality. Reading the sequence from the amino group (N) to the carboxyl group (C), an alpha amino acid after a beta amino acid with S,S configuration has S configuration while an alpha amino acid following a beta amino acid with R,R configuration has R configuration. Also, a gamma amino acid after a beta amino acid with S,S configuration has R,R,S configuration, while a gamma amino acid following a beta amino acid with R,R configuration has S,S,R configuration.

Additionally, a beta amino acid after an alpha amino acid with R configuration has S,S configuration, while a beta amino acid following an alfa amino acid with S configuration has R,R configuration. A beta amino acid after a gamma amino acid with R,R,S configuration has R,R configuration, while a beta amino acid following a gamma amino acid with S,S,R configuration has S,S configuration. A gamma amino acid following an alpha amino acid with S configuration has S,S,R configuration, while a gamma amino acid following an alpha amino acid with R configuration has R,R,S configuration.

In a first group of cyclic peptides of the present invention, an even number of α- amino acids and at least one β-amino acid are used to form a cyclic peptide ("Group I"). In one embodiment of the Group 1 cyclic peptides, the peptides have an amino acid sequence represented by Formula I: c-{[(α-Aa)₂]_n-(β-Aa)_m-} wherein: n = 2, 3, 4, or 5; and m = 1, 2, 3, 4, 5 or 6. The letter "c" in front of the formula is used to indicate that the peptide is cyclic and "α- Aa" and "β-Aa" refer to an alpha amino acid and a beta amino acid, respectively. Figures la-lc illustrate examples of cyclic peptides made according to Formula I, and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-{[(S-α-Aa)-(R-α-Aa)-]_n(S,S-β-Aa)_m-} wherein: n = 2, 3, 4, or 5; and m = 1, 2, 3, 4, 5 or 6. As discussed, adjacent α-amino acids are arranged to have alternating S and R chirality. The R and S configurations are assigned following the sequencing rules described before. For example, as will be apparent to those skilled in the art, the S-configuration of the alpha carbon (Cα) of the β-amino acid is assigned by considering preference 1 to the carbonyl carbon (C=0), preference 2 to the beta carbon (Cβ) of the main chain, and preference 3 to the side chain group (Rⁿα) attached to the alpha carbon. When viewing the molecule from the face opposite the group with lowest priority (H), the direction of the arrow is anticlockwise. The S-configuration of the beta carbon (Cβ) of the β-amino acid is established by assigning preference 1 to N, preference 2 to the alpha carbon (Cα) of the main chain, and preference 3 to the side chain group (Rⁿβ) attached to the beta carbon. When viewing the molecule from the face opposite the group with lowest priority (H), the direction of the arrow is anti-clockwise. In another embodiment of the Group 1 cyclic peptides, the cyclic peptides of the invention have an amino sequence represented by Formula II:

c-[(α-Aa)₂-(β-Aa)_n-]m wherein: n = 1, 2, 3, 4, 5, or 6; and m = 1, 2, 3, 4, 5, or 6. Figures 2a-2d illustrate examples of cyclic peptides made according to Formula II and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-[(S-α-Aa)-(R-α-Aa)-(S,S-β-Aa)_n-]m wherein: n = 1, 2, 3, 4, 5, or 6; m = 1, 2, 3, 4, 5, 6, 7 or 8. Yet in another embodiment of the Group I cyclic peptides, the cyclic peptides of the invention have an amino sequence represented by Formula III: c-[(α-Aa)₃-(β-Aa)_n-]χ wherein: n = 1, 2, 3, 4, 5, or 6; and x = 2 or 4. In this embodiment, three α-amino acids with alternating S and R chirality are followed by one or more β-amino acids ("first group") and then connected to another three α-amino acids with alternating S and R chirality followed by one or more β-amino acids ("second group"). The chirality of α- and β-amino acids in the second group, however, is opposite to that of corresponding α- and β-amino acids in the first group. Yet, all of the β-amino acids in a given group have the same chirality. Thus, a cyclic peptide having ten amino acid residues can, for example, have a sequence of: c-[(R-α-Aa)-(S-α-Aa)-(R-α-Aa)-(S,S-β-Aa)-(S,S-β-Aa)-(S-α-Aa)-(R-α-Aa)-(S-α- Aa)-(R,R-β-Aa)-(R,R-β-Aa)-]. Figure 3 provides another example of cyclic peptides made according to Formula III and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-[(R-α-Aa)-(S-α-Aa)-(R-α-Aa)-(S,S-β-Aa)_nι-(S-α-Aa)-(R-α-Aa)-(S-α-Aa)-(R,R- β-Aa)_n2-]_m wherein: nl = 1, 2, 3, 4, 5, or 6; n2 = 1, 2, 3, 4, 5, or 6; and m = l or 2.

In yet another embodiment of the Group 1 cyclic peptides, the cyclic peptides of the invention can have an amino sequence of Formula IV: c-[(α-Aa)₄-(β-Aa)_n-]m wherein: n = 1, 2, 3, 4, 5, 6, 7, or 8; and m = 1, 2, 3 or 4. In this embodiment, four α-amino acids with alternating S and R chirality are followed by one or more β-amino acids, wherein all of the β-amino acids have the same chirality wherein a β-amino acid after a R-α-amino acid has S,S configuration, while a β-amino acid after an S-α-amino acid has R,R configuration. Figures 4a-4b illustrate examples of cyclic peptides made according to Formula IV, and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-[(S-α-Aa)-(R-α-Aa)-(S-α-Aa)-(R-α-Aa)-(S,S-β-Aa)_n-]_m wherein: n = 1, 2, 3, 4, 5, 6, 7, or 8; and m = 1, 2, 3 or 4.

In still another embodiment of the Group I cyclic peptides, the cyclic peptides of the invention have an amino sequence represented by Formula V: c-[(α-Aa)-(β-Aa)_n-]_x wherein: n =1, 2, 3, 4, 5, 6, 7 or 8; and x = 2, 4, 6, or 8.

In this embodiment, one α-amino acid is followed by one or more β-amino acids ("first group") and then connected to another α-amino acid followed by one or more β-amino acids ("second group"), and so on. The chirality of α- and β-amino acids in the second group, however, is opposite to that of corresponding α- and β-amino acids in the first group. Yet, all of the β-amino acids in a given group have the same chirality. Thus, as shown in Figure 5c, when each group includes two β-amino acids followed by one α- amino acid, the cyclic peptide will have a sequence of: c-[(S-α-Aa)-(R,R-β-Aa)-(R,R-β-Aa)-(R-α-Aa)-(S,S-β-Aa)-(S,S-β-Aa)-].

Figures 5a and 5b also illustrate examples of cyclic peptides made according to Formula V and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-[(S-α-Aa)-(R,R-β-Aa)_nι-(R-α-Aa)-(S,S-β-Aa)_n2-]m wherein: nl = 1, 2, 3, 4, 5, 6, 7, or 8; n2 = 1, 2, 3, 4, 5, 6, 7, or 8; and m = 1, 2, 3, or 4

For each of the cyclic peptides of Formulae I-V discussed above, each of the β- amino acids can have a side chain (Rⁿ) substitution at the α- or β-carbon, or both, or have no side chain (Rⁿ) substitution. Also, each of the side chains (Rⁿ) on the α- and β- amino acids can be independently selected from any side chain of the natural amino acids or the groups consisting of hydroxyl, linear or branched Cl-Cl 0-alkyl, alkenyl, alkynyl, hydroxy- Cl- Cl 0-alkyl, amino-Cl- Cl 0-alkyl, Cl- ClO-alkoxy, Cl- ClO-alkoxy-Cl- Cl 0-alkyl, amino, mono or di-Cl- ClO-alkylamino, carboxamido, carboxamido-Cl- Cl 0-alkyl, sulfonamido, sulfonamide-C 1 - C 10-alkyl, urea, cyano, fluoro, Cl- Cl 0-alkyl-fluoruro, thio, Cl- ClO-alkylthio, mono or bicyclic aryl, and mono or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O, S, mono or bicyclic aryl-Cl- Cl 0-alkyl, heteroaryl- Cl- Cl 0-alkyl and combinations.

In the second group of the cyclic peptides of the present invention, at least one α- amino acid and at least one γ-amino acid are used to form a cyclic peptide wherein the total number of α- and γ-amino acids combined is an even number ("Group II").

In one embodiment of the Group II cyclic peptides, the cyclic peptides of the invention have an amino acid sequence represented by Formula VI: c-[(α-Aa)_n-(γ-Aa)-]_x wherein, n = 1, 3 or 5; and x = 2, 3, 4, 5, 6, 7 or 8; or n = 2, 4 or 6; and x = 2, 4, 6 or 8 Again, the letter "c" in front of the formula is used to indicate that the peptide is cyclic wherein "α-Aa" and "γ-Aa" refer to an alpha amino acid and a gamma amino acid, respectively. Figures 6a and 6b illustrate examples of cyclic peptides made according to Formula VI, and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-[(R-α-Aa)-(R,R,S-γ-Aa)-]_m wherein, m = 2, 3, 4, 5, 6, 7, or 8. As discussed above, the R and S configurations are assigned following the rules described before. As will be apparent to those skilled in the art, the R-configuration of the alpha carbon (Cα) of the γ-amino acid is assigned by considering preference 1 to the carbonyl carbon (C=0), preference 2 to the beta carbon (Cβ) of the main chain and preference 3 to the side chain group (Rⁿ _α) connected to the alpha carbon (Cα). When viewing the molecule from the face opposite the group with lowest priority (H), the direction of the arrow is clockwise. The R-configuration of the beta carbon (Cβ) of the γ-amino acid is established by assigning preference 1 to the gamma carbon (Cγ) of the main chain, preference 2 to the alpha carbon (Cα) of the main chain, and preference 3 to the side chain group (Rⁿ _β) attached to the beta carbon. When viewing the molecule from the face opposite the group with lowest priority (H), the direction of the arrow is clockwise. The S-configuration of the gamma carbon (Cγ) is established by assigning preference 1 to N, preference 2 to the beta carbon (Cβ) of the main chain, and preference 3 to the side chain group (Rⁿ _γ) attached to the gamma carbon. When viewing the molecule from the face opposite the group with lowest priority (H), the direction of the arrow is anti-clockwise.

Figures 6c, 6d and 6e illustrate other examples of cyclic peptides made according to Formula VI. Such cyclic peptides may include three, five or seven α-amino acids with alternating S and R chirality followed by one γ-amino acid, and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-{[(S-α-Aa)-(R-α-Aa)-]_n(R,R,S-γ-Aa)-(R-α-Aa)-}_m wherein: n = 1, 2, 3, 4 or 5; and m = 1, 2, 3, or 4. Figure 6f illustrates yet another example of cyclic peptides made according to Formula VI, and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-{[(S-α-Aa)-(R-α-Aa)-]„ι(R,R,S-γ-Aa)-[(R-α-Aa)-(S-α-Aa)-]_n2(S,S,R-γ-Aa)-}_m wherein: nl = 0, 1, 2, 3, 4 or 5; n2 = 1, 2, 3, 4 or 5; and m = l, 2, 3, or 4. As illustrated in Figure 6f, such cyclic peptides may be arranged to have two or four α- amino acids with alternating S and R chirality followed by one γ-amino acid ("first group") and then connected to another two or four α-amino acids with alternating S and R chirality followed by one or more γ-amino acid ("second group"). The chirality of α- and γ-amino acids in the second group, however, is opposite to that of corresponding α- and γ-amino acids in the first group. In another embodiment of the Group II cyclic peptides, the cyclic peptides of the invention can have an amino sequence represented by Formula VII: c-[(α-Aa)-(γ-Aa)_n-]_m wherein: n = 1, 3 or 5; and x = 1, 2, 3 or 4; or n = 2 or 4; and x = 2, 4 or 6. Figure 7a illustrates an example of cyclic peptides made according to Formula VII, and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-{(R-α-A)-[(R,R,S-γ-Aa)-(S,S,R-γ-Aa)-]„ι(S-α-Aa)-[(S,S,R-γ-Aa)-(R,R,S-γ-

Aa JnHm wherein: nl and n2 are 1 or 2; and m = l, 2, 3 or 4.

Figure 7b illustrates another example of cyclic peptides made according to Formula VII, and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-[(S-α-A)-(S,S,R-γ-Aa)-(R,R,S-γ-Aa)-(S,S,R-γ-Aa)-]_m wherein: m = l, 2, 3, 4, or 5.

For each of the cyclic peptides of formulae VI-VII discussed above, each of the γ- amino acids can have a side chain (Rⁿ) substitution at the α- or β- or γ-carbons, or have no side chain (Rⁿ) substitution. Also, each of the side chains (Rⁿ) on the α- and γ- amino acids can be independently selected from any side chain of the natural amino acids or the groups consisting of hydroxyl, linear or branched Cl- Cl 0-alkyl, alkenyl, alkynyl, hydroxy-C 1 - Cl 0-alkyl, amino-Cl- Cl 0-alkyl, Cl- ClO-alkoxy, Cl- ClO-alkoxy-Cl- Cl 0-alkyl, amino, mono or di-Cl- ClO-alkylamino, carboxamido, carboxamido-Cl- Cl 0-alkyl, sulfonamido, sulfonamide-C 1- Cl 0-alkyl, urea, cyano, fluoro, Cl- ClO-alkyl-fluoruro, thio, Cl- C10- alkylthio, mono or bicyclic aryl, and mono or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O, S, mono or bicyclic aryl-Cl- Cl 0-alkyl, heteroaryl-Cl- Cl 0-alkyl and combinations.

In the third group of the cyclic peptides of the present invention, at least one α- amino acid, at least one β-amino acid, and at least one γ-amino acid are used to form a cyclic peptide wherein the total number of α- and γ-amino acids combined is an even number ("Group III"). In one embodiment of the Group III cyclic peptides, the peptides can have an amino acid sequence represented by Formula VIII: c-[(α-Aa)-(β-Aa)_n-(γ-Aa)-]_m wherein: n = 1, 2, 3 or 4 ; and m = 1, 2, 3, 4, 5, or 6. As in previous formulae, the letter "c" indicates that the peptide is cyclic and "α-Aa," "β- Aa" and "γ-Aa" refer to an alpha amino acid, a beta amino acid, and a gamma amino acid, respectively. Examples of cyclic peptides made according to Formula VIII are shown in Figures 8a-8e, and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-[(R-α-Aa)-(S,S,β-Aa)_n-(R,R,S-γ-Aa)-]_m wherein: n - 1, 2, 3 or 4; and m = 1, 2, 3, 4, 5 or 6.

In another embodiment of the Group III cyclic peptides, the cyclic peptides of the invention can have an amino acid sequence having Formula IX: c-[(α-Aa)-(γ-Aa)-(β-Aa)_n-]_m wherein: n = 1, 2, 3 or 4 ; and m = 1, 2, 3, 4, 5, or 6. Examples of cyclic peptides made according to Formula IX are shown in Figures 9a-9e, and the amino acid sequence of such cyclic peptides can be specifically expressed as: c-[(S-α-Aa) -(S,S,R-γ-Aa)-(S,S-β-Aa)_n-]_m wherein: n = 1, 2, 3 or 4; and m = 1, 2, 3, 4, 5 or 6. For each of the cyclic peptides having formulae VIII-IX discussed above, each of the side chains (Rⁿ) on the α-, β- and γ- amino acids can be independently selected from any side chain of the natural amino acids or the groups consisting of hydroxyl, linear or branched Cl- Cl 0-alkyl, alkenyl, alkynyl, hydroxy-C 1- Cl 0-alkyl, amino-Cl- Cl 0-alkyl, Cl- ClO-alkoxy, Cl- ClO-alkoxy-Cl- Cl 0-alkyl, amino, mono or di-Cl- ClO-alkylamino, carboxamido, carboxamido-Cl- ClO-alkyl, sulfonamido, sulfonamide-C 1- Cl 0-alkyl, urea, cyano, fluoro, Cl- ClO-alkyl-fluoruro, thio, Cl-ClO-alkylthio, mono or bicyclic aryl, and mono or bicyclic heteroaryl having up to 5 heteroatoms selected from N, O, S, mono or bicyclic aryl-Cl -ClO-alkyl, heteroaryl-Cl -ClO-alkyl and combinations.

For each of the embodiments described above, it is contemplated that any alpha amino acid with S configuration may be substituted with a gamma amino acid with R,R,S configuration while any alpha amino acid with R configuration may be substituted with a gamma amino acid with S,S,R configuration. Preparation of Cyclic Peptides

Peptides of the invention may be synthesized, isolated, purified in vitro, e.g., using various methods, for example, solid phase peptide synthesis methods. Solid phase peptide synthetic methods are established and widely used. See, e.g., Stewart et al, Solid Phase Peptide Synthesis, W. H. Freeman Co, San Francisco (1969); Merrifield, J Am. Chem. Soc. 85, 2149 (1963); Meienhofer in Hormonal Proteins and Peptides, ed.; CH. Li, Vol.2 (Academic Press, 1973), pp.48-267; and Bavaay and Merrifield, The Peptides, eds. E. Gross and F. Meienhofer, Vol.2 (Academic Press, 1980) pp. 3-285. Peptides can be further purified by fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on an anion-exchange resin such as DEAE, chromatofocusing, SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75, ligand affinity chromatography, or crystallization or precipitation from non-polar solvent or nonpolar/polar solvent mixtures. Purification by crystallization or precipitation is preferred.

To identify highly active cyclic peptides that have little or no undesired toxicity for mammalian cells, individual cyclic peptides, or libraries of cyclic peptides can be made, and the individual cyclic peptides or cyclic peptides from those libraries can be screened for the desired activity, i.e., antimicrobial, antiviral and anticancerous, and toxicity. For example, libraries of peptides can be made using a one-bead-one-compound strategy provided by Lam et al. 97 Chem. Rev. 411-448 (1997) or synthesized on macrobeads by a split and pool method of Furka, et al. 37 Int. J. Pept. Prot. Res. 487-493 (1991). Mass spectrometric sequence analysis techniques enable rapid identification of every peptide within a given library. See, Biemann, 193 Methods Enzymol 455 (1990). In general, synthetic operations, including peptide cyclization, are performed on solid support to avoid laborious and difficult to automate solution-phase operations. Moreover, the final product of the synthesis regimen is generally sufficiently pure for biological assays without laborious purification procedures. Peptide yields from each synthesis can be sufficient for performing 50 to 100 assays. Rapid, automatable mass spectrometry-based peptide sequence analysis can be performed to identify peptide sequences that have high activity and to discard peptide sequences with low activity.

The synthetic approach employed can provide individually separable and identifiable peptide sequences to avoid the use of combinatorial library mixtures and laborious deconvolution techniques. However, libraries of impure mixtures of peptides can also be generated for testing. Impure preparations of peptides can be used for quick screening of combinations of sequences. When a mixture of peptides shows activity, the peptides in the mixture can either be individually isolated and tested or pure peptides having sequences known to be present in the impure mixture can be individually prepared and tested. Salts of carboxyl groups of a peptide or peptide variant of the invention may be prepared in the usual manner by contacting the peptide with one or more equivalents of a desired base such as a metallic hydroxide base (e.g., sodium hydroxide); a metal carbonate or bicarbonate base such as sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like. N-acyl derivatives of an amino group of the peptide or peptide variants may be prepared by utilizing an N-acyl protected amino acid for the final condensation or by acylating a protected or unprotected peptide. O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N-acylation and O-acylation may be carried out together, if desired.

Acid addition salts of the peptide or variant peptide, or of amino residues of the peptide or variant peptide, may be prepared by contacting the peptide or amine with one or more equivalents of the desired inorganic or organic acid, such as hydrochloric acid. Esters of carboxyl groups of the peptides may also be prepared by any of the usual methods known in the art. Nanotubes and Supramolecular Structures

According to the present invention, the cyclic peptides provided herein are believed to self-assemble into supramolecular structures. Self-assembly means that a collection of cyclic peptides can associate to form a supramolecular structure on or within a cellular membrane without assistance, for example, of materials other than the components of the cellular membrane. In general, the physical and chemical properties of the cellular membrane facilitate self-assembly of the cyclic peptides. The interaction between the components of cellular membranes and the cyclic peptides will determine selectivity for particular cellular membranes.

According to the present invention, cyclic peptide structures of the present invention are believed to adopt or sample a flat ring-shaped conformation in which all backbone amide functionalities lie approximately perpendicular to the plane of the ring structure. In this flat-ring conformation, it is believed that the peptide subunits can stack, under favorable conditions, to furnish a contiguous hydrogen bonded hollow tubular structure that is referred to herein as a nanotube.

The pore size, or internal diameter, of self-assembled nanotubes can be adjusted by the ring size of the peptide subunit employed. A seven-residue cyclic peptide structure, of the type c-[(S-α-Aa)-(R-α-Aa)-(S-α-Aa)-(R-α-Aa)-(S,S-β-Aa)₃-], will have a diameter of approximately 7.5 Δ in diameter.

The flat, ring-shaped cyclic peptides of the present invention are not only structurally predisposed toward intermolecular interaction, but are also energetically favored to self-assemble on selected target cell membranes, including microbial cell membranes, and permeabilize cells through formation of pores or other membrane destabilizing structures. Methods of Use Antimicrobial Agents

The present invention is also directed to methods of treating or preventing microbial infections in a mammal, as well as other animals, such as farm animals and birds. These methods include administering to the animal a therapeutically effective amount of a cyclic peptide of the present invention. Treatment of, or treating, microbial infections is intended to include the alleviation of or diminishment of at least one symptom typically associated with the infection. The treatment also includes alleviation or diminishment of more than one symptom. Ideally, the treatment cures, e.g., substantially kills the microbes and/or eliminates the symptoms associated with the infection.

Microbial infections that can be treated by the present cyclic peptides include infections by any target microbial organisms that can infect a mammal or other animal. Such target microbial organisms include essentially any single cell organism or parasite that has a cellular membrane and that can infect an animal, including mammals. Microbial organisms further include multicellular organisms such as multicellular fungi and other multicellular parasitic organisms. For example, target microbial organisms include bacteria, fungi, yeast strains and other single cell organisms. Cyclic peptides are active against both gram-negative and gram-positive bacteria.

Hence, for example, infections or unwanted levels of the following target microbial organisms can be treated, prevented or addressed by the present cyclic peptides:

Aeromonas spp. (including, for example, Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria), Bacillus spp. (including, for example, Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis), Bacteroides spp. (including, for example, B. fragilis, B. thetaiotaomicron, B. vulgatus, B. ovatus, B. distasonis, B. uniformis, B. stercoris, B. eggerthii, B. merdae, and B. caccae), Burkholderia cepacia, Campylobacter spp.

(including, for example, Campylobacter jejuni, Campylobacter laridis, and Campylobacter hyointestinalis), Clostridium spp. (such as the pathogenic clostridia including all types of Clostridium botulinum (including those in Groups I, II, III and IV, and including those that produce botulism A, B, C, D, E, F and G), all types of Clostridium tetani, all types of Clostridium difficile, and all types of Clostridium perfringens), Enterobacter spp. (including, for example, Enterobacter aerogenes (also sometimes referred to as Klebsiella mobilis), Enterobacter agglomerans (also sometimes referred to as Pantoea agglomerans), Enterobacter amnigenus, Enterobacter asburiae, Enterobacter cancerogenus (also sometimes referred to as Enterobacter taylorae and/or Erwinia cancerogend), Enterobacter cloacae, Enterobacter cowanii, Enterobacter dissolvens (also sometimes referred to as Erwinia dissolvens), Enterobacter gergoviae, Enterobacter hormaechei, Enterobacter intermedium, Enterobacter intermedius (also sometimes referred to as Enterobacter intermedium), Enterobacter kobei, Enterobacter nimipressuralis (also sometimes referred to as Erwinia nimipressuralis), Enterobacter sakazakii, and Enterobacter taylorae (also sometimes referred to as Enterobacter cancerogenus)),

Enterococcus spp. (including, for example, vancomycin Resistant Enterococcus (VRE), Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus gallinarum, and Enterococcus casseliflavus), Escherichia spp. (including the enterotoxigenic (ETEC) strains, the enteropathogenic (EPEC) strains, the enterohemorrhagic (EHEC) strain designated E. coli 0157:H7, and the enteroinvasive (EIEC) strains), Gastrospirillum spp. (including, for example, Gastrospirillum hominis (also sometimes now referred to as Helicobacter heilmannii), Helicobacter spp. (including, for example, Helicobacter pylori and Helicobacter hepaticus, Klebsiella spp. (including, for example, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica), Salmonella spp. (including, for example, S. typhi andS. paratyphi A, B, and C, S. enteritidis, and S. dubliή), Shigella spp. (including, for example, Shigella sonnei, Shigella boydii, Shigella flexneri, and Shigella dysenteriae), Staphylococcus spp. (including, for example, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) strains, Staphylococcus saprophyticus and Staphylococcus saprophyticus (MRSA) strains, and Staphylococcus epidermis and Staphylococcus epidermis (MRSA) strains), Stenotrophomonas (e.g. Stenotrophomonas maltophild), Streptococcus ssp. (including Groups A (one species with 40 antigenic types, Streptococcus pyogenes), B, C, D (five species (Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Streptococcus avium, and Streptococcus bovis)), F, and G, including Streptococcus pneumoniae), Pseudomonas spp. (including, for example, Pseudomonas aeruginosa, Pseudomonas maltophilia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas mallei, Pseudomonas pseudomallei and Pseudomonas putrefaciens), Vibrio spp. (including, for example, Vibrio cholera Serogroup 01 and Vibrio cholera Serogroup Non-Ol, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio furnissii, Vibrio carchariae, Vibrio hollisae, Vibrio cincinnatiensis, Vibrio metschnikovii, Vibrio damsela, Vibrio mimicus, Vibrio vulnificus, and Vibrio fluvialis), Yersinia spp. (including, for example, Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis), Neisseria (e.g. Neisseria meningiditis and Neisseria gonnerheά), Proteus, Citrobacter, Aerobacter, Providencia, Serratia, Brucella, Mycoplasma, Mycobacterium, Francisella tularensis (also sometimes referred to as Pasteurella tularensis, Bacillus tularensis, Brucella tularensis, tularemia, rabbit fever, deerfly fever, Ohara's disease, and/or Francis disease), Mycobacteria, Mycoplasma, and the like. Thus, for example, various bacterial infections or unwanted levels of bacteria that can be treated, prevented or addressed by the present peptides include but are not limited to those associated with anthrax (Bacillus anthracis), staph (Staphylococcus aureus), typhus (Salmonella typhi), food poisoning (Escherichia coli, such as 0157:H7, Bacillus cereus, Staphylococcus aureus, Salmonella, Clostridium perfringens, Campylobacter, Listeria monocytogenes, Vibrio parahaemolyticus), bascillary dysentery (Shigella dysenteria), botulism (Clostridium botulinum), pneumonia (Psuedomonas aerugenosa and/or Burkholderia cepacia), cholera (Vivrio cholerae), ulcers (Helicobacter pylori), smallpox (variola major), listeriosis (Listeria monocytogenes), tularemia (Francisella tularensis), plague (Yersinia pestis; also sometimes referred to as bubonic plague, pneumonic plague, and/or black death) and others. E. coli serotype 0157:H7 has been implicated in the pathogenesis of diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). As indicated herein, the peptides of the invention are also active against drug-resistant and multiply drug-resistant strains of bacteria, for example, multiply resistant strains of Staphylococcus aureus and vancomycin- resistant strains of Enterococcus faecium and Enterococcus faecalis. Antimicrobial activity can be evaluated against these varieties of microbes using methods available to one of skill in the art. Antimicrobial activity, for example, is determined by identifying the minimum inhibitory concentration (MIC) of a cyclic peptide of the present invention that prevents growth of a particular microbial species.

The present invention also provides methods of evaluating therapeutically effective dosages for treating a microbial infection with cyclic peptides described and claimed herein that includes determining the minimum inhibitory concentration of a cyclic peptide at which substantially no microbes grow in vitro. Such a method permits calculation of the approximate amount of cyclic peptide needed per volume to inhibit microbial growth of or to kill 50% of the microbes. Such amounts can be determined, for example, by standard microdilution methods. For example, a series of microbial culture tubes containing the same volume of medium and substantially the same amount of microbes are prepared, and an aliquot of cyclic peptide is added. The aliquot contains differing amounts of cyclic peptide in the same volume of solution. The microbes are cultured for a period of time corresponding to one to ten generations (e.g. 18-24 hrs), and the number of microbes in the culture medium is determined. The optical density of the cultural medium can be used to estimate whether microbial growth has occurred. If no significant increase in optical density has occurred, then no significant microbial growth has occurred. However, if the optical density increases, then microbial growth has occurred. To determine how many microbial cells remain alive after exposure to the cyclic peptide, a small aliquot of the culture medium can be removed at the time when the cyclic peptide is added (time zero) and then at regular intervals thereafter. The aliquot of culture medium is spread onto a microbial culture plate, the plate is incubated under conditions conducive to microbial growth and, when colonies appear, the number of those colonies is counted.

According to the present invention, the cyclic peptides provided herein do not cause substantial or undesired toxicity against non-target mammalian cells or the non-target cells of other animals to be treated. Mammalian or bird red blood cell hemolysis is one way to measure whether a cyclic peptide can cause undesired toxicity against mammalian cells or the cells of other animals to be treated. If a cyclic peptide can self-assemble by association with a mammalian or animal cell membrane, the membrane may be disrupted. Red blood cells are conveniently used to test for membrane disruption because they undergo hemolysis, which can be detected as the release of hemoglobin from the cell. Hemolysis assays can be performed by methods available to one of skill in the art. For example, after exposure to test compounds, the release of hemoglobin can be observed spectrophotometrically by observing the absorbance of light at wavelengths characteristic of hemoglobin, for example, at 543 nm. Control samples can be used, for example, the medium in which the cells are tested or maintained can serve as a zero blank. A second control can be used to determine the absorbance value for 100% lysis or hemolysis that can be a sample that is identical to the test mammalian cell sample but which had been sonicated to completely disrupt the cells. Additionally hemolytic agents such as mellitin or a variety of detergents can also be used to establish 100% hemolysis of test red blood cells. As Antifungal Agents

The present invention is also directed to methods of treating fungal infections in a mammal or other animal, which include administering to the mammal or other animal a therapeutically effective amount of a cyclic peptide of the present invention. According to the present invention, it is believed that the cyclic peptide undergoes self-assembly to form a supramolecular structure that prevents or interrupts target fungal infections, or inactivates target fungi, but does not cause undesired toxicity or substantial hemolysis of non-infected mammalian or animal cells.

Treatment of, or treating, fungal infections is intended to include the alleviation of or diminishment of at least one symptom typically associated with the infection. The treatment also includes alleviation or diminishment of more than one symptom. The treatment may cure the infection, e.g., it may substantially inactivate the fungus and/or eliminate the symptoms associated with the infection.

Fungal infections that can be treated or prevented by the present cyclic peptides include infections by fungi that infect a mammal, including Histoplasma capsulatum, Coccidioides immitis, Cryptococcus neoformans, Candida ssp. including Candida albicans, Aspergillus ssp. including Aspergillus fumigatus, Sporothrix, Trichophyton ssp., Fusarium ssp., Tricosporon ssp., Pneumocystis carinii, and Trichophyton mentagrophytes. Hence, for example, infections or unwanted levels of target fungi can be treated, prevented or addressed by the present cyclic peptides. Such fungi also include fungal pathogens that may have potential for use as biological weapons, including Coccidioides immitis and Histoplasma capsulatum.

A large variety of fungal infections can be treated with the cyclic peptides of the invention. For example, the target fungal infections include systemic (e.g. affecting one or more organ system) subcutaneous, cutaneous, topical or superficial, or mucosal. Fungal infections that can be treated by the present cyclic peptides include infections by any target fungal organisms that can infect a mammal or other animal. Target infections include those caused by single celled and multicellular fungal organisms.

The cyclic peptides described herein are potent antifungal agents for a wide variety of pathogenic fungi or other target fungi. The infection by the target fungal organsm may be associated with another disease or condition in the animal, for example in a patient having a compromised immune system such as AIDS, cancer, diabetes, surgery or transplant patients, and the like. Representative types of fungal organisms that are targets include by way of non-limiting example: Aspergillus spp. (e.g. Aspergillus fumigatus); Bipolaris spp. (e.g. B. australiensis, B. cynodontisc, B. hawaiiensis, B. spiciferd);

Coccidioides immitis; Blastomyces dermatitidis; Cryptococcus neoformans; Candida spp. (e.g. Candida albicans and Candida glabrata); Cladaphialophora bantiana; Fusarium spp. (e.g. F. rubrum, F. solani); Hystoplasmosa capsulatum; Microsporum audouinii; Microsporum canis; Microsporum ferrugineum; Mucormycosis spp. (e.g. Mucormycosis rhizopus, Mucormycosis rizomucor, and Mucormycosis cunninghamelld);

Paracoccidioides brasiliensis; Pneumocystis carinni; Ochroconis gallopavum; Ramichloridium mackenzieϊ); Sporothrix (e.g. S. shenki); and Trichoplryton spp. (including T. concentricum, T. erinacei, T. equinum (equine), T. gourvilii, T. mentagrophytes (rodents); T. mentagrophytes inter digitale, T. megnini, T quinckeanum (mice); T. rubrum (humans), T. schoenleinii, T. simii (monkeys), T soudanense, T. tonsurans, T. verrucosum (bovine), T. violacium, T. yaounde). Such fungi also include fungal pathogens that may have potential for use as biological weapons.

Representative fungal infections further include Chromoblastomycosis (where the etiological agent can be, for example, Phialophora verrucosa, Fonsecaea pedrosi, Fonsecaea compacta, Cladosporium carrionii, Rhinocladiella aquaspersa, Ramichloridium cerophilum); Leukonychia mycotica; Subungual dermatophytosis; fungal allergies; fungal meningitis (e.g. where the etiological agent can be Cryptococcus neoformans); Protothecosis (where the etiological agent can be for example Prototheca spp. (e.g. P. wickerhamii and P. zopfli)); Onychomycosis (Tinea Unguium) (e.g. T. rubrum); Piedra (e.g. Pierda iahortae and Trichosporon beigelii); and Pityriasis versicolor (e.g. Malassezia furfur). See Harrison's Principles of Internal Medicine (15th Edition, Vol. 1, Braunwald et al, McGraw Hill, Inc., New York, 2002, pp 1168-1184); and Clinical Mycology (Sobel et al, Oxford Press, 2003).

In one aspect, the cyclic peptides described herein are useful for the prevention or treatment of topical or superficial fungal infections including those of the skin, stratum corneum, nails and hair. Cutaneous infections are infections of the skin, finger nails and toenails. These infections are often persistent and often require oral antifungal therapy for several months. Examples of these infections include Tinea corporis (ringworm of the body), Tinea pedis (ringworm of the feet; athlete's foot) is common, Onchomycosis or Tinea unguium (fungal infections of the finger and toenails), Candidal infections (e.g., Candida paronchyd) of the axillary, groin, and perineal areas, as well as areas in between fingers and toes. Also contemplated are methods of treating topical or superficial fungal infections in an animal comprising the topical administration of an effective amount of a cyclic peptide described herein to an animal in need of such treatment. In one particular aspect, the cyclic peptides are useful for the treatment of dermatophytosis in humans or animals. Representative etiologic agents of human dermatophytosis can include, but are not limited to, Candida granuloma, Tinea nigra, Tinea capitis, Tinea favosa, Tinea barbae, Tinea corpois, Tinea alba, Tinea versicolor, Tinea flava, Achromia parasitica, Dermatomycosis furfur acea, Piedra iahortae, and Trichosporon beigelii. For human nail infections, etiologic agents include but are not limited to Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum, Microsporum audouinii, Microsporum canis, Microsporum gypseum, Trichophyton schoenleinii, Trichophyton tonsurans, and a variety of molds including but not limited to Acremonium spp, Fusarium oxysporum, Scopulariopsis brevicaulis, Onychocola Canadensis, and Scytalidium dimidiatum.

The cyclic peptides described herein are useful for the prevention or treatment of fungal infections in animals, and in particular, companion animals, livestock and farm animals. Representative fungal organisms that are believed to be etiological in the fungal infections of companion animals, livestock and farm animals include by way of example: Microsporum canis and Microsporum distortum (humans, cats and dogs), Microsporum galinae (pigs), Microsporum nanum (bank voles),and Microsporum cookei (rodents). Specific mycological infections in companion animals can include, but are not limited to, infections caused by Cryptococcus neoformans, Microsporum gallinae, Microsporium canis and various Aspergillus species (e.g., A. fumigatus, A. flavus, A. niger, A. nidulans, and A. terreus). The cyclic peptides described herein are also useful for the prevention or treatment of fungal infections in insects, such as silk worms. Silk worms can be infected by Beauveria spp, and, in particular, Beauvaria bassiana.

As used herein, the terms "antifungal" or "antifungal activity" are used to denote any improvement in a fungal infection to the degree desired. Antifungal activity can be evaluated against the varieties of fungal organisms described above using methods available to one of skill in the art. This may be evidenced by, for example, an observed inhibition in growth of a target fungal organism. Antifungal activity, for example, can be determined by identifying the minimum inhibitory concentration (MIC) of a cyclic peptide that prevents growth of a particular fungal species. Antifungal activity can determined by identifying the minimum fungicidal concentration (MFC) of a cyclic peptide that is fungicidal for a particular fungal species. For example, antifungal activity may be the amount of the peptide that inhibits at least about 50% of the fungi when measured using standard dose or dose response methods, alternatively the amount of the peptide that inhibits at least about 60%, 70%, 80%, 90% or about 100% of the fungi when measured using standard dose or dose response methods. As Antiviral Agents

The present invention is also directed to methods of treating viral infections in a mammal or other animal, which includes administering to the mammal or other animal a therapeutically effective amount of a cyclic peptide of the present invention. According to the present invention, it is believed that the cyclic peptide undergoes self-assembly to form a supramolecular structure that prevents or interrupts target viral infections, or inactivates target viruses, but does not cause undesired toxicity or substantial hemolysis of non-virally infected mammalian or animal cells.

Treatment of, or treating, viral infections is intended to include the alleviation of or diminishment of at least one symptom typically associated with the infection. The treatment also includes alleviation or diminishment of more than one symptom. The treatment may cure the infection, e.g., it may substantially inactivate the virus and/or eliminate the symptoms associated with the infection.

Viral infections that can be treated or prevented by the present cyclic peptides include infections by virus that infect a mammal, including enveloped and non-enveloped viruses, DNA and RNA viruses, viroids, and prions. Hence, for example, infections or unwanted levels of the following target viruses and viral types can be treated, prevented or addressed by the present cyclic peptides: hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), poxviruses, herpes viruses, adenoviruses, papovaviruses, parvoviruses, reoviruses, orbiviruses, picomaviruses, rotaviruses, alphaviruses, rubivirues, influenza virus type A and B, flaviviruses, coronaviruses, paramyxoviruses, morbilliviruses, pneumoviruses, rhabdoviruses, lyssaviruses, orthmyxoviruses, bunyaviruses, phleboviruses, nairoviruses, hepadnaviruses, arenaviruses, retroviruses, enteroviruses, rhinoviruses and the filovirus. Infections or unwanted levels of the following target viruses and viral types that are believed to have potential as biological weapons can be treated, prevented or addressed by the present cyclic peptides: hemorrhagic fever viruses (HFVs), Chikungunya virus, Japanese encephalitis virus, Monkey pox virus, variola virus, Congo-Crimean haemorrhagic fever virus, Junin virus, Omsk haemorrhagic fever virus, Venezuelan equine encephalitis virus, Dengue fever virus, Lassa fever virus, Rift valley fever virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Lymphocytic choriomeningitis virus, Russian Spring-Summer encephalitis virus, White pox, Ebola virus, Machupo virus, Smallpox virus, Yellow fever virus, Hantaan virus, Marburg virus, and Tick-borne encephalitis virus.

In one particular aspect, the cyclic peptides are useful for viral infections caused by Adenoviruses. Adenoviruses have proved a useful model for understanding the multifactorial and multistage processes of virus entry into cells. Many, but not all, adenovirus serotypes bind to cells via a 46 kDa cell surface glycoprotein known as CAR (Coxsackie and Adenovirus Receptor). CAR is a member of the Ig superfamily and is widely expressed on many cell types, thus accounting for the broad tropism of adenovirus in vivo. The adenovirus fiber protein, an elongated and flexible capsid protein, mediates CAR binding. A crystal structure of the adenovirus fiber knob domain in a complex with the first immunoglobulin domain of CAR has provided structural insights for detargeting and retargeting of adenoviral vectors for gene delivery.

Although CAR mediates high-affinity binding of Adenovirus (Ad) particles to host cells, this receptor does not facilitate efficient entry into cells via clathrin-mediated endocytosis. Instead, interaction of the Ad penton base protein with the vitronectin- binding integrins, avb3, avb5 or avbl promotes virus internalization. Interestingly, a growing number of enveloped and non-enveloped animal viruses have been reported to use cell integrins for either attachment and/or internalization. Ad internalization into host cells via av integrins is a complex process that involves activation of several cell signaling molecules including PI3-kinase, the Rho family of small GTPases and pl30CAS. An important downstream target of this signaling complex is the actin cytoskeleton. Polymerization of cortical actin filaments underlying the cell membrane is required for efficient virus internalization. Although the precise role of actin in Ad endocytosis remains unclear, actin filaments may provide needed mechanical force needed to drive endosome formation.

A crucial component of the forming endosome is dynamin, a 100 kDa cytosolic GTPase that regulates endocytic-coated vesicle formation. However, precise mechanism by which dynamin mediates clathrin-coated vesicle formation remains unresolved. Recently, investigators have reported an association between actin and dynamin during the movement of intracellular organelles, suggesting that these cytosolic proteins may work in concert to promote endocytosis. As well as having a GTP binding domain, dynamin also contains a pleckstrin homology (PH) domain. PH domains are commonly found in a number of proteins that require membrane association for their function. Although it is not yet firmly established, the pH domain of dynamin could bind to one or more phosphoinositides present in cell membranes. Artalejo and colleagues found that overexpression of the pH domain inhibited clathrin-mediated endocytosis thus emphasizing the importance of this region in dynamin function. Artalejo, et al, M.A. EMBO J. 16:1565-1574 (1997). These studies raise the possibility that small molecules capable of interacting with the pH domain might interfere with dynamin function and thereby prevent rapid endocytosis.

In addition to dynamin, there are many other cytosolic proteins associated with the forming of endosome that participate in internalization of receptor-ligand complexes. These include adapter proteins (AP2), epsl5, and adapter associated kinases (AAKl). The precise role of these molecules in endocytosis have yet to be fully delineated. The identification of cyclic peptides that selectively interfere with clathrin/dynamin-mediated endocytosis may therefore provide valuable tools for unraveling basic cell biological processes. Moreover, since dynamin-dependent entry pathways have been usurped by a number of different viral and bacterial pathogens, these cyclic peptides could have wide therapeutic applications.

In another aspect, antiviral activity can be evaluated against particular viruses or varieties of viruses using methods available to one of skill in the art. Antiviral activity, for example, is determined by identifying the amount, or amounts, a cyclic peptide of the present invention that alleviates or prevents the infection of a mammalian cell with a virus.

The present invention also provides a method of evaluating a therapeutically effective dosage for treating a viral infection with a cyclic peptide that includes determining the inhibitory concentration (IC50) of the cyclic peptide at which substantially no viruses replicate in vitro. Such a method permits calculation of the approximate amount of cyclic peptide needed per volume to inhibit viral replication or to inhibit 50% of viral infection. Such amounts can be determined, for example, by standard microdilution methods.

For example, a series of culture tubes or plates containing mammalian cells suitable for infection by a target viral type in the same volume of medium and substantially the same amount of viruses are prepared. An aliquot of a test cyclic peptide is added to each culture tube or plate. Each aliquot contains differing amounts of cyclic peptide in the same volume of solution. The culture tubes/plates are cultured for a period of time corresponding to one to ten generations and the number, for example, of mammalian cells or viruses in the culture medium is determined. The number of colonies or the optical density of the cultural medium can be used to estimate whether mammalian cell growth has occurred. Alternatively, the number of viruses in the culture tubes/plates can be estimated by available means. To determine how many active viruses exist after exposure to the cyclic peptide, a small aliquot of the culture medium can be removed at the time when the cyclic peptide is added (time zero) and then at regular intervals thereafter. The aliquot of culture medium is then tested for active viral particles by available procedures.

According to the present invention, the cyclic peptides provided herein do not cause substantial or undesired toxicity against mammalian or other animal cells. As described above, mammalian red blood cell hemolysis is one way to evaluate undesired toxicity. As Anticancer Agents The present invention also contemplates methods of treating cancer in an animal, for example, for human and veterinary uses, which includes administering to a subject animal (e.g., a human or other animal), a therapeutically effective amount of a cyclic peptide of the present invention. According to the present invention, it is believed that the cyclic peptide undergoes self-assembly to form a supramolecular structure that causes cancer cellular membrane permeation, destabilization or depolarization.

Treatment of, or treating, cancer is intended to include the alleviation of or diminishment of at least one symptom^" typically associated with the disease. The treatment also includes alleviation or diminishment of more than one symptom. The treatment may cure the cancer, e.g., it may substantially kill the cancer cells and/or arrest the growth of the cancerous tumor. Cancers to be treated by the present cyclic peptides include solid mammalian tumors as well as hematological malignancies. Solid mammalian tumors include cancers of the head and neck, lung, mesothelioma, mediastinum, esophagus, stomach, pancreas, hepatobiliary system, small intestine, colon, colorectal, rectum, anus, kidney, ureter, bladder, prostate, urethra, penis, testis, gynecological organs, ovarian, breast, endocrine system, skin central nervous system, sarcomas of the soft tissue and bone, and melanoma of cutaneous and intraocular origin. Hematological malignancies includes childhood leukemia and lymphomas, Hodgkin's disease, lymphomas of lymphocytic and cutaneous origin, acute and chronic leukemia, plasma cell neoplasm and cancers associated with AIDS. In addition, a cancer at any stage of progression can be detected, such as primary, metastatic, and recurrent cancers. Information regarding numerous types of cancer can be found, e.g., from the American Cancer Society (www.cancer.org), or from Wilson et al. (1991) Harrison 's Principles of Internal Medicine, 12.sup.th Edition, McGraw-Hill, Inc. Anticancer activity can be evaluated against varieties of cancers using methods available to one of skill in the art. Anticancer activity, for example, is determined by identifying the LDioo or ED50 of a cyclic peptide of the present invention that prevents the growth of a cancer. The present invention also provides a method of evaluating a therapeutically effective dosage for treating a cancer with a cyclic peptide that includes determining the LD_]0o or ED50 of the cyclic peptide in vitro. Such a method permits calculation of the approximate amount of cyclic peptide needed per volume to inhibit cancer cell growth or to kill 50% to 100% of the cancer cells. Such amounts can be determined, for example, by standard microdilution methods. According to the present invention, the cyclic peptides provided herein do not have substantial toxicity against normal mammalian or other animal cells as measured, for example, by a hemolysis assay. Additional Applications

The present invention also contemplates methods of treating fungal infections in an animal, for example, for human and veterinary uses, which include administering to a subject animal (e.g., a human or other animal), a therapeutically effective, antifungal amount of a cyclic peptide of the present invention. Screening Assays

For each of the uses described above, and others, screening or other assays may be used to identify, confirm or evaluate cyclic peptides, for example, that can selectively interact with a microbe, a virus, a cell infected with a virus of interest or a cancer cell of interest. A wide variety of assays may be used for this purpose. In general, such an assay can involve contacting a microbe, a cell infected with a virus of interest, or a cancer cell of interest with at least one cyclic peptide and observing whether the cyclic peptide interacts with a microbe, or inactivates the virus or kills the cancer cell and/or has deleterious effects upon that microbe or the cells of interest. Methods available in the art can be used for determining whether the cyclic peptides of the invention interact with the membrane of a cell type of interest. For example, cyclic peptides can be labeled with a reporter molecule that permits detection of the peptide. After labeling, the cyclic peptides can be contacted with the cell type of interest for a time and under conditions that permit binding or association of the peptide to cellular membranes. The cells can be washed with physiological solutions to remove unbound or unassociated cyclic peptides. The microbes, viruses or cells can then be observed to ascertain whether the reporter molecule is bound or associated with the microbes, viruses, cells or cellular membranes. In another embodiment, one of skill in the art can test whether the cyclic peptide(s) can selectively penetrate the membranes of particular microbes or cellular membranes of selected cells that can be infected with a virus or the membranes of selected cancer cells. This may be done, for example, by examining whether the reporter molecule remains associated with the cellular membranes of the microbe or the cellular membranes of the cell infected with a virus or the cellular membranes of the cancer cell. Reporter molecules that can be employed include any detectable compound or molecule available to one of skill in the art that is conjugated directly or indirectly to a cyclic peptide of the invention. The label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable or molecular epitopes for protein/antibody capture and detection.

Deleterious effects upon the microbe or the cell infected with a virus of interest or the cancer cell of interest can also be detected as an indication of an interaction between a cyclic peptide of the invention and the microbe or the cell. Such deleterious effects may be confirmed by any evidence that the cyclic peptide has had an adverse or cytotoxic effect upon the microbe or cell type of interest. For example, one of skill in the art can test whether the cyclic peptide(s) kill the cell type or cause membrane depolarization or permeabilization of the membranes of the microbes or cell types of interest.

Of particular interest are screening assays for cyclic peptides that have low toxicity for normal human or other animal cells but that have good antimicrobial (depolarizing or pemieabilizing microbial cell membranes, lysing or otherwise killing microbes) or antiviral properties (inactivating viruses, blocking viral infection, blocking receptors recognized by viruses on the surface of host cell membranes, and the like) or anticancer properties.

Generally in the testing of multiple peptides, a plurality of assays are performed in parallel with different cyclic peptides, which may be introduced at different concentrations to obtain a differential response to the various concentrations. Typically, at least one control assay is included in the testing. Such a control can be a negative control involving exposure of the microbes or the cells to a virus of interest or the cancer cell of interest to a physiologic solution containing no cyclic peptide. Another control can involve exposure of the microbe or cell to a virus of interest or the cancer cell of interest to a cyclic peptide that has already been observed to adversely affect the microbe or the virus of interest, or the cancer cell of interest. Another control can involve exposing a microbe or a cell infected with a virus of interest or the cancer cell of interest to a known therapeutic agent that has a desired affect on the microbe or the cell infected with a virus of interest or the cancer cell of interest, for example, an antimicrobial, an antiviral agent, or an anticancer agent with known efficacy at a particular concentration or dosage. One of skill in the art can readily select control compounds and conditions that facilitate such evaluations.

Candidate cyclic peptides are obtained from a wide variety of sources including libraries of cyclic peptides generated as described herein. Cyclic peptides can also be individually or rationally designed and synthesized to have specific structural features selected by one of skill in the art.

Any cell type available to one of skill in the art can be screened by these methods. Mammalian or other animal cells can also be screened to ascertain whether the peptides of the invention interact therewith and/or to determine or confirm whether the peptides of the invention do not interact, bind, lyse, kill or otherwise adversely affect the viability of the mammalian or other animal cell type of interest.

As noted, in one embodiment, mammalian red blood cells are screened with the cyclic peptides to ascertain whether the cyclic peptides have an adverse effect on the red blood cells. When it is established that a cyclic peptide causes little or no undesired hemolysis of red blood cells, it may be tested against other mammalian cell types or used for in vivo testing in standard animal models. Other methods of screening for mammalian cell lysis are available in the art.

Conditions for screening cyclic peptides include conditions that are used by one of skill in the art to grow, maintain or otherwise culture viruses or cell types of interest. Cell types of interest should be assayed under conditions where they would be healthy but for the presence of the cyclic peptide(s). Controls can be performed where the cell types are maintained under the selected culture conditions and not exposed to a cyclic peptide and to assess whether the culture conditions influenced the viability of the cells. One of skill in the art can also perform the assay on cells that have been washed in simple physiological solutions, such as buffered saline, to eliminate, or test for, any interaction between the components in the culture media and the cyclic peptides, cells and/or the target virus. However, culture conditions for the assays generally include providing the cells with the appropriate concentration of nutrients, physiological salts, buffers and other components typically used to culture or maintain cells of the selected type. A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, albumin, and serum (e.g., fetal calf serum) that are used to mimic the physiologic state of the cell types of interest. Conditions and media for culturing, growing and maintaining mammalian cells and bacterial or other microbial cells are available to one of skill in the art. The selected reagents and components are added to the assay in the order selected by one of skill in the art. In general, the cyclic peptides are added last to start the assay. Assays are performed at any suitable temperature, typically between 4 °C and 40° C. Temperatures generally range from about room temperature (about 20 °C) to about 37°C. Incubation periods are selected to ascertain the optimal range of activity or to insure that the cyclic peptides do not adversely affect unwanted cell types. However, incubation times can be optimized to facilitate rapid high-thoroughput screening. Typically, incubation times are between about 1 minute and about 24 hours, other times range from about 5 minutes to about 8 hours.

Cyclic peptides having the desired selectivity and activity during in vitro screening or evaluation may be tested for activity and/or lack of toxicity in vivo in an appropriate animal model. Such animal models include mice, rats, rabbits, cats, dogs, pigs, goats, cattle or horses. For example, the mouse and the rat are convenient animal models for testing whether cyclic peptides of the invention have toxic effects and/or to determine whether the cyclic peptides can combat a microbial infection or a viral infection or inhibit the growth of a cancer cell.

One of skill in the art can readily perform in vivo screening of the cyclic peptides of the invention. For toxicity testing, a series of cyclic peptides at different test dosages can be separately administered to different animals. A single dose or a series of dosages can be administered to the animal. A test period is selected that permits assessment of the effects of the peptide(s) on the animal. Such a test period may run from about one day to about several weeks or months.

The effect of a cyclic peptide(s) on an animal can be determined by observing whether the peptide adversely affects the behavior (e.g., lethargy, food intake, hyperactivity) and physiological state of the animal over the course of test period. The physiological state of the animal can be assessed by standard procedures. For example, during the test period one of skill in the art can draw blood and collect other bodily fluids to test for various enzymes, proteins, metabolites, and the like. One of skill in the art can also observe whether the animal has bloating, loss of appetite, diarrhea, vomiting, blood in the urine, loss of consciousness, and a variety of other physiological problems. After the test period, the animal can be sacrificed and anatomical, pathological, histological and other studies can be performed on the tissues or organs of the animal.

In general, to evaluate the ability of one or more cyclic peptides of the invention to combat a particular microbial infection or a viral infection or inhibit cancer cell growth, mice or other test animals are infected with the selected microbe or the selected virus or treated to have the selected cancer, and a selected test dosage of one or more cyclic peptides is administered thereafter at predetermined elapsed time periods or intervals. Test animals are observed over the course of several days to weeks to ascertain whether the cyclic peptide protects the animals from the microbial infection or the viral infection or cancer. At the end of the test period, the test animals can be sacrificed and examined to ascertain whether the cyclic peptide has optimally protected the test animals from infection or cancer and/or to determine whether any adverse side effects have occurred.

Controls are used to establish the effects of the microbe or the virus or cancer when the cyclic peptide is not administered. Other controls can also be performed, for example, the safety and efficacy of the present cyclic peptides can be compared to that of known antimicrobial agents (e.g., penicillin, kanamycin, vancomycin, erythromycin, etc.) or known antiviral agents or known anticancer agents.

The invention further provides a method of identifying or evaluating a cyclic peptide of selective association with a target biomolecule. Such target biomolecules can include, for example, intracellular, extracellular or membrane-associated proteins, enzymes, nucleic acids, receptors, organelles and the like. This method can involve contacting a solution of cyclic peptides with the target biomolecule under hydrogen bond- promoting conditions and determining whether the peptides selectively associate with the desired biomolecules and possess biological activity of interest. The target biomolecule can be displayed, for example, on the surface of a living cell, on the surface of a genetically engineered cell, or on the surface of a liposome. Alternatively, the peptide can be contacted with the target biomolecule under other desired assay conditions available to one of skill in the art.

Cyclic peptides having desired antimicrobial or antiviral properties or anticancer properties in vitro and/or in vivo that also have substantially no undesired toxicity against unwanted cell types may be used in the preparation of appropriate dosage forms, as described in more detail below. Dosages, Formulations and Routes of Administration for the Peptides

The peptides of the invention, including their salts, are administered so as to achieve a reduction in at least one symptom associated with a microbial infection, a fungal infection, a viral infection and/or a cancer, indication or disease, or a decrease in the amount of antibody associated with the indication or disease.

To achieve the desired effect(s), the peptide, a variant thereof, or a combination thereof may be administered as single or divided dosages. The amount administered will vary depending on various factors including, but not limited to, the cyclic peptide chosen, the disease, the weight, the physical condition, the health, the age of the mammal, whether prevention or treatment is to be achieved, and if the peptide is chemically modified. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art. Administration of the therapeutic agents in accordance with the present invention may be in a continuous or intermittent manner in single or multiple doses, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the peptides of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. To prepare the composition, peptides are synthesized or otherwise obtained, purified as necessary or desired, and then lyophilized and stabilized as necessary. The peptide is adjusted to the appropriate concentration, and optionally combined with other agents. The absolute weight of a given peptide included in a unit dose can vary widely. Daily doses of the cyclic peptides of the invention can vary as well.

Both local and systemic administration is contemplated through normal routes of administration, including parenteral and non-parenteral. One or more suitable unit dosage forms comprising the therapeutic peptides of the invention can be administered by a variety of routes, including but not limited to oral, buccal, sublingual, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, topical (e.g., dermal, transdermal and ocular routes of administration), intrathoracic, intrapulmonary, intranasal (respiratory), pulmonary, spinal, and anal routes.

Peptides of the invention may also be formulated for sustained release (for example, using microencapsulation, see WO 94/ 07529, and U.S. Patent No. 4,962,091). The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts. Such methods may include the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

When the therapeutic peptides of the invention are prepared for oral administration, they are generally combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. For oral administration, the peptides may be present as a powder, a granular formulation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum. The active peptides may also be presented as a bolus, electuary or paste. Orally administered therapeutic peptides of the invention can also be formulated for sustained release, e.g., the peptides can be coated, micro- encapsulated, or otherwise placed within a sustained delivery device. The total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation. Pharmaceutical formulations containing one or more peptides of the invention can be prepared by procedures known in the art using well-known and readily available ingredients. For example, the peptide can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives. Binding agents can also be included, such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone. Moisturizing agents can be included, such as glycerol, and disintegrating agents such as calcium carbonate and sodium bicarbonate. Agents for retarding dissolution, such as paraffin, can also be included. Resorption accelerators, such as quaternary ammonium compounds can also be included. Surface active agents, such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carriers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added. The compositions of the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They may also contain gums such as xanthan, guar or carbo gum, or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites and the like. For example, tablets or caplets containing the cyclic peptides of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Caplets and tablets can also include inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate and the like. Hard or soft gelatin capsules containing at least one cyclic peptide of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coated caplets or tablets containing one or more peptides of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.

Peptides of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance, by intramuscular, subcutaneous, intraperitoneal or intravenous routes.

Pharmaceutical formulations of peptides of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively, the form of an emulsion or suspension or salve.

Thus, the peptides of the invention may be formulated for parenteral administration (e.g., by injection, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi- dose containers. As noted above, preservatives can be added to help maintain the shelve life of the dosage form. The active peptides and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active peptides and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water before use.

These formulations of the present invention can contain pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts, vehicles and adjuvants that are well known in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non- limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0. It is also possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol," polyglycols and polyethylene glycols, C1 -C4 alkyl esters of short-

chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol," isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.

It is possible to add, if necessary, an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes, flavorings and colorings. Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and α-tocopherol and its derivatives can be added.

Also contemplated are combination products that include one or more cyclic peptides of the present invention and one or more other antimicrobial agents. A variety of antibiotics can be included in the pharmaceutical compositions of the invention, such as aminoglycosides (e.g., streptomycin, gentamicin, sisomicin, tobramycin and amicacin), ansamycins (e.g. rifamycin), antimycotics (e.g. polyenes and benzofuran derivatives), β- lactams (e.g., penicillins and cephalosporins), chloramphenical (including thiamphenol and azidamphenicol), linosamides (lincomycin, clindamycin), macrolides (e.g., erythromycin, oleandomycin, spiramycin), ketolides, polymyxins, bacitracins, tyrothycin, capreomycin, glycopeptides (e.g. vancomycin, ticoplanin) tetracyclines (including oxytetracycline, minocycline, doxycycline), phosphomycin and fusidic acid.

Additionally, as noted the peptides are suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the active peptide, for example, in a particular part of the intestinal or respiratory tract, possibly over a period of time. Coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, draining devices and the like. For topical administration, the therapeutic agents may be formulated as is known in the art for direct application to a target area. Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap. Other conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Thus, the therapeutic peptides of the invention can be delivered via patches or bandages for dermal administration. Alternatively, the peptide can be formulated to be part of an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it might be desirable to use microporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized. The backing layer can be any appropriate thickness that will provide the desired protective and support functions. A suitable thickness will generally be from about 10 to about 200 microns. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The active peptides can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122; 4,383,529; or 4,051,842. The percent by weight of a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01% to 95% of the total weight of the formulation, and typically 0.1-85% by weight.

Drops, such as eye drops or nose drops, may be formulated with one or more of the therapeutic peptides in an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure. The therapeutic peptide may further be formulated for topical administration in the mouth or throat. For example, the active ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier. Peptides of the invention can also be administered to the respiratory tract. Thus, the present invention also provides aerosol pharmaceutical formulations and dosage forms for use in the methods of the invention. In general, such dosage forms comprise an amount of at least one of the agents of the invention effective to treat or prevent the clinical symptoms of a specific infection, indication, or disease. Any statistically significant attenuation of one or more symptoms of an infection, indication or disease that has been treated pursuant to the method of the present invention is considered to be a treatment of such infection, indication or disease within the scope of the invention.

Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatine or blister packs from which the powder may be administered with the aid of an inhalator, insufflator, or a metered-dose inhaler (see, for example, the pressurized metered dose inhaler (MDI) and the dry powder inhaler disclosed in Newinan, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds, pp. 197-224, Butterworths, London, England, 1984).

Peptides of the present invention can also be administered in an aqueous solution when administered in an aerosol or inhaled form. Thus, other aerosol pharmaceutical formulations may comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.1 mg/ml and about 100 mg/ml of one or more of the peptides of the present invention specific for the indication or disease to be treated. Dry aerosol in the form of finely divided solid peptide or nucleic acid particles that are not dissolved or suspended in a liquid are also useful in the practice of the present invention. Peptides of the present invention may be formulated as dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 μm, alternatively between 2 and 3 μm. Finely divided particles may be prepared by pulverization and screen filtration using techniques well known in the art. The particles may be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular infection, indication or disease since the necessaiy effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations. For administration to the upper (nasal) or lower respiratory tract by inhalation, the therapeutic peptides of the invention are conveniently delivered from a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Nebulizers include, but are not limited to, those described in U.S. Patent Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co, (Valencia, CA). For intra-nasal administration, the therapeutic agent may also be administered via nose drops or a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker). Furthermore, the active ingredients may also be used in combination with other therapeutic agents, for example, pain relievers, anti-inflammatory agents, antihistamines, bronchodilators and the like, whether for the conditions described or some other condition. The present invention further pertains to a packaged pharmaceutical composition for controlling microbial or viral infections, or cancer, such as a kit or other container. The kit or container holds a therapeutically effective amount of a peptide or peptides of the invention and instructions for use. The pharmaceutical composition includes at least one cyclic peptide of the present invention, in a therapeutically effective amount.

The invention is further illustrated by the following non-limiting Examples. EXAMPLE 1

Materials and Methods - Solid Phase Peptide Synthesis - General Solvents and reagents. Acetonitrile (ACN, optima grade), dichloromethane (DCM, ACS grade), N,N-dimethylformamide (DMF, sequencing grade), N-metylpyrrolidinone (NMP, peptide synthesis grade), diethylether (Et₂0, ACS grade), N,N- diisopropylethylamine (DIEA, peptide synthesis grade) were purchased from Fisher and used without further purification. Trifluoroacetic acid (TFA, New Jersey Halocarbon), 2- ( 1 H-benzotriazol- 1 -y 1)- 1 , 1 ,3 ,3 -tetramethy luronium hexafluόrophosphate (HBTU, Advanced Chemtech), benzotriazole- 1 -yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP, Novabiochem), 1,3-Diisopropylcarbadiimide (DIC, Aldrich), and N-[(Dimethylamino)-lH-l,2,3-triazolo[4,5-b]pyridin-l-ylmethylene]- methylmethanaminium hexafluorophosphate N-oxide (HATU, Applied Biosystems) were used as obtained. Commercially available amino acids and resins were used as obtained from Bachem, Novabiochem, Peptech or Advanced Chemtech. The side-chain protections were as follows. For Fmoc synthesis: Arg and hArg (Pbf), Lys and hLys (Boc), Ser and hSer (t-Bu), His (Boc), Gin (Trt), Trp (Boc). All other chemicals were used as obtained from Aldrich, Acros, Sigma or Fluka.

Peptide synthesis. Solid phase peptide synthesis was performed on polystyrene resin functionalized with a TFA labile linker, which considerably facilitated the synthesis, handling, solid-phase cyclization, and final side chain deprotection and peptide isolation. The growing peptide chain was linked through the first amino acid side chain (for example lysine or homolysine) to the trytil moiety allowing for selective "head to tail" cyclization of the completed peptide sequence on solid support. The α-carboxyl group of the first N- α -Fmoc amino acid was protected as an allyl ester. Resin loading and peptide chain elongation was perfomed under standard Fmoc solid phase peptide synthesis conditions (Stewart, Solid Phase Peptide Synthesis, 1984) using chlorotrytil polystyrene resin as the solid support, with HBTU or DIG as coupling reagents and 25% piperidine in NMP for Fmoc deprotection. After completion of the final amino acid coupling, the resin was exposed to Palladium (0) and N-methyl morpholine (NMM) to remove the C-terminal allyl protecting group. Subsequent N-terminal Fmoc deprotection followed directly by cyclization with HATU provided the desired cyclic peptide. The protected cyclic peptide was released from the solid support and deprotected in one step using an 82.5% TFA (17.5% cation scavengers) solution for 1-3 h. After cleavage, ether was added to precipitate the peptide followed by centrifugation of the organic supernadant. The purity of crude peptides was assessed by HPLC and SSI-MS. The crude peptides can be partially purified by dissolving in boiling ACN/water/HCl mixture (30/70/0.1) and cooling the turbid solution in a fridge. In case of peptide high solubility in this mixture, the precipitate can be obtained by adding acetone (3 vol. eq.) to the above solution. Further purification was achieved by preparative reverse phase HPLC using, for example, a gradient of eluent A (0.1 TFA in H₂0 (v/v) and eluent B (0.1 TFA in 90% ACN/10% H₂0 (v/v)) using flow rate of 12 ml/min.

EXAMPLE 2 Preparation of c-(R-K-hW-L-W-hK-) Peptide synthesis was carried out by generally following the procedures described in Example 1 above. The following procedures for peptide c-(R-K-hW-L-W-hK-) are representative of the synthesis methods used for other cyclic peptides shown in Examples 3 to 15. The underlining indicates that the α-amino acid is a R- α -amino acid, and the hX (homoamino acid) indicates a β-amino acid of S-configuration at C β as for example hW represents S- β -homotriptophan (S- β -homoTrp) or hK represents S- β -homolysine (S- β - homoLys)].

Resin loading. Trityl chloride resin was swollen in dichloromethane for 20 min. A solution of Fmoc-hLys-OAllyl (prepared following Kates' protocol as set forth In Kates, et al, F. Tetrahedron Lett, 34, 1549-1552 (1993) for the preparation of Fmoc-Lys-OAllyl). In dicholromethane was added to the resin, followed by 4 eq. DIEA

(diisopropylethylamine). After 2 hrs. shaking, the resin was washed with dichloromethane (DCM), and then shaken with a mixture of methanol DIEA/DCM (1:1:8) for 30 min. After 3 washes with DCM the resin was dried in vacuo, and the resin loading was evaluated based on Fmoc relase monitored by UV adsorption at 290 nm. Preparation of Fmoc- β-homolysine-0 Allyl (Fmoc-hLys-OAllyl). Fmoc-hLys-

OAllyl was made following the protocol used by Kates, et al. Tetrahedron Lett., 34, 1549- 1552 (1993) for the preparation of Fmoc-Lys(Boc)-OAllyl. Fmoc-hLys(Boc)-OH (2 g, 4.1 mmol) was added to allyl bromide (25 mL, 0.29 mol), followed by DIEPA (1.5 mL). This mixture was heated at 90°C for 1 h. Then, the reaction was allowed to cool, concentrated by rotary evaporation, and after dilution with ethyl acetate was washed with 0.1 N HCI, saturated sodium bicarbonate and finally brine. The organic layer was filtered through a pad of silica gel and concentrated to afford a solid that was washed with ether and used directly in the next step. In a round bottom flask, 1 g of resulting Fmoc-hLys(Boc)-OAUyl in 10% TFA/dichloromethane was dissolved . After stirring for 1 hr, the solution was evaporated and the residue of TFA salt of Fmoc-hLys-OAllyl was dried in vacuo.

Peptide synthesis. Peptides were synthesized using standard solid-phase Fmoc protocols (Wellings, et al. Methods Enzymol, 289, 44-67 (1997)) on the Fmoc-hLys-OAll loaded trityl. Following the synthesis of the linear peptide, the resin was swollen in dichloromethane and then added to a degassed solution of 0.2 eq of palladium acetate [Pd(OAc)₂], 1 eq of triphenylphosphine, 10 eq of NMM and 20 eq of phenylsilane in DCM. After 3hrs. shaking under nitrogen, the resin was washed with DCM (2x 5 ml), NMP (2x5 ml) and 1% DIEA in DMF (2x5 ml). After final deprotection of Fmoc group with 25% piperidine in NMP, the resin was washed thoroughly with NMP, DCM and 1% DIEA in NMP. The resin was then treated with 2 eq of HATU and 5 eq of DIPEA in 0.8 M LiCl/NMP for lh (twice). After washing with NMP (3x 5 ml) and DCM (2x5 ml) followed by MeOH (1x5 ml), the peptide was cleavaged from the resin and deprotected with a mixture of TFA/Cresol/H₂0/thioanisole/EDT (82.5:5:5:5:2.5) (-100 mL/g of peptide) at rt for 1.5 hrs. After precipitation by adding ice-cold ether and centrifugation, the supernatant solution was removed and the resulting solid was purified by HPLC (Jupiter 4u proteo 90A, Phenomenex) using a gradient of eluent A (0.1 % TFA in H₂0(v/v)) and eluent B (0.1 % TFA in 90 % ACN/10 H₂0 (v/v)) using a flow rate of 12 mL/min. The purity of dried crude peptide was assessed by HPLC and SSI-MS. MS calculated was 926.5 and MS found was 926.6.

EXAMPLE 3 Preparation of c-(R-W-hL-hW-R-K-) Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptide was assessed by HPLC and SSI-MS. MS calculated was 954.6 and MS found was 954.1.

EXAMPLE 4 Preparation of c-(TC-W-hL-hW-H-K-)

Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 907.5 and MS found was 907.4.

EXAMPLE 5 Preparation of c-(S-W-hL-hW-H-K-)

Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 866.4 and MS found was 866.4.

EXAMPLE 6 Preparation of c-(Q-W-hL-h W-R-I

Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 926.4 and MS found was 926.1.

EXAMPLE 7 Preparation of c-(R-hW-hL-Q-K-H-)

Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 877.5 and MS found was 877.1. EXAMPLE 8 Preparation of c-(S-W-hW-L- -hK-)

Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 915.5 and MS found was 915.0.

EXAMPLE 9 Preparation of c-(Q-R-h W-L- -hK) Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 926.5 and MS found was 926.3.

EXAMPLE 10 Preparation of c-(H-K-hW-L-W-hK) Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 907.5 and MS found was 907.3.

EXAMPLE 11 Preparation of c-(S-hK-hW-L- W-L- W-K) Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 1156.6 and MS found was 1157.3.

EXAMPLE 12 Preparation of c-(K-hK-hW-L-W-L-W-K) Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 1197.7 and MS found was 1197.3. EXAMPLE 13 Preparation of c-(S-hK-hK-L- -L-W-K)

Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 1098.7 and MS found was 1098.5.

EXAMPLE 14 Preparation of c-(H-hK-hN-L-W-L-W-K) Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 1134.6 and MS found was 1133.9.

EXAMPLE 15 Preparation of c-(R-h W-h W-hL-Q-K-H) Solid phase synthesis of this peptide was carried out by generally following the procedures described in Examples 1 and 2 above. The purity of dried crude peptides was assessed by HPLC and SSI-MS. MS calculated was 1077.6 and MS found was 1077.1.

EXAMPLE 16 In Vitro Screening for Antibacterial Activity Antimicrobial Assay. Cyclic peptides of Examples 2-15 were screened for antimicrobial activity. Antimicrobial activity was evaluated by an initial screen using a broth dilution assay essentially as described in the guidelines of the National Committee for the Control of Laboratory Standards, (NCCLS) [National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Fourth edition. Approved Standard (1997). Document M7-A4. (NCCLS, Villanova, Pennsylvania, 1997). Test tubes (macrodilution method) or microtiter plates (microdilution method) containing two-fold serial dilution of peptides were inoculated with various bacterial cultures. Controls included non-inoculated medium (sterility), vehicle control, and various commercially available antibiotics for which minimal inhibitory concentrations against tested organisms were known. In vitro results using both methods were reproducible and in all cases M Cs from the macrodilution assays were equal to, or one dilution below, the inhibitory concentrations determined by microdilution tests (See, V. Lorian, Antibiotics in Laboratory Medicine, Williams and Wilkins, Baltimore 1991). The microdilution method has the advantages of requiring less amount of peptide for each assay and the possibility of multiple simultaneous inoculations and was used in most studies. Various strains of bacteria tested are described below.

Species Strains Type Special properties

Escherichia coli ATCC 700336 Gram Negative Prototypic uropathogenic strain from human urinary infection Escherichia coli ATCC29425(K12) Gram Negative Reference strain Staphylococcus aureus ATCC33591 Gram Positive Methicillin-resistant (MRSA)

Enterococcus faecalis ATCC 51575 Gram positive Vancomycin resistant (VRE); resistant to vancomycin, gentamicin and streptomycin

MIC (Minimum Inhibitory Concentration) Determination; Broth Dilution Method Preparation of peptide solutions. Stock peptide solutions were prepared in 5%

DMSO in aq. sucrose (9%). Determination of peptide concentrations was done by quantitative HPLC analysis using known concentrations of internal standards and/or by

measuring UV absorption of tryptophane-containing peptide solutions in H₂0 (λ=280 St_rp:

5690). Serial 2-fold dilutions were made in the above DMSO/aq. sucrose (9%) mixture with concentrations ranging approximately 400-2 μg/ml, and aliquots were dispensed in test tubes (100 μl) for the macrodilution test or in microtiter plates (20 μl) for the microdilution assays. Inoculum preparation. Overnight cultures of different microorganisms grown in suitable media were diluted 4000 times to an approximate inoculum size of 2.5 x 10⁵ cfu/ml.

Macrodilution method. Macrodilution methods were performed using procedures similar to those described in the National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial SusceptibilityTests for Bacteria That Grow Aerobically. Fourth edition. Approved Standard (1997). Document M7-A4. (NCCLSNillanova, Pennsylvania, 1997). Two ml aliquots of the above inoculum were dispensed to test tubes containing different peptide solutions. After incubation at 37 °C with shaking for 18 hours the lowest concentration at which no bacterial growth was observed was recorded as the MIC.

Microdilution method. Microdilution methods were performed using procedures similar to those described in the National Committee for Clinical Laboratory Standards. "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically," Fourth Edition. Approved Standard (1997). Document M7-A4.

(NCCLSNillanova, Pennsylvania, 1997. Eighty μl aliquots of the above inoculum were dispensed into 96-well microtiter plates containing different peptide solutions and incubated for 18 hrs. at 37 °C with shaking (plates were sealed with parafilm to avoid excessive evaporation of culture medium) and MICs were recorded. Each assay was performed at least twice and errors were typically plus or minus one dilution.

Following initial screening of the Example 2-15 cyclic peptides for antimicrobial activity and initial MIC evaluation, cyclic peptide c-(S-W-hW-L-W-hK) was determined to display antimicrobial activity against Enterococcus faecalis (MIC (ug/mL) value of 12). The MICs for the other three strains tested, E. coli, E. coli (K12) and Staphylococcus aereus, were evaluated at greater than 100. Cyclic peptide c-(S-hK-hW-L-W-L-W-K) was determined to display antimicrobial activity against E. coli [MIC (ug/mL) value of 50] while MICs for the other three strains tested, Enterococcus faecalis, E. coli (K12) and Staphylococcus aereus,were evaluated at greater than 100. Cyclic peptide c-(K-hK-hW-L- W-L-W-K) was determined to display antimicrobial activity against three strains tested, E. coli, E. coli (K12) and Staphylococcus aereus,with MICs of 25, 12, and 50 respectively. The MIC for Enterococcus faecalis was evaluated at greater than 100.

Cyclic peptides showing MICs of less than 100 in initial screening are considered suitable for further studies to test their antimicrobial activity. Preferred cyclic peptides are those with MICs of less than 50 in initial screening. In another more preferred embodiment, cyclic peptides having 12 or less MICs are preferred in initial screening.

Those skilled in the art will readily appreciate that the present invention is adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The cyclic peptides, and the methods for making and using the same described herein, are presently representative, preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes and modifications to such peptides, and methods of making and using the same, will occur to those skilled in the art upon reading this specification. It is understood that any and all such changes and modifications are encompassed within the scope of the invention. The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.

The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc, shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with any proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

WHAT IS CLAIMED:

1. A cyclic peptide having a sequence of from four to about twenty amino acids, wherein said sequence includes an even number of α-amino acids and at least one β-amino acid.

2. The cyclic peptide of claim 1, wherein the cyclic peptide has an amino acid sequence represented by Formula I : c-{ [(α-Aa)₂-]_n-(β-Aa)_m-} wherein: n = 2, 3, 4 or 5; and ' m = 1, 2, 3, 4, 5 or 6.

3. The cyclic peptide of claim 2, wherein the amino acid sequence is: c-{[(S-α-Aa)-(R-α-Aa)-]_n(S,S-β-Aa)_m-} wherein: n = 2, 3, 4 or 5; and m = 1, 2, 3, 4, 5 or 6.

4. The cyclic peptide of claim 2, wherein the cyclic peptide has the amino acid sequence that is an enantiomer of the sequence in claim 3.

5. The cyclic peptide of claim 1, wherein the cyclic peptide has an amino acid sequence represented by Formula II: c-[(α-Aa)₂-(β-Aa)_n-]_m wherein: n = 1, 2, 3, 4, 5 or 6; m = 1, 2, 3,4, 5 or 6.

6. The cyclic peptide of claim 5, wherein the amino acid sequence is: c-[(S-α-Aa)-(R-α-Aa)-(S,S-β-Aa)_n-]m wherein: n = 1, 2, 3, 4, 5, or 6; m = 1, 2, 3,4, 5 or 6.

7. The cyclic peptide of claim 5, wherein the cyclic peptide has an amino acid sequence that is an enantiomer of the sequence in claim 6.

8. The cyclic peptide of claim 1, wherein the cyclic peptide has an amino acid sequence represented by Formula III: c-[(α-Aa)₃-(β-Aa)_n-]_x wherein: n = 1, 2, 3, 4, 5, or 6; and x = 2 or 4.

9. The cyclic peptide of claim 8, wherein the amino acid sequence is: c-[(R-α-Aa)-(S-α-Aa)-(R-α-Aa)-(S,S-β-Aa)_nl-(S-α-Aa)-(R-α-Aa)-(S-α-Aa)-(R,R-

β-Aa)_n2-]_m wherein: nl = 1, 2, 3, 4, 5 or 6; n2 = 1, 2, 3, 4, 5 or 6; and m = 1 or 2.

10. The cyclic peptide of claim 1, wherein the cyclic peptide has an amino acid sequence represented by Formula IV: c-[(α-Aa)₄-(β-Aa)_n-]_m wherein: n = 1, 2, 3, 4, 5, 6, 7 or 8; and m = l, 2, 3 or 4.

11. The cyclic peptide of claim 10, wherein the amino acid sequence is: c-[(S-α-Aa)-(R-α-Aa)-(S-α-Aa)-(R-α-Aa)-(S,S-β-Aa)_n-]_m wherein: n = 1, 2, 3, 4, 5, 6, 7 or 8; and m = 1, 2, 3 or 4.

12. The cyclic peptide of claim 10, wherein the cyclic peptide has an amino acid sequence that is an enantiomer of the sequence in claim 11.

13. The cyclic peptide of claim 1, wherein the cyclic peptide has an amino acid sequence represented by Formula V: c-[(α-Aa)-(β-Aa)_n-]_x wherein: n = l, 2, 3, 4, 5, 6, 7 or 8; and x = 2, 4, 6 or 8.

14. The cyclic peptide of claim 13, wherein the amino acid sequence is: c-[(S-α-Aa)-(R,R-β-Aa)_nl-(R-α-Aa)-(S,S-β-Aa)_n2-]_m wherein: nl = 1, 2, 3, 4, 5, 6, 7 or 8; n2 = 1, 2, 3, 4, 5, 6, 7 or 8; and m = 1, 2, 3 or 4.

15. A cyclic peptide having a sequence of from four to about twenty amino acids, wherein said sequence includes at least one α-amino acid and at least one γ-amino acid and wherein the total number of α- and γ-amino acids combined is an even number.

16. The cyclic peptide of claim 15, wherein the cyclic peptide has an amino acid sequence represented by Formula VI: c-[(α-Aa)_n-(γ-Aa)-]_x wherein, n = 1, 2, 3, 4, 5, or 6; and x = 2, 3, 4, 5, 6, 7, or 8.

17. The cyclic peptide of claim 16, wherein the amino acid sequence is: c-[(R-α-Aa)-(R,R,S-γ-Aa)-]_m wherein, m = 2, 3, 4, 5, 6, 7 or 8.

18. The cyclic peptide of claim 16, wherein the cyclic peptide has an amino acid sequence that is an enantiomer of the sequence in claim 17.

19. The cyclic peptide of claim 16, wherein the amino acid sequence is: c-{[(S-α-Aa)-(R-α-Aa)-]_n(R,R,S-γ-Aa)-(R-α-Aa)-}_m wherein: n = 1, 2 or 3; and m = 1, 2, 3 or 4.

20. The cyclic peptide of claim 16 wherein the cyclic peptide has an amino acid sequence that is an enantiomer of the sequence in claim 19.

21. The cyclic peptide of claim 16, wherein the amino acid sequence is: c-{[(S-α-Aa)-(R-α-Aa)-]_nι(R,R,S-γ-Aa)-[(R-α-Aa)-(S-α-Aa)-]_n2(S,S,R-γ-Aa)-}_m wherein: nl and n2 are 1 or 2; and m = 1, 2, 3 or 4.

22. The cyclic peptide of claim 16 wherein the cyclic peptide has an amino acid sequence that is an enantiomer of the sequence in claim 21.

23. The cyclic peptide of claim 15, wherein the cyclic peptide has an amino acid sequence represented by Formula VII: c-[(α-Aa)-(γ-Aa)_n-]_x wherein: n = 2, 3 or 4; and x = 1, 2, 3 or 4.

24. The cyclic peptide of claim 23, wherein the amino acid sequence is: c-{(R-α-Aa)-[(R,R,S-γ-Aa)-(S,S,R-γ-Aa)-]_nl(S-α-Aa)-[(S,S,R-γ-Aa)-(R,R,S-γ-Aa)-

]n₂}m wherein: nl and n2 are 1 or 2; and m = 2 or 4.

25. The cyclic peptide of claim 22, wherein the amino acid sequence formula is: c-[(R-α-A)-(R,R,S-γ-Aa)-(S,S,R-γ-Aa)-(R,R,S-γ-Aa)-]_m wherein: m = l, 2, 3, 4 or 5.

26. The cyclic peptide of claim 22 wherein the cyclic peptide has an amino acid sequence that is the enantiomer of the sequence in claim 25.

27. A cyclic peptide having a sequence of from four to about twenty amino acids, wherein said sequence includes at least one α-amino acid, at least one β-amino acid, and one least one γ-amino acid, and wherein the total number of α- and γ- amino acids combined is an even number.

28. The cyclic peptide of claim 27, wherein the cyclic peptide has an amino acid sequence represented by Formula VIII: c-[(α-Aa)-(β-Aa)_n-(γ-Aa)-]_m wherein: n = 1, 2, 3 or 4 ; and m = l, 2, 3, 4, 5 or 6.

29. The cyclic peptide of claim 28, wherein the amino acid sequence is: c-[(R-α-Aa)-(S,S-β-Aa)_n-(R,R,S-γ-Aa)-]_m wherein: n = 1, 2, 3 or 4; and m = 1, 2, 3, 4, 5 or 6.

30. The cyclic peptide of claim 28, wherein the cyclic peptide has an amino acid sequence that is an enantiomer of the sequence in claim 29.

31. The cyclic peptide of claim 27, wherein the cyclic peptide has an amino acid sequence represented by Formula IX: c-[(α-Aa)-(γ-Aa)-(β-Aa)_n-]_m wherein: n = 1, 2, 3 or 4 ; and m = l, 2, 3, 4, 5 or 6.

32. The cyclic peptide of claim 27, wherein the amino acid sequence is: c-[(S-α-Aa)-(S,S,R-γ-Aa)-(S,S-β-Aa)_n-]_m wherein: n = 1, 2, 3 or 4; and m = 1, 2, 3, 4, 5 or 6.

33. The cyclic peptide of claim 27, wherein the cyclic peptide has an amino acid sequence that is the enantiomer of the sequence in claim 32.

34. The cyclic peptide of claims 1, 15 or 27 wherein at least one of the alpha amino acids with S configuration is substituted by a gamma amino acid with R,R,S configuration or wherein at least one of the alpha amino acids with R configuration is substituted by a gamma amino acid with S,S,R configuration.

35. The cyclic peptide of claims 1, 15, 27 or 34, wherein the cyclic peptide self- assembles into a supramolecular structure.

36. The cyclic peptide of claim 35, wherein the supramolecular structure comprises a nanotube, a barrel of associated, axially parallel nanotubes, a carpet of associated nanotubes, or mixtures thereof.

37. The cyclic peptide of claim 1, 15, 27 or 34, wherein the cyclic peptide or a supramolecular structure thereof induce depolarization of membranes of target microbial organisms selectively over animal cell membrane depolarization.

38. The cyclic peptide of claim 1, 15, 27 or 34, wherein the cyclic peptide or a supramolecular structure thereof induce lysis of target microbial organisms selectively over animal cell lysis.

39. The cyclic peptide of claim 1, 15, 27 or 34, wherein the cyclic peptide or a supramolecular structure thereof induce lysis of a target virus selectively over animal cell lysis.

40. The cyclic peptide of claim 1, 15, 27 or 34, wherein the cyclic peptide or a supramolecular structure thereof induce lysis of a cancer cell selectively over animal cell lysis.

41. A composition comprising a pharmaceutically acceptable carrier and a cyclic peptide of claims 1, 15, 27 or 34.

42. A method for identifying or evaluating a cyclic peptide with antiviral activity comprising: (a) contacting a target viral organism with a test cyclic peptide; and (b) determining whether the test cyclic peptide has antiviral activity; wherein the test cyclic peptide comprises the cyclic peptide of claims 1, 15, 27 or 34.

43. A method of identifying a cyclic peptide selectively cytotoxic to a target cancer cell type comprising:

(a) contacting said target cancer cell type with a test cyclic peptide comprising the cyclic peptide of claims 1, 15, 27 or 34; and (b) determining whether said test cyclic peptide induces cell death of said target cancer cell type without inducing substantial or undesired cell death in a second cell type.

4. A method for identifying or evaluating a cyclic peptide with antimicrobial activity comprising:

(a) contacting a target microbial organism with a test cyclic peptide; and

(b) determining whether the test cyclic peptide has antimicrobial activity; wherein the test cyclic peptide comprises the cyclic peptide of claims 1, 15, 27 or 34.