WO2005068492A2

WO2005068492A2 - Peptide inhibitors of retroviral integrase useful for the treatment of retroviral infection

Info

Publication number: WO2005068492A2
Application number: PCT/US2004/042726
Authority: WO
Inventors: Peter P. Roller; Krzysztof Krajewski; Christophe Marchand; Yves Pommier; Vinay Pathak; Yijun Zhang
Original assignee: The United States Of America As Represented By The Secretary Of Health And Human Services, Nih
Priority date: 2004-01-06
Filing date: 2004-12-21
Publication date: 2005-07-28
Also published as: WO2005068492A3

Abstract

The present invention relates to peptides and peptide derivatives and their use to prevent the onset of retroviral infection or attenuate the severity of the symptoms of retroviral infection. The invention particularly relates to the use of peptide derivatives to prevent the onset of retroviral infection or attenuate the severity of the symptoms of Acquired Immunodeficiency Syndrome (AIDS).

Description

Title of the Invention:

Peptide Inhibitors of Retroviral Integrase Useful for the Treatment of Retroviral Infection Field of the Invention: The present invention relates to peptides and their derivatives that inhibit retroviral integrase and to their use in the treatment of retroviral infection. The invention particularly relates to the use of such molecules to prevent the onset of retroviral infection or to attenuate the severity of the symptoms of Acquired Immunodeficiency Syndrome (AIDS).

Cross-Reference to Related Applications This application claims priority to U.S. Patent Applications Serial Nos. 60/534,378 (filed on January 6, 2004); 60/547,067 (filed on February 25, 2004), and 60/599,856 (filed: August 10, 2004), each of which applications is herein incorporated by reference in its entirety.

Statement of Governmental Interest This invention was funded by NCI Intramural Research Program CCR at the National Institutes of Health. The United States Government has certain rights to this invention.

Background of the Invention: Retroviruses comprise a large family of RNA viruses that synthesize a DNA copy of their RNA genome after infection into a host cell (Segura-Totten, M. et al. (2001) "Virology. HIV—breaking the rules for nuclear entry," Science. 294(5544): 1016-7.; Gotte, M. et al. (1999) "HIV-1 REVERSE TRANSCRIPTION: A BRIEF OVERVIEW FOCUSED ON STRUCTURE-FUNCTION RELATIONSHIPS AMONG MOLECULES INVOLVED IN INITIATION OF THE REACTION," Arch Biochem Biophys. 365(2): 199-210; Mikkelsen, J.G. et al. (2004). "COMPLEMENTARITY-DIRECTED RNA DlMER-LlNKAGE PROMOTES RETROVIRAL RECOMBINATION IN VlVO," Nucleic Acids Res. 32(1): 102-14; Gonda, M.A. (1988) "MOLECULAR GENETICS AND STRUCTURE OF THE HUMAN IMMUNODEFICIENCY VIRUS," J Electron Microsc Tech. 8(1): 17-40; Buchbinder, A. (1990) "VIROLOGY OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1," Ear Nose Throat J. 69(6): 376, 379-84; Kulkosky, J. et al. (1994) "MOLECULAR MECHANISM OF RETROVIRAL DNA INTEGRATION," Pharmacol Ther. 61(1-2): 185-203; Bushman, F.D. et al. (1990) "RETROVIRAL DNA INTEGRATION DIRECTED BY HIV INTEGRATION PROTEIN IN VITRO," Science. 249(4976): 1555-8). Human Immunodeficiency Virus (HIV) is a retrovirus that has been found to be the causal agent of Acquired Immunodeficiency Syndrome (AIDS) (Wood, R.W. et al. (1987) "ACQUIRED IMMUNODEFICIENCY SYNDROME," Infect Dis Clin North Am. (1): 145-63; Gallo, R.C. et al. (2003) "THE DISCOVERY OF HIV As THE CAUSE OF AIDS," N Engl J Med. 349(24): 2283-5; Holtgrave, D.R. (2004) "ESTIMATION OF ANNUAL HIV TRANSMISSION RATES IN THE UNITED STATES,

1978-2000," J Acquir Immune Defic Syndr. 35(1): 89-92; Ebbesen, P.(1986) "THE GLOBAL EPIDEMIC OF AIDS," AIDS Res. (2 Suppl) 1 : S23-8; Gallo, R.C.(1987) "THE AIDS VIRUS," Sci Am. 256(1): 46-56; Morrow, CD. et al. (1994) "VIRAL GENE PRODUCTS AND REPLICATION OF THE HUMAN IMMUNODEFICIENCY TYPE 1 VIRUS," Am J Physiol. 266(5 Pt 1): Cl 135-56).

The etiology of retroviruses has been extensively studied in order to identify inhibitor compounds that could be used to prevent or attenuate the symptoms of AIDS (Clumeck, N. et al. (1993) "CURRENT USE OF ANTI-HIV DRUGS IN AIDS," J. Antimicrob. Chemother. (Suppl. A) 32, 133-138; Witvrouw, M. et al. (2003) "THE INTEGRASE OF THE HUMAN IMMUNODEFICIENCY VIRUS AS A NOVEL TARGET FOR THE ANTIVIRAL THERAPY OF AIDS," Verh K Acad Geneeskd Belg 65(5): 325-34; Docherty, A.J. et al. (2003) "PROTEASES As DRUG TARGETS," Biochem Soc Symp. (70): 147-61; Nair, V. (2003) "NOVEL INHIBITORS OF HIV INTEGRASE: THE DISCOVERY OF POTENTIAL ANTI-HIV THERAPEUTIC AGENTS," Curr Pharm Des.: 9(31): 2553-65; Thaker, H.K. et al. (2003) "HIV VIRAL SUPPRESSION IN THE ERA OF ANTIRETROVIRAL THERAPY," Postgrad Med J. 79(927): 36-42; De Clercq, E. (2002) "NEW ANTI-HIV AGENTS AND TARGETS," Med. Res. Rev. 6:531-65; Mitsuya, H. et al. (1987) "STRATEGIES FOR ANTIVIRAL THERAPY IN AIDS," Nature 325(6107): 773-8; Gallo, R.C. et al. (1987) "ETIOLOGY OF AIDS RESEARCH," Nature 326(6112): 435-6). These efforts have led to the recognition of three retroviral enzymes that are required for viral propagation: Reverse transcriptase, which mediates the production of a DNA copy of the initial RNA viral genome; HIV protease, which mediates the site- specific cleavage of the HIV polyprotein to yield functionally active viral proteins; and HIV integrase, which mediates the incorporation of the viral genome into the genome ofa host cell.

Research on the mechanism of action of these enzymes has led to the identification of numerous inhibitors of reverse transcriptase and HIV protease (see "PROTEASES INHIBITORS AND BEYOND," PI Perspective 21:16-16 (1997) (Anonymous); James, J.S. (2002) "RETROVIRUSES CONFERENCE: SOME NEW DRUGS IN THE PIPELINE," AIDS Treat News 12 (379):2-4). Anti-retroviral drugs currently approved for use in the United States include protease inhibitors: Amperanir (APV) (Agenerase; GlaxoSmithKline/Vertex); Atazanavir Sulfate (Reyataz; Bristol-Myers Squibb); Fosamprenavir Calcium(Lexiva; GlaxoSmithKline); Indinavir (IDV) (Crixivan; Merck); Lopinavir/ritonavir (LPV/r) (Kaletra; Abbott Laboratories); Nelfmavir (NFV) (Viracept; Agouron Pharmaceuticals); Ritonavir (RTV) (Norvir; Abbott Laboratories); Saquinavar mesylate (Invirase; Hoffmann-La Roche); and Saquinavir (SQV) (Fortovase; Hoffmann-La Roche); nucleoside reverse transcriptase inhibitors: Abacavir (Ziagen; GlaxoSmithKline); Abacavir, Zidovudine, And Lamivudine (Trizivir; GlaxoSmithKline); Didanosine, ddl, Dideoxyinosine (Videx; Bristol Myers- Squibb); Enteric Coated Didanosine (Videx Ec; Bristol Myers-Squibb); FTC, Emtricitabine (Emtriva; Gilead Sciences); Lamivudine And Zidovudine (Combivir; GlaxoSmithKline); Lamivudine, 3TC (Epivir; GlaxoSmithKline); Nelfmavir Mesylate, NFV (Viracept; Agouron Pharmaceuticals); Stavudine, D4t (Zerit; Bristol Myers-Squibb); Tenofovir Disoproxil Fumarate (Viread; Gilead);

Zalcitabine, Ddc, Dideoxycytidine (Hivid; Hoffmann-La Roche); and Zidovudine, AZT, Azidothymidine, ZDV (Retrovir GlaxoSmithKline); non-nucleoside reverse transcriptase inhibitors: Delaviridine (DLV) (Rescriptor; Pharmacia); Efavirenz (EVF) (Sustiva; Bristol-Myers Squibb); and Nevirapine (NVP) (Viramune; Boehringer Ingelheim); and fusion inhibitors (Enfuvirtide, T-20 (Fuzeon; Hoffmann-La Roche/Trimeris) (O'Brien, W.A. (2003) "NEW CLASSES OF HIV DRUGS ON THE HORIZON," AIDS Read. 13(3 Suppl.):S4-8; http://www.fda.gov/oashi/aids/pedlbl.html).

The recent success of uglily active antiretroviral therapy (HAART) using combinations of inhibitors of viral enzymes have altered the natural course of AIDS progression by suppressing viral load for long periods of time (Preston, S.L. et al. (2003) "PHARMACODYNAMICS AND CLINICAL USE OF ANTI-HIV DRUGS," Infect Dis Clin North Am. 17(3): 651-674; Sherer, R. (2003) "HIV, HAART, AND HYPERLIPIDEMIA: BALANCING THE EFFECTS," J Acquir Immune Defic Syndr. 34 Suppl 2: S 123-129; Gathe, J. Jr. (2003) "ADHERENCE AND POTENCY WITH ANTIRETRO VIRAL THERAPY: A COMBINATION FOR SUCCESS," J Acquir Immune

Defic Syndr. 34 Suppl 2: SI 18-122; Daar, E.S. (2003) "Potency And Durability Of Antiretroviral Therapy," J Acquir Immune Defic Syndr. 34 Suppl 2: SI 11-117; Katzenstein, T.L. (2003) "MOLECULAR BIOLOGICAL ASSESSMENT METHODS AND UNDERSTANDING THE COURSE OF THE HIV INFECTION," APMIS Suppl. 114:1 -37; Pozniak, A. (2003) "OPTIMIZING EFFICACY AND TOLERABILITY IN TODAY'S HAART," AIDS Read. S4-8; Faragon, J.J. et al. (2003) "DRUG INTERACTIONS ASSOCIATED WITH HAART: Focus ON TREATMENTS FOR ADDICTION AND RECREATIONAL DRUGS," AIDS Read. 13(9):433-434, 437-41, 446-50).

However, despite the success of inhibitors of HIV reverse transcriptase and HIV protease, difficult obstacles to managing retroviral infection remain. A significant fraction of patients fail to respond to such treatment. Retroviruses show a remarkable ability to mutate into variant forms that are resistant to the therapeutic agents. While these problems can be addressed using multiple inhibitors, such "polytherapy" is unfortunately associated with numerous adverse side effects (see, van Sighem, A.I. et al. (2003) "MORTALITY AND PROGRESSION TO AIDS AFTER STARTING HIGHLY ACTIVE ANTIRETROVIRAL THERAPY," AIDS 17(15):2227-2236; Re, M.C. et άl. (2003) "MUTATION PATTERNS OF THE REVERSE TRANSCRIPTASE GENES IN HIV-1 INFECTED PATIENTS RECEIVING COMBINATIONS OF NUCLEOSIDE AND NON NUCLEOSIDE INHIBITORS," Int J Antimicrob Agents. 22(4):388-394; Selwyn, P.A. et al. (2003) "PALLIATIVE CARE FOR AIDS AT A LARGE URBAN TEACHING HOSPITAL: PROGRAM DESCRIPTION AND PRELIMINARY OUTCOMES," J Palliat. Med. 6(3):461-474; Druillennec, S. et al. (2000) "HIV-1 NCP7 As A TARGET FOR THE DESIGN OF NOVEL ANTIVIRAL AGENTS," Drug News Perspect. 13(6):337-349). The severity of these side effects frequently leads to patient non- compliance and interruption of treatment. The retroviral integrase may provide a useful therapeutic target for a new class of retroviral inhibitors (Neamati, N. et al. (2000) "HIV-1 INTEGRASE INHIBITOR: PAST, PRESENT, FUTURE," Advances in Pharmacology 49:147-165; De Clercq, E. et al. (2002) "NEW ANTI-HIV AGENTS AND TARGETS," Med. Res. Rev. 6:531-65; Nair, V. et al. (2002) "HIV INTEGRASE AS A TARGET FOR ANTIVIRAL CHEMOTHERAPY," Rev Med Virol. 12(3): 179-93). First, there is no cellular equivalent to the integrase and the integrase is essential for viral replication (Pommier, Y. et al. (2000) "RETROVIRAL INTEGRASE INHIBITORS YEAR 2000: UPDATE AND PERSPECTIVES," Antiviral Research 47:139-148; Nair, V. (2002) "HIV INTEGRASE AS A TARGET FOR ANTIVIRAL CHEMOTHERAPY," Rev Med Virol. 12(3): 179-93; de Soultrait, V.R.. et al. (2002) "A NOVEL SHORT PEPTIDE IS A SPECIFIC INHIBITOR OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 INTEGRASE," J. Mol. Biol. 318:45-58). As such, a therapy based on inhibiting the retroviral integrase would be less susceptible to viral mutational selection than therapies based on inhibiting the retroviral protease or reverse transcriptase (see, "PROTEASES INHIBITORS AND BEYOND," PI Perspective 21 : 16-16 (1997

(Anonymous)). In addition, rapid and sensitive assays exist that test integrase enzyme activity and inhibitors (Nair, V. (2002) "HIV INTEGRASE As A TARGET FOR ANTIVIRAL CHEMOTHERAPY," Rev Med Virol. 12(3): 179-93; Debyser, Z. et al. (2002) "IN SEARCH OF AUTHENTIC INHIBITORS OF HIV-1 INTEGRASE INHIBITORS," Antivir Chem Chemother. 13(l):45-58). Oligonucleotides can be created that mimic the retroviral long terminal repeats (LTR) that are the substrates of the retroviral integrase, thereby facilitating drug discovery (De Clercq, E. (2002) "NEW ANTI-HIV AGENTS AND TARGETS," Med. Res. Rev. 6:531-65). Additionally, the crystal and NMR structures of the integrase are available, thereby facilitating rational inhibitor design (Craigie R.

(2001) "HIV INTEGRASE, A BRIEF OVERVIEW FROM CHEMISTRY TO THERAPEUTICS," J Biol Chem 276(26): 23213-23216; de Soultrait, V.R. et al.

(2002) "A NOVEL SHORT PEPTIDE IS A SPECIFIC INHIBITOR OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 INTEGRASE," J. Mol. Biol. 318:45-58). Several HIV integrase inhibitors have already been identified and show promise in the treatment of retroviral infection (O'Brien, W.A. (2003) "NEW CLASSES OF HIV DRUGS ON THE HORIZON. A REVIEW OF THE PRESENTATION AT THE SATELLITE SYMPOSIUM "NEW HOPE: ADVANCING CARE IN HIV INFECTION" at the 15th Annual Association Of Nurses In AIDS Care Conference, November 2002," AIDS Read. 13(3 Suppl): S4-8; Billich, A. (2003) "S-1360 SHIONOGI-

GLAXOSMITHKLINE," Curr Opin Investig Drugs. 4(2): 206-9; Gulick, R.M. (2003) "NEW ANTIRETROVIRAL DRUGS," Clin Microbiol Infect. 9(3): 186-93; Chen, I.J. et al. (2002) "STRUCTURE-BASED INHIBITOR DESIGN TARGETING HIV-1 INTEGRASE," Curr Drug Targets Infect Disord. 2(3): 217-34; Debyser, Z. et al. (2002) "IN SEARCH OF AUTHENTIC INHIBITORS OF HIV- 1 INTEGRATION," Antivir Chem Chemother. 13(1): 1-15; Tarrago-Litvak, L. et al. (2002) "INHIBITORS OF HIV-1 REVERSE TRANSCRIPTASE AND INTEGRASE: CLASSICAL AND EMERGING THERAPEUTICAL APPROACHES," Curr. Pharm. Des. 8:595-614; De Clercq, E. (2002) "NEW DEVELOPMENTS IN ANTI-HIV CHEMOTHERAPY," Biochim Biophys Acta. 1587(2-3): 258-275; Raulin, J. (2002) "HUMAN IMMUNODEFICIENCY VIRUS AND HOST CELL LIPIDS. INTERESTING PATHWAYS IN RESEARCH FOR A NEW HIV THERAPY," Prog Lipid Res. 41(1): 27-65; d'Angelo, J. et al. (2001) "HIV-1 INTEGRASE: THE NEXT TARGET FOR AIDS THERAPY?" Pathol Biol (Paris) 49(3): 237-46; Neamati, N. (2001) "STRUCTURE-BASED HIV-1 INTEGRASE INHIBITOR DESIGN: A FUTURE PERSPECTIVE," Expert Opin Investig Drugs. 10(2): 281-296; Mathe, G. (1999) "WHY HAVE TEN OR SO NONTOXIC, RETROVIRUS INTEGRASE INHIBITORS NOT BEEN MADE AVAILABLE FOR AIDS TREATMENT? A TEN- YEAR EXPERIENCE [CORRECTION OF EXPERIMENT] MUST LIBERATE THEM," Biomed Pharmacother. 53(10): 484-486; Erratum in: Biomed Pharmacother (2000) 54(1):60; Wlodawer, A. (1999) "CRYSTAL STRUCTURES OF CATALYTIC CORE DOMAINS OF RETROVIRAL INTEGRASES AND ROLE OF DIVALENT CATIONS IN ENZYMATIC ACTIVITY," Adv Virus Res. 52: 335-50; Farnet, CM. et al. (1996) "HIV cDNA INTEGRATION: MOLECULAR BIOLOGY AND INHIBITOR DEVELOPMENT," AIDS 10 Suppl. A: S3-11).

However, despite all such efforts, resistance to antiretroviral agents remains a major problem affecting the epidemiology and treatment of AIDS and its feline and simian counterparts. Thus, a need exists for an anti-retroviral composition that would be able to prevent the onset of retroviral infection or attenuate the severity of the symptoms of retroviral infection, especially HIV infection. The present invention is directed to such needs.

Summary of the Invention: The present invention relates to peptides and their derivatives that inhibit retroviral integrase and to their use in the treatment of retroviral infection. The invention particularly relates to the use of such molecules to prevent the onset of retroviral infection or to attenuate the severity of the symptoms of Acquired Immunodeficiency Syndrome (AIDS). More specifically, the present invention relates generally to the use of indolicidin peptide derivatives to attenuate the severity and/or onset of retroviral infection, especially by inhibiting the functioning of the HIV-1 integrase. The present invention thus permits the use of such novel peptides for the treatment of retroviral infection, including AIDS. The invention also provides a method of assaying for HIV-1 integrase.

In detail, the invention provides a peptide having the formula:

a peptide comprising the formula: Aaι-Aa₂-Aa₃-Aa₄-Aa₅-Aa6-Aa₇-W-W-Aa8-Aa₉-Aaιo-Aaιι-Z; wherein "Aa" denotes an amino acid residue; wherein such peptide is at least 6 amino acid residues in length; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aaπ is R, P or absent, and wherein Z is the terminal carboxyl group of the peptide and is either unmodified or modified; with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa₉, Aaio, and Aan are all absent, Aa₈ will also be absent; or a pharmaceutically acceptable salt thereof.

The invention particularly concerns the embodiment of such peptide, wherein the peptide is selected from the group consisting of: SEQ ID NO. 2 ILPWKWPWWPWPP-Z SEQ ID NO. 3 ILAWKWAWWAWPP-Z SEQ ID NO. 4 LPWKWPWWPWPP-Z SEQ ID NO. 5 PWKWPWWPWPP-Z SEQ D3 NO. 6 WKWPWWPWPP-Z SEQ ID, NO. 7 KWPWWPWPP-Z SEQ ID NO. 8 WPWWP PP-Z SEQ D3 NO. 9 PWWPWPP-Z SEQ D3 NO. 10 WWPWPP-Z SEQ π> NO. 11 ILPWKWPWWPWP -z and SEQ D3 NO. 13 ILPWKWPWW - Z

The invention is further directed to a peptide comprising the formula:

wherein X₂ is absent, or Xi and X₂ are independently selected peptides having the formula: Aaι-Aa₂-Aa₃-Aa₄-Aa5-Aa6-Aa₇-W-W-Aa₈-Aa₉-AaiQ-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa₉, Aaio, and Aan are all absent, Aa₈ will also be absent; wherein each such peptide is at least 6 amino acid residues in length; wherein J is a linker, and wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; or a pharmaceutically acceptable salt thereof.

The invention particularly concerns the embodiments of such peptide, wherein wherein J is a lysine or arginine amino acid residue, and wherein the peptide comprises the formula: SEQ ro NO. 14: (ILPWKWP WPWPP) ₂κ-z . The invention particularly concerns the embodiments of all such peptides wherein the terminal carboxyl group Z of the peptide is modified, the modification being amidation.

The invention further concerns a peptide comprising the formula:

wherein X₂ is absent, or Xi and X₂ are independently selected peptides having the formula: Aai -Aa₂-Aa₃-Aa -Aa₅-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, wherein each such peptide Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; or a pharmaceutically acceptable salt thereof.

The invention further concerns the embodiments of such peptides or pharmaceutically acceptable salts thereof wherein J is a lysine or arginine amino acid residue; wherein n is 2; and/or wherein Z is a lysine or arginine amino acid residue.

The invention particularly concerns the embodiment of such peptides or pharmaceutically acceptable salts thereof, wherein the peptide is selected from the group consisting of: SEQ D3 NO.2 ILPWKWPWWPWPP- -z SEQ ID NO.3 ILAWKWAWWAWPP- -z SEQ ID NO.4 LPWKWPWWP PP- -z SEQ D3 NO.5 PWKWP PWPP- -z SEQ π> NO.6 WKWPWWPWPP- -z SEQ D3 NO.7 KWPWWPWPP- -z SEQ TD NO.8 PW PWPP- -z SEQ DD NO.9 PWWP PP- -z SEQ D3 NO.10 WWPWPP- -z SEQ ID NO.11 ILPWKWPWWP P - -z and SEQ D3 NO.13 ILPWKWPWW -z

The invention particularly concerns the embodiments of such peptides or pharmaceutically acceptable salts thereof, wherein the peptide comprises the formula: SEQ ID NO. 18: ( ( ILPWKWPWWPWPP ) ₂K) ₂-K . The invention further concerns a peptide comprising the formula:

wherein X is absent, or Xi and X₂ are each peptides that are independently selected having the formula: Aai -Aa₂-Aa₃-Aa₄-Aa5-Aa6-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i ; wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aa is R, P or absent; wherein each such peptide, Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J and at least one reactive group capable of bonding to B ; wherein m is an integer greater than 0; wherein B is a substituent having at least m reactive groups, each capable of bonding to Z; or a pharmaceutically acceptable salt thereof. The invention particularly concerns the embodiments of such peptide, wherein J is a lysine or arginine amino acid residue, wherein n is 2, wherein m is 2, and/or wherein Z is a lysine or arginine amino acid residue.

The invention particularly concerns the embodiment of such peptides or pharmaceutically acceptable salts thereof, wherein the peptide is selected from the group consisting of: SEQ ED NO. 2 ILPWKWPWWPWPP- Z ; SEQ ED NO. 3 ILAWKWAWWAWPP- Z ; SEQ D3 NO. 4 LPWKWPWWPWPP-Z SEQ ID NO. 5 PWKWPWWPWPP-Z ; SEQ ID NO. 6 WK PWWPWPP-Z ; SEQ ED NO. 7 K PWWPWPP-Z ; SEQ ED NO. 8 WPWWPWPP-Z ; SEQ ED NO. 9 P WPWPP-Z ; SEQ ED NO. 10 WWPWPP-Z ; SEQ ED NO. 11 ILPWKWPWWPWP -Z and SEQ ED NO. 13 ILPWKWPWW -Z The invention further concerns an anti-retroviral pharmaceutical composition comprising a peptide comprising the formula: Aaι-Aa₂-Aa₃-Aa₄-Aa5-Aa6-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aan-Z; wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aas is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aai ι is R, P or absent, wherein such peptide is at least 6 amino acid residues in length; and wherein Z is the terminal carboxyl group of the peptide and is either unmodified or modified; with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa₉, Aaio, and Aan are all absent, Aa₈ will also be absent; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein the peptide is present in the composition at a concentration that is effective for attenuating the severity of, or preventing, a retroviral infection.

The invention further concerns the embodiments of such anti-retroviral pharmaceutical composition wherein the terminal carboxyl group Z of the peptide is modified, the modification being amidation. The invention particularly concerns the embodiments of such anti-retroviral pharmaceutical composition, wherein, wherein the peptide is selected from the group consisting of: SEQ ID NO. 2 ILPWKWPWWPWPP-Z , SEQ ID NO. 3 I LAWK AWWAW P P - Z , SEQ ED NO. 4 LPWKWPWWP PP-Z ; SEQ ED NO. 5 PWKWPWWPWPP-Z , SEQ ED NO. 6 WKWPWWPWPP-Z , SEQ ID NO. 7 KWPWWPWPP-Z , SEQ ID NO. 8 WPWWPWPP-Z , SEQ ID NO. 9 PWWPWPP-Z SEQ ID NO. 10 WWPWPP-Z SEQ ED NO. 11 ILPWKWPWWPWP -Z and SEQ ED NO. 13 ILPWKWPWW -Z The invention further concerns an anti-retroviral pharmaceutical composition comprising a peptide comprising the formula: Xi — J — Z χ₂ wherein X₂ is absent, or Xi and X₂ are independently selected peptides having the formula: Aaι-Aa -Aa₃-Aa₄-Aa5-Aa6-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa₉, Aaio, and Aan are all absent, Aa₈ will also be absent; wherein each such peptide is at least 6 amino acid residues in length; wherein J is a linker, and wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein the peptide is present in the composition at a concentration that is effective for attenuating the severity of, or preventing, a retroviral infection.

The invention particularly concerns the embodiment of such anti-retroviral composition, wherein the peptide is selected from the group consisting of: SEQ ID NO.2 ILPWKWPWWPWPP-Z SEQ ED NO.3 ILAWKWAWWAWPP-Z SEQ ED NO.4 LPWKWPWWPWPP-Z SEQ ED NO.5 PWKWPWWPWPP-Z; SEQ ED NO.6 WKWPWWPWPP-Z SEQ ED NO.7 KWPWWPWPP-Z SEQ ED NO.8 WPWWPWPP-Z SEQ ED NO.9 PWWPWPP-Z SEQ ED NO.10 WWPWPP-Z SEQ ED NO.11 ILPWKWPWWPWP -z and SEQ ED NO.13 ILPWKWPWW -Z

The invention particularly concerns the embodiments of such anti-retroviral pharmaceutical composition wherein J is a lysine or arginine amino acid residue, and/or wherein the peptide has the formula: SEQ ED NO. 14: (ILPWKWPWWPWPP) ₂K-Z . The invention further concerns an anti-retroviral pharmaceutical composition comprising a peptide having the formula:

wherein X₂ is absent, or Xj and X₂ are independently selected peptides having the formula: Aai -Aa₂-Aa₃-Aa -Aa₅-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, wherein each such peptide X] and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein the peptide is present in the composition at a concentration that is effective for attenuating the severity of, or preventing, a retroviral infection.

The invention particularly concerns the embodiment of such anti-retroviral composition, wherein the peptide is selected from the group consisting of: SEQ D3 NO.2 ILPWKWPWWPWPP-Z SEQ ED NO.3 ILAWKWAWWAWPP-Z SEQ D3 NO.4 LPWKWPWWPWPP-Z SEQ DD NO.5 PWKWPWWPWPP-Z SEQ ED NO.6 WKWPWWPWPP-Z SEQ DD NO.7 KWPWWPWPP-Z SEQ ED NO.8 WPWWPWPP-Z SEQ ID NO.9 PWWPWPP-Z SEQ ED NO.10 WWPWPP-Z SEQ ID NO.11 ILPWKWPWWPWP -z and SEQ ID NO.13 ILPWKWPWW -Z

The invention concerns the embodiments of such anti-retroviral pharmaceutical composition, wherein J is a lysine or arginine amino acid residue, wherein n is 2, wherein Z is a lysine or arginine amino acid residue. The invention particularly concerns the embodiments of such anti-retroviral pharmaceutical composition, wherein the peptide comprises the formula: SEQ ED NO. 18: ((ILPWKWPWWPWPP)₂K)₂-K. The invention also concerns an anti-retroviral pharmaceutical composition comprising a peptide comprising the formula:

wherein X₂ is absent, or Xi and X₂ are each peptides that are independently selected having the formula: Aai -Aa₂-Aa₃-Aa -Aa₅-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i ; wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent; wherein each such peptide, Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J and at least one reactive group capable of bonding to B; wherein m is an integer greater than 0; wherein B is a substituent having at least m reactive groups, each capable of bonding to Z; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein the peptide is present in the composition at a concentration that is effective for attenuating the severity of, or preventing, a retroviral infection.

The invention concerns the embodiments of such anti-retroviral pharmaceutical composition, wherein J is a lysine or arginine residue, wherein n is 2, wherein m is 2. The invention particularly concerns the embodiment of such antiretroviral composition, wherein the peptide is selected from the group consisting of: SEQ ED NO. 2 ILPWKWPWWPWPP-Z SEQ ED NO. 3 I L WKWAWW AW P P - Z ^• SEQ ED NO. 4 LPWKWPWWPWPP-Z SEQ ED NO. 5 P WKW PWW PW P P - Z SEQ ED NO. 6 WKWPWWPWPP-Z SEQ ED NO. 7 KWPWWPWPP-Z SEQ ED NO. 8 WPWWPWPP-Z SEQ ID NO. 9 PWWPWPP-Z SEQ ED NO. 10 WWPWPP-Z SEQ D3 NO. ll ILPWKWPWWPWP -Z ; and SEQ ED NO. 13 ILPWKWPWW -Z

The invention further concerns a method for preventing, or attenuating the severity of, a retroviral infection, which comprises providing to an individual having such infection, an effective amount of an anti-retroviral pharmaceutical composition comprising a peptide having the formula: Aaι-Aa₂-Aa₃-Aa -Aa₅-Aa6-Aa -W-W-Aa8-Aa₉-Aaιo-Aan-Z; wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa is W or absent; Aas is K or absent; Aa₆ is W or absent; Aa is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, wherein such peptide is at least 6 amino acid residues in length; and wherein Z is the terminal carboxyl group of the peptide and is either unmodified or modified; with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa , Aaio, and Aan are all absent, Aa₈ will also be absent; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein the peptide is present in the composition at a concentration that is effective for preventing, or attenuating the severity of, a retroviral infection.

The invention particularly concerns the embodiment of such method, wherein the peptide is selected from the group consisting of: SEQ ID NO. 2 ILPWKWPWWPWPP-Z SEQ ED NO. 3 I L WKWAWWAW P P - Z SEQ ED NO. 4 LPWKWPWWPWPP-Z SEQ ED NO. 5 PWKWPWWPWPP-Z SEQ ED NO. 6 WKWPWWPWPP-Z SEQ ED NO. 7 KWPWWPWPP-Z SEQ ED NO. 8 WPWWPWPP-Z SEQ D3 NO. 9 PWWPWPP-Z SEQ D3 NO. 10 WWPWPP-Z SEQ ED NO. 11 ILPWKWPWWPWP -z and SEQ ED NO. 13 ILPWKWPWW -Z The invention further concerns a method for preventing, or attenuating the severity of, a retroviral infection, which comprises providing to an individual having such infection, an effective amount of an anti-retroviral pharmaceutical composition comprising a peptide having the formula:

X₂ wherein X₂ is absent, or Xj and X₂ are independently selected peptides having the formula:

Aaι-Aa₂-Aa₃-Aa -Aa₅-Aa6-Aa₇-W-W-Aa₈-Aa -Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa is W or absent; Aas is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa₉, Aaio, and Aan are all absent, Aa₈ will also be absent; wherein each such peptide is at least 6 amino acid residues in length; wherein J is a linker, and wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; wherein Z is the terminal carboxyl group of the peptide and is independently either unmodified or modified; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein the peptide is present in the composition at a concentration that is effective for preventing, or attenuating the severity of, a retroviral infection.

The invention particularly concerns the embodiment of such method, wherein the peptide is selected from the group consisting of: SEQ ED NO.2 I PWKWPWWPWPP-Z SEQ ED NO.3 I AWKWAWWAWPP-Z SEQ ED NO.4 LPWKWPWWPWPP-Z SEQ ED NO.5 PWKWPWWPWPP-Z SEQ ED NO.6 WKWPWWPWPP-Z SEQ ID NO.7 KWPWWPWPP-Z SEQ ED NO.8 WPWWPWPP-Z SEQ ID NO.9 PWWPWPP-Z SEQ ID NO.10 WWPWPP-Z SEQ ED NO.11 ILPWKWPWWPWP -z and SEQ ED NO.13 ILPWKWPWW -Z

The invention also concerns the embodiments of such method, wherein J is a lysine or arginine amino acid residue, and/or wherein the peptide has the formula: SEQ ED NO. 14: (ILPWKWPWWPWPP) ₂K-Z .

The invention also concerns the embodiments of such method, wherein the terminal carboxyl group Z of the peptide is modified, the modification being amidation.

The invention also concerns a method for preventing, or attenuating the severity of, a retroviral infection, which comprises providing to an individual having such infection, an effective amount of an anti-retroviral pharmaceutical composition comprising a peptide having the formula:

wherein X₂ is absent, or Xi and X₂ are independently selected peptides having the formula: Aaι-Aa₂-Aa₃-Aa -Aa₅-Aa₆-Aa₇-W-W-Aas-Aa₉-Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aai is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, wherein each such peptide X] and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein the peptide is present in the composition at a concentration that is effective for preventing, or attenuating the severity of, a retroviral infection.

The invention particularly concerns the embodiment of such method, wherein the peptide is selected from the group consisting of: SEQ ID NO. 2 ILPWKWPWWPWPP-Z SEQ ED NO. 3 I L AWKWAWW W P P - Z SEQ ED NO. 4 LPWKWPWWPWPP-Z SEQ ED NO. 5 PWKWPWWPWPP-Z SEQ ED NO. 6 WKWPWWPWPP-Z SEQ ED NO. 7 KWPWWPWPP-Z SEQ ID NO. 8 WPWWPWPP-Z SEQ ED NO. 9 PWWPWPP-Z SEQ ED NO. 10 WWPWPP-Z SEQ ED NO. 11 ILPWKWPWWPWP -Z ; and SEQ ID NO. 13 ILPWKWPWW -Z The invention also concerns the embodiments of such method, wherein J is a lysine or arginine amino acid residue, wherein n is 2, wherein Z is a lysine or arginine amino acid residue, and/or wherein the peptide comprises the formula: SEQ ED NO. 18: ( ( ILPWKWPWWPWPP ) ₂K) ₂-K. The invention also concerns a method for preventing, or attenuating the severity of, a retroviral infection, which comprises providing to an individual having such infection, an effective amount of an anti-retroviral pharmaceutical composition comprising a peptide having the formula:

wherein X₂ is absent, or X_\ and X₂ are each peptides that are independently selected having the formula: Aai -Aa₂-Aa₃-Aa₄-Aa5-Aa6-Aa₇-W-W-Aa₈-Aa₉-Aaι ₀-Aaι ι ; wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent; wherein each such peptide, Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J and at least one reactive group capable of bonding to B; wherein m is an integer greater than 0; wherein B is a substituent having at least m reactive groups, each capable of bonding to Z; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein the peptide is present in the composition at a concentration that is effective for preventing or attenuating the severity of, a retroviral infection.

The invention particularly concerns the embodiment of such method, wherein the peptide is selected from the group consisting of: SEQ ED NO. 2 ILPWKWPWWPWPP-Z SEQ ED NO. 3 ILAWKWAWWAWPP-Z SEQ ED NO. 4 LPWKWPWWPWPP-Z SEQ ED NO. 5 PWKWPWWPWPP-Z SEQ ED NO. 6 WKWPWWPWPP-Z SEQ ED NO. 7 KWPWWPWPP-Z SEQ ED NO. 8 WPWWPWPP-Z SEQ ED NO. 9 PWWPWPP-Z SEQ ED NO. 10 WWPWPP-Z SEQ ED NO. 11 ILPWKWPWWPWP -Z ; and SEQ ED NO. 13 I L PWKW PWW - Z .

The invention also concerns the embodiments of such method, wherein J is a lysine or arginine amino acid residue, wherein n is 2, wherein Z is a lysine or arginine amino acid residue, and/or wherein the peptide comprises the formula: SEQ ED NO. 18: ( ( ILPWKWPWWPWPP ) ₂K) ₂-K.

The invention particularly concerns the embodiments of such compositions and methods, wherein the retroviral infection is feline immunodeficiency virus (FIV), or human immunodeficiency virus (HIV).

The invention is also particularly directed to the embodiments of all such methods wherein the retroviral infection is an infection of HIV.

Brief Description of the Figures: Figure 1 shows that the Strand Transfer (ST) step leads to higher and lower molecular weight species migrating slower and faster respectively, than the original 21-mer substrate. Figure 2 shows a typical experiment corresponding to the inhibition of

HIV-1 integrase by increasing concentration of peptide SEQ ID NO. 2 in the presence of MgCl₂ reveals an IC5₀ of 2 μM for the 3' end processing (3'-P) and strand transfer (ST) reactions.

Figure 3 shows the synthesis of a dimeric peptide; i: 1. HBTU, HOBt, DIPEA in NMP; 2. 20% piperidine in NMP, ii: 11 cycles of Fmoc SPPS with HBTU/HOBt/DIPEA coupling, iii: 1. 1% TIS, 2.5% EDT, 2.5% H₂0 in TFA (2 h, r.t).

Figure 4 shows the synthesis of a dimeric peptide. i: HBTU, HOBt, DIPEA in NMP, ii: 1. 20% piperidine in NMP; 2. 11 cycles of Fmoc SPPS with HBTU/HOBt/DIPEA coupling, iii: 1. 1% TIS, 2.5% EDT, 2.5% H₂0 in TFA (2 h, r.t.).

Figure 5 shows the circular dichroism (CD) spectra of peptides of Table 2. Shown are the CD spectra of peptides 2, 8, and 10 (solid lines), indolicidin (1, dashed line) and peptide 7 ( dotted line). Concentrations: 0.1 mM for 1, 2, 7, 0.05 mM for 8, and 0.025 mM for 10, in a TFE / water (4:1 vol.) mixture, spectra collected at 22 °C.

Figure 6 illustrates two multimeric constructs of the present invention.

Description of the Preferred Embodiments: The present invention relates to peptides and their derivatives that inhibit retroviral integrase and to their use in the treatment of retroviral infection. The invention particularly relates to the use of such molecules to prevent the onset of retroviral infection or to attenuate the severity of the symptoms of Acquired Immunodeficiency Syndrome (AIDS). As used herein, such "attenuation" is preferably of a magnitude sufficient to mediate a reduction of at least 50%, more preferably 60%, and still more preferably 80%, or more greater in the replication, propagation or transmission of HIV. As used herein, the term "treatment" of retroviral infection is intended to encompass the administration of a pharmaceutical agent prophylactically so as to prevent the onset of retroviral infection in an individual at risk of such infection and/or therapeutically, so as to attenuate the severity of the symptoms of an existing retroviral infection.

The present invention derives, in part, from the recognition that retroviral integrases provide a target for anti-retroviral therapy, and that peptide molecules having a sequence related to that of indolicidin are able to inhibit retroviral integrases. Indolicidin is a naturally occurring linear peptide produced in the granules of bovine neutrophils (Staubitz P. et al. (2001) "STRUCTURE-FUNCTION RELATIONSHIPS IN THE TRYPTOPHAN-RICH, ANTIMICROBIAL PEPTIDE INDOLICIDIN," J

Pept Sci. 7(10):552-564; Selsted, M.E. et al. (1992) "INDOLICIDIN, A NOVEL BACTERICIDAL TRIDECAPEPTIDE AMIDE FROM NEUTROPHILS," J. Biol. Chem. 267(7):4292-4295). Indolicidin has a unique composition that consists of five tryptophan residues, three proline residues, and three basic amino acid residues (Osapray K. et al. (2000) "FORMATION AND CHARACTERIZATION OF A SINGLE TRP-

TRP CROSS-LINK IN INDOLICIDIN THAT CONFERS PROTEASE STABILITY WITHOUT ALTERING ANTIMICROBIAL ACTIVITY," J. Biol. Chem. 275 (16):12017-12022). The carboxyl terminus of indolicidin is naturally amidated (Lee DG, et al. (2003) "FUNGICIDAL EFFECT OF INDOLICIDIN AND ITS INTERACTION ITH PHOSPHOLIPID MEMBRANES," Biochem. Biophys. Res. Com. 305:305-310). Indolicidin possesses the sequence: SEQ ED NO. 1: ILPWKWPWWPWRR-NH₂

Because of its small size and potent antimicrobial activity, indolicidin has been considered as a possible therapeutic agent. The use of indolicidin and certain indolicidin derivatives to provide antimicrobial activity against HIV has been described (Selsted et al, U.S. Patent No. 6,303,575). Indolicidin's antimicrobial activities are generally thought to be related to membrane-disruptive properties of the peptide; the peptide is believed to be capable of mediating cell lysis through the dissolution of the membrane and the forming of membrane pores (Osapray K. et al.

(2000) "FORMATION AND CHARACTERIZATION OF A SINGLE TRP-TRP CROSS-LINK IN INDOLICIDIN THAT CONFERS PROTEASE STABILITY WITHOUT ALTERING ANTIMICROBIAL ACTIVITY," J. Biol. Chem. 275 (16): 12017-12022; Zhao H. et al.

(2001) "COMPARISON OF THE MEMBRANE ASSOCIATION OF Two ANTIMICROBIAL PEPTIDES, MAGAININ 2 AND INDOLICIDIN," Biophys J. 81:2979-2991). The present invention concerns indolicidin and peptides whose sequences are variants of the sequence of indolicidin (SEQ ID NO. 1: ILPWKWPWWPWRR- NH₂). In preferred embodiments, such peptides have the sequence: Aaι-Aa₂-Aa₃-Aa -Aa5-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i -Z; wherein "Aa" denotes an amino acid residue, wherein such peptide is at least 6 amino acid residues in length, and wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; A^ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, and wherein Z is the terminal carboxyl group of the peptide and is either unmodified or modified. Preferably the terminal carboxyl group of the peptide will be modified; the most preferred modification being amidation. Preferably, when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa9, Aaio, and Aan are all absent, Aa₈ will also be absent.

The present invention additionally concerns multimers and branched aggregates of the above peptides, such as those of the formula:

X₂ wherein X is absent, or Xi and X₂ are independently selected peptides having the formula: Aai -Aa₂-Aa₃-Aa₄-Aa5-Aa₆-Aa₇-W-W-Aas-Aa₉-Aaιo-Aaι i . wherein, subject to the above-indicated definitions of Aai through Aan, the amino acid sequences of Xi and X₂ are independently selected; and J is a linker, and wherein Z is an atom or terminal group of J, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J.

Preferably, J is an amino acid residue having functional groups that are capable of bonding to Xi and/or to X] and X₂. Preferably, J is either arginine or lysine.

The multimers and branched aggregates of the above peptides include those having the formula:

wherein X₂ is absent, or Xi and X₂ are independently selected peptides having the formula: Aaι-Aa₂-Aa3-Aa -Aa5-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i .

wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aai is W or absent; Aa₅ is K or absent; Aaβ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, wherein each such peptide Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or terminal group of J, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J.

J may comprise any moiety having 1, 2, 3 or more functional groups that may be employed to bond to a peptide molecule. Examples of suitable J substituents include amino acid residues (e.g., one or more lysine, arginine, glutamine, aspartate, glutamate, etc. residues), binary-, tertiary- or poly- amines, binary, tertiary or poly-carboxylic acids, etc. In a preferred embodiment J, is an amino acid, especially lysine, arginine, glutamine, aspartate or glutamate, and Z is a terminal group thereof. In a preferred embodiment, n will be 1, 2, 3, 4, 5, or more. Where n>l, the multiple [Xι-(X )-J] substituents be bound to the same atom of Z, or to up to n different atoms of Z.

The multimers and branched aggregates of the above peptides may additionally include those having the formula:

wherein X₂ is absent, or Xi and X₂ are each peptides that are independently selected having the formula:

Aai -Aa₂-Aa₃-Aa₄-Aa5-Aa6-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i ;

wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent; wherein each such peptide, Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or group of J, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J and at least one reactive group capable of bonding to B; wherein m is an integer greater than 0; wherein B is a substituent having at least m reactive groups, each capable of bonding to Z.

Substituent B may be a multivalent molecule, a polymer, membrane, solid support, absorbent matrix, gel, foam, etc. In a preferred embodiment, J is an amino acid residue, especially a lysine, arginine, glutamine, aspartate or glutamate amino acid residue, and Z is a terminal group thereof. The Xi, X₂, J and Z substituents of such formula may be identical or may vary independently of one another. In a preferred embodiment, m will be 1, 2, 3, 4, 5, or more. Where m>l, the multiple [[Xι-(X₂)-J]-Z] substituents may be bound to the same atom of B, or to up to m different atoms of B. In one embodiment, substituent B is a condom or vaginal insert, to which the bonded [[Xι-(X₂)-J]Z] substituent(s) impart a microcidal activity, such microcidal activity being sufficient to prevent or attenuate the infectivity or transmissibility of HIV.

Substituents J, Z or B may independently be selected to be stable under aqueous or physiological conditions or to be degradable or erodible under such conditions. In an alternate embodiment, the bond(s) between substituent J and Xi (and/or X₂) will be stable under aqueous or physiological conditions. In a further embodiment, such bond(s) will be labile or cleavable under such conditions so as to effect the release of Xi (and/or X₂) from J. Likewise, the invention also contemplates that the bond(s) between any or all substituents J and Z, and/or between any or all substituents Z and B may be either stable under aqueous or physiological conditions. The use of labile or cleavable bonds permits the formulations of the present invention to serve as prolonged drug delivery vehicles for Xi (and/or X₂). Suitable labile or cleavable bonds may include ionic bonds, bonds that can be cleaved in response to enzymic (e.g., protease) action, pH changes (see, e.g., U.S. Patent No. 6,630,351), etc.

More preferably, the present invention concerns indolicidin and peptides whose sequences are variants of the seqence of indolicidin (SEQ ED NO. 1: ILPWKWPWWPWRR-NH₂) having the sequence: SEQ ID NO. 1 ILPWKWPWWPWRR- [NH₂ ] SEQ ID NO. 2 ILPWKWPWWPWPP- [NH₂ ] SEQ ID NO. 3 ILAWKWAWWAWPP- [NH₂ ] SEQ ID NO. 4 LPWKWPWWPWPP- [NH₂ ] SEQ ID NO. 5 PWKWPWWPWPP- [NH₂ ] SEQ ID NO. 6 WKWPWWPWPP- [NH₂ ] SEQ ID NO. 7 KWPWWPWPP- [NH₂ ] SEQ ID NO. 8 WPWWPWPP- [NH₂ ] SEQ ID NO. 9 PWWPWPP- [NH₂ ] SEQ ID NO. 1( ) : WWPWPP- [NH₂ ] SEQ ID NO. 11 L : ILPWKWPWWPWP - [NH₂ ] SEQ ID NO. Ω ! : ILPWKWPWWP [NH₂ ] SEQ ID NO. 12 S : ILPWKWPWW [NH₂ ] in which [NH2] denotes the optional (and preferred) amidation of the carboxy terminus of the peptide. Such peptides are preferred Xi and X₂ peptides. Still more preferably, the present invention concerns peptides whose sequences are variants of the sequence of indolicidin (SEQ ID NO. 1:

ILPWKWPWWPWRR-NH₂) having the sequence: SEQ ID NO. 2 ILPWKWPWWPWPP- [NH₂ ] SEQ ID NO. 3 ILAWKWAWWAWPP- [NH₂ ] SEQ ID NO. 4 LPWKWPWWPWPP- [NH₂ ] SEQ ID NO. 5 PWKWPWWPWPP- [NH₂ ] SEQ ID NO. 6 WKWPWWPWPP- [NH₂ ] SEQ ID NO. 7 KWPWWPWPP- [NH₂ ] SEQ ID NO. 8 WPWWPWPP- [NH₂ ] SEQ ID NO. 9 PWWPWPP- [NH₂ ] SEQ ID NO. 1( ) : WWPWPP- [NH₂ ] SEQ ID NO. 11 L : ILPWKWPWWPWP - [NH₂ ] SEQ ID NO. 12 5 : ILPWKWPWW [NH₂ ] in which [NH₂] denotes the optional (and preferred) amidation of the carboxy terminus of the peptide. Such peptides are more highly preferred Xi and X₂ peptides.

Such peptides can be obtained in any of a variety of ways: through purification and derivatization of indolicidin; via peptide synthesis, via recombinant means, etc. Preferably, such peptides are synthesized via solid phase peptide synthesis with Fmoc-chemistry and with Fmoc-amino acid derivatives on a peptide synthesizer. Upon formation of the necessary sequence, the peptides are cleaved from the solid phase resin. If desired, in order to achieve a desired purity, the synthesized peptides may be washed and purified, e.g., via preparative RP HPLC, etc. The purity of peptides produced in this manner may exceed 90%. Molecular masses of all peptides may be verified by MALDI-TOF-MS spectral analysis.

Any of a variety of methods can be used to obtain the carboxy amidated forms of the above-described peptides. For example, carboxy amidation can be accomplished through the use of recombinant enzymes or by expression in the milk of transgenic animals. Methods employing recombinant enzymes are appropriate for small-scale production, whereas transgenic milk expression is particularly suitable suitable for making complex disulfide-containing peptides required in large quantity. (Cottingham, I.R. et al. (2001) "A METHOD FOR THE AMIDATION OF RECOMBINANT PEPTIDES EXPRESSED AS INTEΓN FUSION PROTEINS IN ESCHERICHIA con," Nat Biotechnol. 19(10):974-7).

Pharmaceutical Compositions of the Present Invention One or more of the peptides of the present invention, either alone or with other active agents may be used to prepare pharmaceutical compositions for treating retroviral infection, and in particular for inhibiting the functioning of retroviral integrase enzymes. Such compositions may be used to treat HIV (especially HIV-1) infection, FIV (Feline Immunodeficiency Virus) and/or SIV (Simian Immunodeficiency Virus).

The pharmaceutical composition of the present invention may be in the form of an emulsion, gel, solution, suspension, etc. In addition, the pharmaceutical composition can also contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives. Administration of pharmaceutically acceptable salts described herein is preferred. Such salts can be prepared from pharmaceutically acceptable non-toxic bases including organic bases and inorganic bases. Salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, basic amino acids, and the like. Preferred salts include but are not limited to sodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, sodium pyruvate, potassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, potassium pyruvate, disodium DL-α-glycerol-phosphate, and disodium glucose-6-phosphate. "Phosphate" salts of sodium or potassium can be either the monobasic form, e.g., NaHP0₄, or the dibasic form, e.g., Na₂HP0₄, but a mixture of the two, resulting in a desired pH, is most preferred.

As used herein a "salt" is a substance produced from the reaction between acids and bases which comprises a metal (cation) and a nonmetal (anion). Salt crystals may be "hydrated" i.e., contain one or more water molecules. Such hydrated salts, when dissolved in an aqueous solution at a certain molar concentration, are equivalent to the corresponding anhydrous salt dissolved in an aqueous solution at the same molar concentration. For the present invention, salts which are readily soluble in an aqueous solution are preferred.

Further, the pharmaceutical composition may be prepared in the form of admixture with one or more pharmaceutically acceptable excipients so long as such additional excipients do not interfere with the effectiveness of the peptides and the side effects and adverse reactions are not increased additively or synergistically. The pharmaceutical compositions of the present invention can be associated with chemical moieties which may improve the composition's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the pharmaceutical compositions, eliminate or attenuate any undesirable side effect of the pharmaceutical compositions, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 19^th Edition, A. R.

Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995). Procedures for coupling such moieties to a molecule are well known in the art.

As used herein a pharmaceutical "excipient" is a substance other than the pharmacologically active drug or prodrug which is included in the manufacturing process or are contained in a finished pharmaceutical product dosage form. Some, for example, comprise the product's delivery system. In the preferred embodiment pharmaceutical excipients transport the active drug to the site in the body where the drug is intended to exert its action. In more preferred embodiment, excipients will keep the drug from being released too early in the assimilation process in places where it could damage tender tissue and create gastric irritation or stomach upset. In even more preffered embodiment, excipients will help the drug to disintegrate into particles small enough to reach the blood stream more quickly and still others protect the product's stability so it will be at maximum effectiveness at time of use. In order to improve patient compliance, these excipients can be used simply to make the pharmaceutical composition taste and look better (International Pharmaceutical Excipients Council of the Americas; http://www.ipecamericas.org/public/faqs).

Suitable excipients include Magnesium Stearate, Lactose, Microcrystalline Cellulose, Starch (corn), Silicon Dioxide, Titanium Dioxide, Stearic Acid, Sodium Starch Glycolate, Gelatin, Talc, Sucrose, Calcium Stearate, Povidone,

Pregelatinized Starch, Hydroxy Propyl Methylcellulose, OPA products (coatings & inks), Croscarmellose, Hydroxy Propyl Cellulose, Ethylcellulose, Calcium Phosphate (dibasic), Crospovidone, Shellac (and Glaze).

Administration of the Pharmaceutical Compositions of the Present Invention The pharmaceutical compositions of the present invention may be administered by any suitable means, for example, inhalation, or interdermally, intracavity (e.g., oral, vaginal, rectal, nasal, peritoneal, ventricular, or intestinal), intradermally, intramuscularly, intranasally, intraocularly, intraperitoneally, intrarectally, intratracheally, intravenously, orally, subcutaneously, transdermally, or transmucosally (i.e., across a mucous membrane) in a dose effective for the production of neutralizing antibody and resulting in protection from infection or disease. The present pharmaceutical compositions can generally be administered in the form of a spray for intranasal administration, or by nose drops, inhalants, swabs on tonsils, or a capsule, liquid, suspension or elixirs for oral administration. The pharmaceutical compositions may be in the form of single dose preparations or in multi-dose flasks. Reference is made to Remington's Pharmaceutical Sciences, 19^th Edition, A. R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995). Administration can be into one or more tissues including but not limited to muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, e.g., myocardium, endocardium, and pericardium; lymph nodes, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, or connective tissue. Furthermore, in the methods of the present invention, the pharmaceutical compositions may be administered to any internal cavity of a mammal, including, but not limited to, the lungs, the mouth, the nasal cavity, the stomach, the peritoneal cavity, the intestine, any heart chamber, veins, arteries, capillaries, lymphatic cavities, the uterine cavity, the vaginal cavity, the rectal cavity, joint cavities, ventricles in brain, spinal canal in spinal cord, and the ocular cavities. Administration may be by needle injection, catheter infusion, biolistic injectors, particle accelerators (e.g., pneumatic "needleless" injectors), gelfoam sponge depots, other commercially available depot materials (e.g., hydrogels), osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, topical skin creams, and decanting, use of polynucleotide coated suture (Qin, J.Y. et al. (1999) "GENE SUTURE-A NOVEL METHOD FOR INTRAMUSCULAR GENE TRANSFER AND ITS APPLICATION IN HYPERTENSION THERAPY," Life Sciences 65:2193-2203) or topical applications during surgery. Any mode of administration can be used so long as the mode results prophylactic or therapeutic efficacy. Methods to detect such a response include serological methods, e.g., western blotting, staining tissue sections by immunohistochemical methods, and measuring the activity of the polypeptide.

In one embodiment, DNA compositions will be used to provide the preferred peptides of the present invention. Pharmaceutical DNA compositions and methods for their manufacture and delivery that may be used in accordance with the present invention are disclosed in US Patents Nos. 5,589,466; 5,620,896; 5,641,665; 5,703,055; 5,707,812; 5,846,946; 5,861,397; 5,891,718; 6,022,874; 6,147,055; 6,214,804; 6,228,844; 6,399,588; 6,413,942; 6,451,769, European Patent Documents EP1165140A2; EP1006796A1 and EP0929536A1; and PCT Patent Publications WO00/57917; WO00/73263; WO01/09303; WO03/028632; W094/29469; WO95/29703; and W098/14439.

The compositions of the present invention can be lyophilized to produce pharmaceutical compositions in a dried form for ease in transportation and storage. The pharmaceutical compositions of the present invention may be stored in a sealed vial, ampoule or the like. In the case where the pharmaceutical composition is in a dried form, the composition is dissolved or suspended (e.g., in sterilized distilled water) before administration. An inert carrier such as saline or phosphate buffered saline or any such carrier in which the pharmaceutical compositions has suitable solubility, may be used.

The pharmaceutical compositions can be solubilized in a buffer prior to administration. Suitable buffers include, for example, phosphate buffered saline (PBS), normal saline, Tris buffer, and sodium phosphate vehicle (100-150 mM preferred). Insoluble polynucleotides can be solubilized in a weak acid or base, and then diluted to the desired volume with a neutral buffer such as PBS. The pH of the buffer is suitably adjusted, and moreover, a pharmaceutically acceptable additive can be used in the buffer to provide an appropriate osmolarity within the lipid vesicle. Preferred salt solutions and auxiliary agents are disclosed herein.

Compositions used in of the present invention can be formulated according to known methods. Suitable preparation methods are described, for example, in Remington's Pharmaceutical Sciences, 19^th Edition, A. R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995), incorporated herein by reference in its entirety. Although the composition is preferably administered as an aqueous solution, it can be formulated as an emulsion, gel, solution, suspension, lyophilized form, or any other form known in the art. According to the present invention, if the composition is formulated other than as an aqueous solution, it will require resuspension in an aqueous solution prior to administration. Such compositions may be formulated into any of the various compositions and may be used in any of the methods disclosed herein. For aqueous compositions used in vivo, use of sterile pyrogen-free water is preferred. Such formulations will contain an effective amount of such peptide together with a suitable salt and/or pharmaceutically acceptable excipient as disclosed herein, in order to prepare pharmaceutically acceptable compositions suitable for optimal administration to a vertebrate.

The effective amount of a peptide, or a pharmaceutically acceptable salt thereof included in a pharmaceutical composition depends on factors including the age and weight of the subject, the delivery method and route, the type of treatment desired, and the type of peptide or composition being administered. In general, a pharmaceutical composition of the present invention that includes peptides or peptide compositions will contain from about 1 ng to about 30 mg of such peptides or peptide compositions, more preferably, from about 100 ng to about 10 mg of such peptides or peptide compositions. Certain preferred compositions of the present invention may include about 1 ng of such peptides or peptide compositions, about 5 ng of such peptides or peptide compositions, about 10 ng of such peptides or peptide compositions, about 50 ng of such peptides or peptide compositions, about 100 ng of such peptides or peptide compositions, about 500 ng of such peptides or peptide compositions, about 1 μg of such peptides or peptide compositions, about 5 μg of such peptides or peptide compositions, about 10 μg of such peptides or peptide compositions, about 50 μg of such peptides or peptide compositions, about 100 μg of such peptides or peptide compositions, about 150 μg of such peptides or peptide compositions, about 200 μg of such peptides or peptide compositions, about 250 μg of such peptides or peptide compositions, about 300 μg of such peptides or peptide compositions, about 350 μg of such peptides or peptide compositions, about 400 μg of such peptides or peptide compositions, about 450 μg of such peptides or peptide compositions, about 500 μg of such peptides or peptide compositions, about 550 μg of such peptides or peptide compositions, about 600 μg of such peptides or peptide compositions, about 650 μg of such peptides or peptide compositions, about 700 μg of such peptides or peptide compositions, about 750 μg of such peptides or peptide compositions, about 800 μg of such peptides or peptide compositions, about 850 μg of such peptides or peptide compositions, about 900 μg of such peptides or peptide compositions, about 950 μg of such peptides or peptide compositions, about 1 mg of such peptides or peptide compositions, about 5 mg of such peptides or peptide compositions, about 10 mg of such peptides or peptide compositions, about 15 mg of such peptides or peptide compositions, about 20 mg of peptides or peptide compositions, about 25 mg of such peptides or peptide compositions, or about 30 mg of such peptides or peptide compositions. In one embodiment of the present invention, alternatively, or conjunctively, one or more of the above-described pharmaceutical compositions will comprise a peptide or peptide composition in admixture with one or more pharmaceutically acceptable excipients that may be administered to a recipient prior to the commencement of HIV infection, or subsequent to the onset of such infection. In accordance with the methods of the present invention, a single antiretroviral pharmaceutical composition, peptide or peptide composition containing more than one peptide sequence may be administered. Alternatively, more than one peptide, peptide composition, pharmaceutically acceptable salt thereof or pharmaceutically acceptable composition may be co-administered or sequentially administered.

The present invention also provides kits for use in treating retroviral infection comprising an administration means and a container means containing a pharmaceutical composition of the present invention. Preferably, the container in which the composition is packaged prior to use will comprise a hermetically sealed container enclosing an amount of the lyophilized formulation or a solution containing the formulation suitable for a pharmaceutically effective dose thereof, or multiples of an effective dose. The composition is packaged in a sterile container, and the hermetically sealed container is designed to preserve sterility of the pharmaceutical formulation until use. Optionally, the container can be associated with administration means and/or instruction for use.

Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified. Example 1 Synthesis and Purification of Indolicidin Peptide Derivatives

Indolicidin peptide derivatives are synthesized by Solid Phase Peptide Synthesis and assembled on an ABI 9-fluorenylmethoxycarbonyl (Fmoc)-amide resin on 0.6 mmol/g scale. All amino acids are coupled by O-Benzotriazole- N,N,N',N'-tetramthyl-uronium-hexafluoro-phosphate/ 6-Chloro- 1 -Hydroxy- lBenzotriazole/ N,N-diisopropyIethylamine (HBTU/HOBt/DIPEA) activation in N-methylpyrrolidone (NMP), utilizing AB1433A peptide synthesizer. The following 9-fluorenylmethoxycarbonyl (Fmoc) and t-butoxycarbonyl (Boc) protecting groups are used for amino acids before coupling: Fmoc-Ala-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Pro-OH and Fmoc-Trp (Boc)- OH, Fmoc-Lys (Fmoc)-OH.

After assembly of the peptide chain, the resin is washed 3 times with dichloromethane (DCM) and 6 times with methanol, then dried for 24 h in vacuo. Newly synthesized indolicidin peptide derivatives are cleaved from the resin and side chain is deprotected with a mixture of 2.5% 1,2-ethanedithiol (EDT), 2.5% water, 1% triisopropylsilane (TIS) in trifluoroacetic acid (TFA) (1.5 ml/100 mg resin) for 2 h. A solution is filtered into 20 ml of a cold ethanol. After 30 min at - 10°C, the precipitate is separated by centrifugation and washed 4 times with ethanol. The precipitate is then dried under vacuum for 1 h over KOH pellets. The peptide is dissolved in a water/acetonitrile mixture with 0.05% TFA. The solution is kept at room temperature for 6 h frozen and lyophilized.

The crude peptides are purified by preparative reverse phase-high performance liquid chromatography (RP HPLC). The peptides are analyzed on a Vydac C , C₈ or Cis column using water-acetonitrile solvents in water containing 0.05% TFA. The purity of all peptides is between 90 and 100%, (RP HPLC on C₈ and C columns). Molecular mass is determined by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF) mass spectrometry. The observed monoisotopic masses are in good agreement with theoretical ones. For example: analytical data for SEQ ED NO. 2:

ILPWKWPWWPWPP-NH₂) is as follows: Analytical HPLC (Vydac C₈ column, gradient: from 20%> to 70% in 25 min): l peak, HPLC retention time: 15.8 min MALDI-TOF-MS: 1787.7 (1787.95 M+H⁺). The following peptides are produced:

SEQ ID NO. 1 ILPWKWPWWPWRR-NH₂ SEQ ID NO. 2 ILPWKWPWWPWPP-NH₂ SEQ ED NO. 3 ILAWKWAWWAWPP-NH₂ SEQ ID NO. 4 LPWKWPWWPWPP-NH₂ SEQ ID NO. 5 PWKWPWWPWPP-NH₂ SEQ ID NO. 6 WKWPWWPWPP-NH₂ SEQ ID NO. 7 KWPWWPWPP-NH₂ SEQ ID NO. 8 WPWWPWPP-NH₂ SEQ ID NO. 9 PWWPWPP-NH₂ SEQ ED NO. 10 WWPWPP-NH SEQ ID NO. 11 ILPWKWPWWPWP -NH₂ SEQ ED NO. 12 ILPWKWPWWP -NH₂ SEQ ID NO. 13 ILPWKWPWW -NH₂

Example 2 Inhibition Assay on HIV-1 replication in T-Iymphoid cell Virus Stock Preparation. 293T cells (ATCC) are maintained in the presence of Dulbecco's Modified Eagle's Medium (Cellgro), 10% fetal calf serum (HyClone Laboratories), penicillin (50 U/ml; Gibco) and streptomycin (50 μg/ml; Gibco) and plated at density of 5 x 10 ⁶ per 100-mm-diameter culture dish. After overnight incubation in the incubator at 37° C containing 5% C0₂, cells are transfected with a mixture of 10 μg plasmid HIV-1 NL4-3 (env^"luc⁺) (see, Sakamoto, T. et al. (2003) "ESTABLISHMENT OF AN HIV CELL-CELL FUSION ASSAY BY USING TWO GENETICALLY MODIFIED HELA CELL LINES AND REPORTER GENE," J Virol Methods 114(2): 159-66; Fikkert, V. et al. (2002) "ENV CHIMERIC VIRUS TECHNOLOGY FOR EVALUATING HUMAN IMMUNODEFICIENCY VIRUS SUSCEPTIBILITY TO ENTRY INHIBITORS," Antimicrob Agents Chemother. 46(12): 3954-62; Erratum in: Antimicrob Agents Chemother. 2003 Mar;47(3):1177). This construct expresses the HIV_NL4-₃ provirus (Connor RI, et al. (1996)

"CHARACTERIZATION OF THE FUNCTIONAL PROPERTIES OF ENV GENES FROM LONG-TERM SURVIVORS OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 INFECTION," Journal of Virology 70(8)5306-5311; Connor RI, et al. (1997) "CHANGE IN CORECEPTOR USE CORRELATES WITH DISEASE PROGRESSION IN HIV- 1-INFECTED INDIVIDUALS," J. Exp. Med.l85(4):621-628; Popik W, et al. (2002) "HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 USES LIPID RAFT-COLOCALIZED CD4 AND CHEMOKINE RECEPTORS FOR PRODUCTIVE ENTRY INTO CD4+ T CELLS," Journal of Virology 76(10) 4709-4722) and 1 g (packaging construct), vesicular stomatitis virus glycoprotein envelope (pCMV VSV-G) (Yee et al. (1994) "A GENERAL METHOD FOR THE GENERATION OF HIGH-TITER, PANTROPIC RETROVIRAL VECTORS - HIGHLY EFFICIENT INFECTION OF PRIMARY HEPATOCYTES" Proc. Natl. Acad. Sci. (U.S.A.) 91:9564-9568) using MBS Mammalian Transfection Kit (Stratagene). Cell culture supernatant is replaced with fresh medium 4 hours after transfection and further cultured for a total of 48 hours. Thereafter, cell supernatants containing HIV-1 virus are harvested, clarified and were used in inhibition assay.

Inhibition assay on HIV-1 replication in T-Iymphoid cell. CEM x 174 T- lymphoid cells (ATCC) are maintained in the presence of RPMI 1640 medium, 10%) fetal calf serum, penicillin (50 U/ml) and streptomycin 50 (μg/ml). Cells are plated into 96-well flat bottom culture plate (Costar) at a concentration of 1 x 10⁵ per well. Inhibitors diluted to different concentrations are added to cells (three cultures each) and incubated for one hour.

Subsequently, HIV-1 virus giving about 5 x 10⁴ relative light units of luciferase signal is added and cultured for a total of 48 hours. Ten wells in each 96-well plate are set aside as negative and positive controls. For the negative control, five wells with cells that do not contain the HIV-1 integrase inhibitor or the HIV-1 virus were used. For the positive control, five wells with cells containing HIV-1 virus are used. Infected cells in a volume of 250 μl are lysed by adding 60 μl of 5 times concentrated reporter lysis buffer (Promega) for 10 minutes at room temperature and subjected to one freeze-thaw cycle. Luciferase activity is measured following addition of 100 μl of substrate (Promega) to 20 μl of cell lysate using TD20/20 Luminometer (Promega). Concentrations of compounds that resulted in 50% inhibition of HIV-1 infection (ICsos) is determined as compared with the negative and positive controls included in the assay.

Example 3 In vitro HIV-1 recombinant integrase assay Indolicidin peptide derivatives are tested for their ability to inhibit recombinant HIV-1 integrase in vitro. In the assay, a 21-mer double-stranded DNA oligonucleotide (oligonucleotide *A 5 '-end-labeled annealed to oligonucleotide B), corresponding to SEQ ID NO. 15 , is used to follow both 3'- processing (3'-P) and strand transfer (ST) steps of the integration reaction.

"A" is SEQ ID NO.15: GTGTGGAAAA TCTCTAGCAG T "B" is SEQ ID NO.16: ACTGCTAGAG ATTTTCCACA C

Oligonucleotide A is 5 '-end-labeled by T4-Polynucleotide Kinase (Invitrogen, Carlsbad, CA). 10 pmoles of oligonucleotide A is incubated at 37°C for 30 min in 50 μl of IX kinase buffer containing 10 μCi of γ-ATP (Amersham Pharmacia Biotech, Piscataway, NJ) and 10 units of kinase. The labeling solution is then applied to the top of a G25 Quick Spin column (Boehringer Mannheim, Indianapolis, IN) and the filtrate is annealed with 20 pmoles of oligonucleotide B (complementary strand) for 5 min at 95°C and 30 min at 37°C.

In the 3'-P reaction, integrase liberates a GT dinucleotide at the 3 '-end of the labeled strand resulting in the generation of a 19-mer labeled product. The strand transfer (ST) reaction consists of the insertion of a 3'-processed oligonucleotide into another DNA target. This ST step leads to higher and lower molecular weight species migrating slower and faster respectively, than the original 21-mer substrate (Figure 1). The higher molecular weight species (STP) are generally used to evaluate strand transfer. For inhibitor testing, a peptide-enzyme complex is preformed by mixing

400 nM of HIV-1 integrase and the peptide at the desired concentration in a buffer containing 50 mM MOPS, pH 7.2, 7.5 mM MnCl₂ or MgCl₂ and 14.3 mM 2- mercaptoethanol for 15 min at room temperature. The integration reaction is then initiated by addition of 5 nM of 5'-labeled double-stranded DNA template (*A/B) and continued in a total volume of 10 μl for 60 min at 37°C. The reaction is terminated by the addition of the same volume of electrophoresis denaturing dye containing 99% formamide, 1%> SDS, 0.2 mg/ml bromophenol blue and 0.2 mg/ml xylene cyanol blue. Samples are loaded onto a 20%> 19/1 acrylamide denaturing gel Accugel (National Diagnostics, Atlanta, GA) containing 7 M urea in 1 X TBE. Gels are exposed overnight and analyzed using a Molecular Dynamics Phosphorimager (Sunnyvale, CA).

A typical experiment corresponding to the inhibition of HIV-1 by increasing concentration of peptide RJN 25 (Figure 2) in the presence of MgCl_2] reveals IC₅o values of 2 μM for the 3'-P and ST reactions. Therefore, RIN 25 inhibits both steps of the integration reaction in vitro with the same potency whereas, the two diketo acid derivatives currently in clinical evaluation are selective for ST inhibition. The IC₅₀ values obtained for the inhibition of HIV-1 integrase in vitro and replication are summarized in Table 1.

The above Examples demonstrate that retroviral integrases provide a target for anti-retroviral therapy, and that peptide molecules having a sequence related to that of indolicidin are able to inhibit retroviral integrases, and provide a method for treating retroviral infection. Example 4

Synthesis and HIV-1 Integrase Inhibitory Activity of Dimeric and Tetrameric Analogues of Indolicidin It has been previously reported that dimeric analogues of the hexapeptide (SEQ ED NO. 17) HCKFWW-NH₂ (Lutzke, R.A.P. et al. "IDENTIFICATION OF A HEXAPEPTIDE INHIBITOR OF THE HUMAN IMMUNODEFICIENCY VIRUS INTEGRASE PROTEIN BY USING A COMBINATORIAL CHEMICAL LIBRARY," Proc. Natl. Acad. Sci. USA 1995, 92, 11456) exhibited greater HIV-1 integrase inhibitory activity than the hexapeptide monomers (Krajewski, K. etal. (2003) "DESIGN AND SYNTHESIS OF DIMERIC HIV-1 INTEGRASE INHIBITORY PEPTIDES," Bioorg. Med. Chem. Lett. 13:3203-3205).

To investigate whether dimeric and higher order (e.g., tetrameric, octomeric, etc.) analogs of the peptides of the present invention exhibit enhanced inhibitory activity, dimeric analogues of such peptides are synthesized. Because there are no cysteine residues in such analogues, a linker residue was employed. Suitable linker residues include any moiety having 2, 3 or more functional groups that may be employed to bond to an analogue molecule. Examples of suitable moieties include amino acid residues (e.g., one or more lysine, arginine, glutamine, aspartate, glutamate, etc. residues), binary-, tertiary- or poly- amines, binary, tertiary or poly-carboxylic acids, etc. Lysine is a particularly preferred linking group, and is used as a linker for coupling the backbones of peptides (coupled at their carboxy ends to the two NH₂ groups of lysine (Figure 3). The peptides employed were analogues of SEQ ED NO. 2, so as to form a branched peptide derivative of formula:

SEQ ID NO: 18 ( ( ILPWKWPWWPWPP) ₂K) ₂-K The dimeric peptides are synthesized by an automated solid phase peptide synthesis (SPPS) (ABI 433A Peptide Synthesizer), using a Rink amide resin and a Fmoc chemistry (the coupling step with HBTU/HOBt/DIPEA in NMP). Because the peptides contain large number of tryptophan residues, a Fmoc Trp(Boc)-OH derivative is used for SPPS, to prevent unwanted side-reactions during peptide cleavage and deprotection. The method of synthesis of the dimeric peptide is shown in Figure 3. In this synthesis Fmoc-Lys(Fmoc)-OH is coupled to the Rink resin, and then, after removing the Fmoc protecting groups, additional amino acids are coupled to the two NH₂ groups of lysine. For the synthesis of the tetrameric peptide (Figure 4) a template is designed composed of three residues of lysine (tri-lysine template, [K]₂K). The tri- lysine template is synthesized by a coupling reaction of Fmoc Lys(Fmoc)-OH with a fully deprotected lysine bonded to the Rink resin. Then, after deprotection, additional amino acids are coupled to four NH₂ groups of tri-lysine template. After synthesis the peptide bound resins are washed six times with NMP,

DCM, and MeOH, then dried under reduced pressure overnight. The peptides are cleaved from the resin and fully deprotected by treatment with 1% TIS, 2.5%> EDT, 2.5%ι H20 in TFA (10 ml/g resin), after 2 h the mixture is filtered into cold diethyl ether. After 30 min at -10°C the precipitated peptides are separated by centrifugation, washed four times with diethyl ether, dried under reduced pressure (1 h over KOH), dissolved in water/acetonitrile 1:1 mixture, kept at r.t. (room temperature) 6 h (to complete tryptophan residues deprotection), and lyophilized.

The peptides are purified by preparative RP HPLC (Vydac C4 or Cl 8 column). Purity of all peptides is between 90% and 95% (RP HPLC on an analytical C8 column). MALDI TOF MS spectra (Kratos Axima-CFR instrument, matrix: α-cyjano-4-hydroxycinnaminic acid) verifies molecular masses of all peptides. The observed monoisotopic masses are in good agreement with theoretical masses (shown in parentheses): 1 1905.8 (1906.0 M+H⁺) Da; 2 1787.7 (1787.9 M+H⁺) Da; 3 1674.5 (1674.9 M+H*) Da; 4 1690.4 (1689.9 M+H⁺) Da; 5 1716.5 (1716.9 M+H⁺) Da; 6 1834.7 (1835.0 M+H⁺) Da; 7 1709.6 (1709.9 M+H⁺) Da; 8 3685.7 (3686.0 M+ϊt) Da; 9 3686.1 (3686.0 M+H⁺) Da; 10 7486.6 (an , average mass) (7487.0 M+H⁺, an average mass) Da.

HIV-1 integrase assays are performed as described by Marchand, C.et al. (1999) Methods Enzymol. 340:624, with the following modifications. The peptides are pre-incubated with 500 nM wild type (wt) HIV-1 integrase for 15 min at room temperature in a buffer containing 50 mM MOPS, pH 7.2, 7.5 mM NaCl, 7.5 mM MnCl₂, and 14.3 mM 2-mercaptoethanol. Reactions are started by adding 20 nM of the 5' end ³²P labeled 21 -mer double-stranded DNA template in a final volume of 10 μl, and reactions are carried out for 1 h at 37 °C. Reactions are quenched by adding 10 μl of denaturing loading dye (formamide 99%>, SDS 1%>, bromophenol blue 0.2 mg/ml, xylene cyanol FF 0.2 mg/ml). Samples are loaded onto 20% (19:1) denaturing polyacrylamide gels. Gels are dried, exposed overnight and analyzed using Molecular Dynamics Phosphorlmager (Sunnyvale, CA). The densitometric analysis is performed using ImageQuant v5.2 from Molecular Dynamics software package. Each lane is quantified to determine the amount of 3'-processing and strand transfer products. The results of HIV 1 integrase inhibitory assay are presented in Table 2.

* Peptide 1 is indolicidin; ** all D-amino acid sequence.

In sum, the above-described HIV-1 integrase inhibitory assays demonstrate that the analogue having the sequence of SEQ ID NO:2, with two proline residues at its C-end in the place of two arginine residues of indolicidin, is a substantially more potent inhibitor of both 3 '-processing (7 times more potent) and strand transfer (3 times more potent) than indolicidin, and that even a small changes in sequence of peptide 2, such as removal of the N-terminal residue (SEQ ED NO:4) or the C-terminal residue (SEQ ID NO: 11) reduced inhibitory activity. The peptide having SEQ ED NO:3 is an analog of CP10A (ILAWKWAWWAWRR- NH₂) (Friedrich, C. L. et al. (2001) "STRUCTURE AND MECHANISM OF ACTION OF AN INDOLICIDIN PEPTIDE DERIVATIVE WITH IMPROVED ACTIVITY AGAINST GRAM- POSITIVE BACTERIA," J. Biol. Chem. 276:24015-24022). CP10A is more potent antimicrobial agent than indolicidin, and has a much higher tendency to form an ordered α-helical structure.

As evidenced by the CD spectra of the peptides, only peptide 7 of Table 2 exhibits a double minimum at 202 and 223 nm along with the maximum at 187 nm, indicating the presence of an α-helical structure. The CD spectrum of peptide 7 in a 4: 1 MeOH/water mixture also shows the presence of an α helical structure, indicating a strong preference of this peptide to adopt the α helical conformation. However, peptide 7 is significantly less active in HIV-1 integrase inhibitory assays, than peptide 2. This indicates that the α helical conformation of peptide inhibitor is not favored for interaction with HIV-1 integrase. The overall shape of the CD spectra (Figure 5) for peptides 3 and 4 is similar to that of peptides 2, 8, and 10 (solid lines). The CD spectrum of indolicidin is slightly different from the spectra of peptides 2, 8, and 10, the main difference being the presence of weak positive Cotton effect at 231 nm. This Cotton effect can be associated with the presence of tryptophan residues (Woody, R. W. (1994) "CONTRIBUTIONS OF TRYPTOPHAN SIDE CHAINS TO THE FAR-ULTRAVIOLET CIRCULAR DICHROISM OF PROTEINS," Eur. Bioph. J. 23:253). The absence of this maximum in the spectra of 2, 8, and 10 suggested a less defined tryptophan side chain orientation, and more flexible conformations of these peptides.

A comparison of assay results for peptides 1, 2, 5 and 6 shows that the presence a basic amino acid residue (Arg or Lys) is crucial for the inhibitory activity of the peptide. This is supported by the inactivity of peptide 5. The activity data for peptide 6 shows that the presence of arginine residues at the C-terminal end may compensate for the absence of Lys residue, and in fact peptide 6 is even more potent than indolicidin (peptide 1).

The assay results for peptides 2 and 8 show that the dimeric peptide is much more potent than the monomeric one. The inhibitory potencies of enantiomeric peptides 8 and 9 are similar, suggesting that interactions between HIV-1 integrase and peptide inhibitors are not enantiospecific. The tetrameric peptide 10 is the most potent inhibitor of both 3'-processing (13 times more potent than 2) and strand transfer (20 times more potent than 2). The higher potency of dimeric and tetrameric peptides in comparison to the monomeric ones, was also observed in the case of other integrase inhibitory peptide (HCKFWW-NH₂; SEQ ID NO: 17) (Krajewski, K. et al. (2003) "DESIGN AND SYNTHESIS OF DIMERIC HIV-1 INTEGRASE INHIBITORY PEPTIDES," Bioorg. Med. Chem. Lett. 13:3203- 3205) supports the conclusion that multimeric inhibitory peptides may act as multivalent inhibitors, simultaneously occupying two or four neighboring catalytic sites within the integrase oligomeric complex, with an entropic advantage. From that point of view it is especially interesting, that in human cells HIV-1 integrase exists in the form of homotetramers, and probably at least an octamer of integrase is required to accomplish an effective integration (Cherepanov, P. et al. (2003) "HIV-1 INTEGRASE FORMS STABLE TETRAMERS AND ASSOCIATES WITH

LEDGF/P75 PROTEIN IN HUMAN CELLS," J. Biol. Chem. 278:372). Figure 6 illustrates two multimeric constructs of the present invention.

All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within lαiown or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Claims

What Is Claimed Is: Claim 1. A peptide comprising the formula: Aai -Aa₂-Aa₃-Aa₄-Aa₅-Aa₆-Aa₇-W- W-Aa₈-Aa₉-Aaιo-Aaι i -Z; wherein "Aa" denotes an amino acid residue; wherein such peptide is at least 6 amino acid residues in length; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, and wherein Z is the terminal carboxyl group of the peptide and is either unmodified or modified; with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa₉, Aaio, and Aan are all absent, Aa₈ will also be absent; or a pharmaceutically acceptable salt thereof.

Claim 2. The peptide or pharmaceutically acceptable salt thereof of claim 1, wherein said peptide is selected from the group consisting of: SEQ ED NO. 2 ILPWKWPWWPWPP-Z SEQ ID NO.3 ILAWKWAWWAWPP-Z SEQ ID NO.4 LPWKWPWWPWPP-Z SEQ ID NO. 5 PWKWPWWPWPP-Z SEQ ED NO.6 WKWPWWPWPP-Z SEQ ED NO. 7 KWPWWPWPP-Z SEQ ID NO.8 WPWWPWPP-Z SEQ D) NO.9 PWWPWPP-Z SEQ ED NO.10 WWPWPP-Z SEQ ID NO.11 ILPWKWPWWPWP -z and SEQ ED NO.13 ILPWKWPWW - Z

Claim 3. A peptide comprising the formula: X, X₂ wherein X₂ is absent, or Xi and X₂ are independently selected peptides having the formula: Aaι-Aa -Aa₃-Aa₄-Aa5-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa_g is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa , Aaio, and Aan are all absent, Aa₈ will also be absent; wherein each such peptide is at least 6 amino acid residues in length; wherein J is a linker, and wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; or a pharmaceutically acceptable salt thereof.

Claim 4. The peptide or pharmaceutically acceptable salt thereof of claim 3, wherein J is a lysine or arginine amino acid residue.

Claim 5. The peptide or pharmaceutically acceptable salt thereof of claim 4, wherein said peptide comprises the formula: SEQ ED NO. 14: (ILPWKWPWWPWPP) ₂K-Z .

Claim 6. The peptide or pharmaceutically acceptable salt thereof of any of claims 1-5, wherein said terminal carboxyl group Z of said peptide is modified, said modification being amidation.

Claim 7. A peptide comprising the formula:

X, — J

X wherein X is absent, or Xi and X are independently selected peptides having the formula: Aaι-Aa₂-Aa3-Aa₄-Aa₅-Aa6-Aa7-W-W-Aa₈-Aa₉-Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aaδ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aa is R, P or absent, wherein each such peptide Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; or a pharmaceutically acceptable salt thereof.

Claim 8. The peptide of claim 7, wherein J is a lysine or arginine amino acid residue.

Claim 9. The peptide of claim 7, wherein n is 2.

Claim 10. The peptide of claim 9, wherein Z is a lysine or arginine amino acid residue.

Claim 11. The peptide or pharmaceutically acceptable salt thereof of claim 10, wherein said peptide comprises the formula: SEQ ED NO. 18: ((ILPWKWPWWPWPP)₂K)₂-K.

Claim 12. A peptide comprising the formula:

wherein X₂ is absent, or Xi and X₂ are each peptides that are independently selected having the formula: Aaι-Aa₂-Aa₃-Aa₄-Aa₅-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aan; wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aaβ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent; wherein each such peptide, Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J and at least one reactive group capable of bonding to B; wherein m is an integer greater than 0; wherein B is a substituent having at least m reactive groups, each capable of bonding to Z; or a pharmaceutically acceptable salt thereof.

Claim 13. The peptide of claim 12, wherein J is a lysine or arginine amino acid residue.

Claim 14. The peptide of claim 12, wherein n is 2.

Claim 15. The peptide of claim 14, wherein m is 2.

Claim 16. The peptide of claim 15, wherein Z is a lysine or arginine amino acid residue.

Claim 17. An anti-retroviral pharmaceutical composition comprising a peptide comprising the formula: Aai -Aa₂-Aa₃-Aa₄-Aa₅-Aa₆-Aa₇- W-W-Aa₈-Aa₉-Aaι ₀-Aaι i -Z; wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aa_]0 is R, P or absent; and Aan is R, P or absent, wherein such peptide is at least 6 amino acid residues in length; and wherein Z is the terminal carboxyl group of the peptide and is either unmodified or modified; with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa , Aaio, and Aan are all absent, Aa₈ will also be absent; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein said peptide is present in said composition at a concentration that is effective for attenuating the severity of, or preventing, a retroviral infection.

Claim 18. The anti-retroviral pharmaceutical composition of claim 17, wherein said peptide is selected from the group consisting of: SEQ ED NO. 2 ILPWKWPWWPWPP-Z; SEQ ID NO. 3 ILAWKWAWWAWPP-Z ; SEQ ED NO. 4 LPWKWPWWPWPP-Z; SEQ ED NO. 5 PWKWPWWPWPP-Z SEQ ED NO. 6 WKWPWWPWPP-Z SEQ ID NO. 7 KWPWWPWPP-Z SEQ ID NO. 8 WPWWPWPP-Z SEQ ED NO. 9 PWWPWPP-Z SEQ ED NO. 10 WWPWPP-Z SEQ ED NO. 11 IILLPPWWKKWWPPWWWWPPWWPP --Zz; and SEQ ED NO.13 ILPWKWPWW -Z

Claim 19. An anti-retroviral pharmaceutical composition comprising a peptide comprising the formula: X, χ₂ wherein X₂ is absent, or Xi and X₂ are independently selected peptides having the formula: Aai -Aa₂-Aa₃-Aa₄-Aa5-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa is L or absent; Aa₃ is P, A or absent; Aa^ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, with the proviso that when Aai is I, Aaio and Aai _I will independently be either P or absent, and when Aa , Aaio, and Aan are all absent, Aa₈ will also be absent; wherein each such peptide is at least 6 amino acid residues in length; wherein J is a linker, and wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein said peptide is present in said composition at a concentration that is effective for attenuating the severity of, or preventing, a retroviral infection.

Claim 20. The anti-retroviral pharmaceutical composition of claim 19, wherein J is a lysine or arginine amino acid residue.

Claim 21. The anti-retroviral pharmaceutical composition of claim 20, wherein said peptide has the formula: SEQ ED NO. 14: (ILPWKWPWWPWPP ) ₂K- Z .

Claim 22. The anti-retroviral pharmaceutical composition of claim 17, wherein said terminal carboxyl group Z of said peptide is modified, said modification being amidation.

Claim 23. An anti-retroviral pharmaceutical composition comprising a peptide having the formula:

wherein X₂ is absent, or Xi and X₂ are independently selected peptides having the formula: Aai -Aa₂-Aa₃-Aa -Aa₅-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aas is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, wherein each such peptide Xi and X , if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein said peptide is present in said composition at a concentration that is effective for attenuating the severity of, or preventing, a retroviral infection.

Claim 24. The anti-retroviral pharmaceutical composition of claim 23, \ wherein J is a lysine or arginine amino acid residue.

Claim 25. The anti-retroviral pharmaceutical composition of claim 24, wherein n is 2.

Claim 26. The anti-retroviral pharmaceutical composition of claim 25, wherein Z is a lysine or arginine amino acid residue.

Claim 27. The anti-retroviral pharmaceutical composition of claim 26, wherein said peptide comprises the formula: SEQ ED NO. 18: ((ILPWKWPWWPWPP)₂K)₂-K.

Claim 28. An anti-retroviral pharmaceutical composition comprising a peptide comprising the formula:

wherein X₂ is absent, or Xi and X₂ are each peptides that are independently selected having the formula: Aaι-Aa₂-Aa₃-Aa -Aa₅-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i ; wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa is W or absent; Aa₅ is K or absent; Aas is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent; wherein each such peptide, Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J and at least one reactive group capable of bonding to B; wherein m is an integer greater than 0; wherein B is a substituent having at least m reactive groups, each capable of bonding to Z; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein said peptide is present in said composition at a concentration that is effective for attenuating the severity of, or preventing, a retroviral infection.

Claim 29. The anti-retroviral pharmaceutical composition of claim 28, wherein J is a lysine or arginine amino acid residue.

Claim 30. The anti-retroviral pharmaceutical composition of claim 29, wherein n is 2.

Claim 31. The anti-retroviral pharmaceutical composition of claim 30, wherein m is 2.

Claim 32. The pharmaceutical composition of any of claims 17, 19, 23 or 28, wherein said retroviral infection is an infection of HIV.

Claim 33. A method for preventing, or attenuating the severity of, a retroviral infection, which comprises providing to an individual having such infection, an effective amount of an anti-retroviral pharmaceutical composition comprising a peptide having the formula: Aai -Aa₂-Aa3-Aa -Aa5-Aa6-Aa7-W-W-Aa₈-Aa₉- Aaio- Aai i-Z; wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent, wherein such peptide is at least 6 amino acid residues in length; and wherein Z is the terminal carboxyl group of the peptide and is either unmodified or modified; with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa₉, Aaio, and Aan are all absent, Aa₈ will also be absent; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein said peptide is present in said composition at a concentration that is effective for preventing, or attenuating the severity of, a retroviral infection.

Claim 34. The method of claim 33, wherein said peptide is selected from the group consisting of: SEQ ED NO. 2 ILPWKWPWWPWPP-Z ; SEQ ED NO. 3 I L AWK AW AW P P - Z ; SEQ ED NO. 4 LPWKWPWWPWPP-Z; SEQ ED NO. 5 PWKWPWWPWPP-Z; SEQ ED NO. 6 WKWPWWPWPP-Z; SEQ ED NO. 7 KWPWWPWPP-Z; SEQ ED NO. 8 WPWWPWPP-Z; SEQ ED NO. 9 PWWPWPP-Z; SEQ ED NO. 10 WWPWPP-Z; SEQ ED NO. 11 ILPWKWPWWPWP, -Z; and SEQ ED NO. 13 ILPWKWPWW -Z .

Claim 35. A method for preventing, or attenuating the severity of, a retroviral infection, which comprises providing to an individual having such infection, an effective amount of an anti-retroviral pharmaceutical composition comprising a peptide having the formula:

χ₂ wherein X₂ is absent, or Xi and X₂ are independently selected peptides having the formula: Aai -Aa₂-Aa₃-Aa -Aa5-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aa₆ is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aa is R, P or absent, with the proviso that when Aai is I, Aaio and Aan will independently be either P or absent, and when Aa₉, Aaio, and Aan are all absent, Aa₈ will also be absent; wherein each such peptide is at least 6 amino acid residues in length; wherein J is a linker, and wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; wherein Z is the terminal carboxyl group of the peptide and is independently either unmodified or modified; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein said peptide is present in said composition at a concentration that is effective for preventing, or attenuating the severity of, a retroviral infection.

Claim 36. The method of claim 35, wherein J is a lysine or arginine amino acid residue.

Claim 37. The method of claim 36, wherein said peptide has the formula: SEQ ED NO. 14: (ILPWKWPWWPWPP ) K-Z .

Claim 38. The method of claim 35, wherein said terminal carboxyl group Z of said peptide is modified, said modification being amidation.

Claim 39. A method for preventing, or attenuating the severity of, a retroviral infection, which comprises providing to an individual having such infection, an effective amount of an anti-retroviral pharmaceutical composition comprising a peptide having the formula:

wherein X₂ is absent, or Xi and X₂ are independently selected peptides having the formula: Aai -Aa₂-Aa₃-Aa -Aa5-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i . wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aas is W or absent; Aa₇ is P or absent; Aa₈ is P or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aa is R, P or absent, wherein each such peptide Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker; wherein Z is an atom or terminal group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein said peptide is present in said composition at a concentration that is effective for preventing, or attenuating the severity of, a retroviral infection.

Claim 40. The method of claim 39, wherein J is a lysine or arginine amino acid residue.

Claim 41. The method of claim 40, wherein n is 2.

Claim 42. The method of claim 41, wherein Z is a lysine or arginine amino acid residue.

Claim 43. The method of claim 42, wherein said peptide comprises the formula: SEQ ID NO. 18: ( ( ILPWKWPWWPWPP ) ₂K) ₂-K .

Claim 44. A method for preventing, or attenuating the severity of, a retroviral infection, which comprises providing to an individual having such infection, an effective amount of an anti-retroviral pharmaceutical composition comprising a peptide having the formula:

wherein X₂ is absent, or Xi and X₂ are each peptides that are independently selected having the formula: Aai -Aa₂-Aa₃-Aa -Aa₅-Aa₆-Aa₇-W-W-Aa₈-Aa₉-Aaιo-Aaι i ; wherein "Aa" denotes an amino acid residue; wherein: Aai is I or absent; Aa₂ is L or absent; Aa₃ is P, A or absent; Aa₄ is W or absent; Aa₅ is K or absent; Aas is W or absent; Aa₇ is P or absent; Aa₈ is P

10 or absent; Aa₉ is W or absent; Aaio is R, P or absent; and Aan is R, P or absent; wherein each such peptide, Xi and X₂, if present, is at least 6 amino acid residues in length; wherein n is an integer greater than 0; wherein J is a linker;

15 wherein Z is an atom or group of J, which may be modified or unmodified, or is a multivalent substituent having at least n reactive groups, each capable of bonding to J and at least one reactive group capable of bonding to B; wherein m is an integer greater than 0;

20 wherein B is a substituent having at least m reactive groups, each capable of bonding to Z; or a pharmaceutically acceptable salt thereof; in admixture with one or more pharmaceutically acceptable excipients, wherein said peptide is present in said composition at a

25 concentration that is effective for preventing or attenuating the severity of, a retroviral infection.

Claim 45. The method of claim 44, wherein J is a lysine or arginine amino acid residue.

Claim 46. The method of claim 45, wherein n is 2.

Claim 47. The method of claim 46, wherein Z is a lysine or arginine amino acid residue.

Claim 48. The method of any of claims33, 35, 39 or 44, wherein said retroviral infection is an infection of HIV.