WO1988009338A1 - Peptides synthetiques et procede de traitement du sida - Google Patents

Peptides synthetiques et procede de traitement du sida Download PDF

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Publication number
WO1988009338A1
WO1988009338A1 PCT/US1988/001674 US8801674W WO8809338A1 WO 1988009338 A1 WO1988009338 A1 WO 1988009338A1 US 8801674 W US8801674 W US 8801674W WO 8809338 A1 WO8809338 A1 WO 8809338A1
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WIPO (PCT)
Prior art keywords
ala
ser
peptide
met
gly
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Ceased
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PCT/US1988/001674
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English (en)
Inventor
Miroslav Trampota
Matthew R. Pincus
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SYNTBIOTECH Inc
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SYNTBIOTECH Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to synthetic peptides, and more particularly to pentapeptides, hexapeptides, heptapeptides and octapeptides which are useful for treating acquired immune deficiency syndrome and its related diseases (collectively AIDS).
  • the present invention is also directed to a method of treating AIDS.
  • HIV human immunodeficiency virus
  • HAV human immunodeficiency virus
  • HTLV-III human T-lymphotropic virus type III
  • LAV lymphadenopathy-associated virus
  • ARV AIDS-associated retrovirus
  • AIDS is generally recognized as epidemic in several areas of the world, including the United States. At present, the groups at highest risk of infection with HIV include homosexual and bisexual men and abuses of injected drugs. It is also known, however, that AIDS is transmitted heterosexually.
  • HIV acts by crippling the body's immune system. Particularly, HIV selectively attacks T4 cells, a subpopulation of helper/inducer lymphocytes which constitute part of the immune system. Infection with HIV results in both a reduction in the number and a change in function of the targeted T4 lymphocytes with eventual collapse of the immune system. Thus, the disease manifests itself as severe imrnunosuppression typically resulting in devastating opportunistic infections and neoplasias.
  • Present methods of treating AIDS are limited and largely ineffective. There is no known cure for AIDS, and in fact, effective treatment of a retroviral infection in man is unprecedented.
  • Known therapies are generally limited to regimens designed to treat the secondary infections and neoplasias associated with AIDS.
  • AIDS has been treated with immunomodulators, such as cimetidine and interleukin-2, indomethacin, an antiinflammatory and prostaglandin inhibitor, and azidithymidine (AZT). None of the known therapies has been totally effective.
  • immunomodulators such as cimetidine and interleukin-2, indomethacin, an antiinflammatory and prostaglandin inhibitor, and azidithymidine (AZT). None of the known therapies has been totally effective.
  • peptide T (Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr). It has also been reported that peptide T and certain analogs thereof block the binding of HIV to T4 cells. C.B. Pert et al., Proc. Natl. Acad. Sci., Vol. 83, pp. 9254-9258, December 1986. Further, a correlation between the binding of peptide T to the T4 (CD4) surface molecule and the ability of the peptide to promote monocyte migration has also been reported. M.R. Ruff et al., FEBS Letters, Vol. 211, Number 1, pp. 17-22, January 1987.
  • the present invention provides novel synthetic penta, hexa, hepta and octapeptides useful for the treatment of AIDS and a method for treating AIDS by administering the peptides of the invention.
  • Summary of the Invention The present invention involves synthetic penta, hexa, hepta and octapeptides useful as antagonists for blocking the binding of HIV to the T4 receptor molecule on human T-lymphocyte cells.
  • the peptides of the invention may be linear or cyclic and have the formula
  • A is L-Ala, DL-Ala, D-Ala, Gly, or Val;
  • B is L-Ala, DL-Ala, D-Ala, Gly, or Val;
  • C is L-Met, L-Met (0), Cys, Thr or Gly; and x is an integer of from 0-1.
  • the present invention also includes a method for treating AIDS by administering therapeutically effective doses of the peptides of the invention.
  • the presently preferred peptide is a linear octapeptide having the amino acid sequence:
  • amino acids in the peptides may be substituted N-alkyl amino acids instead of primary amino acids; methyl and ethyl substituents are preferred.
  • hydroxyl group (OH) side chains of one or more amino acids may be substituted with an ether or ester.
  • Any substituted alkyl ester or ether may be used, for example, phenylethylester, benzylester, thiophene ethylester, phenylethylether, benzylether or thiophene ethylether.
  • the presently preferred esters are methyl, ethyl and propylethers, and the presently preferred esters are methyl, ethyl and propylesters.
  • the C terminal carboxyl group of the amino acid may be substituted with either an ester or an amide.
  • Suitable esters include those previously mentioned.
  • Suitable amides include foramide, N-methylamide and N-methylphenylamide.
  • linear peptides of the present invention may be prepared by conventional solid or semisolid peptide synthetic techniques such as, for example, the methods described by B. Merrifield, Science, Vol. 232, pp. 341-347, 1986 and R. Schwyzer et al., Bioorganic Chemistry, Vol. 8, pp. 429-442, 1979, respectively.
  • Classical stepwise or fragment condensation solution techniques may also be employed to synthesize the peptides of the invention. Solid phase synthesis is presently preferred.
  • cyclic peptides of the invention may be prepared by known techniques, such as, for example, the method described by Y. Hamada, Tetrahedron Letters, Vol. 26, p. 5155, 1985.
  • Solid phase synthesis of the linear peptides of the invention comprises the steps of:
  • Suitable polymers include aminomethyl resin, polyamide peptide resin, phenylacetamidomethyl resin (PAM), and benzhydrylamine resin. While any linking agent known to those skilled in the art may be used, it is presently preferred to use dicyclohexyl- carbodiamide (DCC).
  • DCC dicyclohexyl- carbodiamide
  • the C-terminal amino acid may be protected with either t-butoxycarbonyl (BOC) or fluoronylmethoxycarbonyl (FMOC);
  • a protected amino acid preferably either a BOC protected symmetric anhydride amino acid or a FMOC protected pentafluorophenyl (PFP) active ester of an amino acid, at -20oC-60oC in a suitable solvent.
  • a suitable solvent include dimethylformamide (DMF), diethylformamide, ethylacetate, tetrahydrofuran and dichloromethane, preferably DMF.
  • the symmetric anhydride amino acid may be obtained by converting a BOC protected amino acid in accordance with known techniques, such as, for example, the method described by B. Merrifield, Science, Vol. 232, pp. 341-347, 1986. Conversion of an FMOC protected amino acid to its PFP active ester may be carried out by conventional methods such as, for example, the method described by R.P. Andrews, Nature, Vol. 319, pp. 429-30, 1986;
  • Suitable cleaving agents include trifluoromethane sulfonic acid, trifluoroacetic acid, anhydrous hydrogen fluoride or by catalytic hydrogenolysis. Palladium or platinum, particularly supported on carbon, are suitable catalysts.
  • Suitable protecting groups are those which do not interfere with subsequent reactions and which can be removed under conditions which do not cause undesired reactions at other sites on the amino acids or products produced therefrom.
  • the preferred protecting groups are benzyl or substituted benzyl wherein the substituent is, for example, alkyl or alkoxy having from one to four carbon atoms, or halo (Cl, Br, F, I). It should be understood that the exact chemical structure of the protecting group is not critical. The important consideration is that the protecting group can be readily removed, leaving the protected group unchanged. Accordingly, the selection and identification of an appropriate protecting group is considered with the purview of those skilled in the art.
  • the coupling reaction of step 2 may be carried out manually or semiautomatically in accordance with known techniques. Typically, coupling requires about 30 minutes to 6 hours, with rapid shaking or other suitable means of agitation.
  • the resin is washed with appropriate solvents, such as DMF, tetradhydrofuran, and ethylacetate.
  • the amino acid protecting group is removed by treating with the appropriate reagent for 30-90 minutes at -5oC - +25oC to obtain thereby a free amino group.
  • the reagent used depends on the protective group to be removed. For example, for removing BOC, it is preferred to use trifluoroacetic acid in methylene chloride. FMOC is preferably removed wtih 20% piperidine in DMF.
  • the peptide is cleaved from the polymeric support by treating with anhydrous hydrogen fluoride, methanesulfonic acid/trifluoroacetic acid or other suitable reagents as are known to those skilled in the art.
  • the peptide thus obtained is then typically purified by conventional methods such as lyophilization, crystallization, HPLC or other known purification techniques.
  • the activity of the peptides synthesized is generally evaluated by chemotaxis assay, such as described by Ruff, et al., FEBS, Vol. 211, Number 1, page 17-22, 1987. Briefly, monocyte migration is directly observed in a chemotaxis chamber. Additionally, quantitative binding of radiolabelled peptides of the invention to T4 receptors on T-lymphocytes is a useful tool for evaluating the activity of the peptides.
  • H-MET-PAM resin (3.50 gm) was coupled with a symmetric anhydride prepared from 10 mmol of BOC-TYR(OBz) and 5 mmol DCC in DMF at room temperature for 60 minutes with vigorous shaking.
  • the resin thus obtained was washed with 20 ml methylene chloride (with shaking) for 10 minutes, shaken with 10 ml 1:1 methylene chloride-trifluoroacetic acid (30 minutes), washed with n-ethyl morpholine (5% in methylene chloride for 5 minutes) and again washed wtih 25 ml methylene chloride for 5 minutes to obtain H-TYR(OBz)-MET-PAM resin (3.52 gm).
  • the resin thus obtained was repeatedly coupled, deprotected and washed (steps 2 and 3).
  • the coupling reagents used, in order of use were BOC-ASN-sym. anhydride(+3 equiv. HOBZ), BOC-SER(OB z )-sym. anhydride-3 times, BOC-ALA-sym. anhydride and BOC-D-ALA-sym. anhydride.
  • the yield of the final BOC-octapeptide-PAM resin was 99%.
  • Intermediate penta, hexa, and hepta BOC-peptides were isolated in yields of 99.9%, 99.8%, and 99.9%, respectively.
  • Example 2 Solid Phase Preparation of H-SER-SER-ASN-TYR-MET BOC-SER-(OBz)-SER(OBz)-ASN-TYR(OBz)-MET-PAM resin (1.0 gm) was placed in a 150 ml one neck round bottom flask equipped with a stirring bar 2.0 gm ethane dithiol-thioanisole (1:2) was added to the contents of the flask and stirred at room temperature for 15 minutes. Then, 15 ml of a solution of trifluoromethane sulphonic acid in trifluoroacetic acid was added and the reaction mixture was stirred for 2 hours to obtain the penta peptide.
  • Examples 4-8 A comparative study comparing the chemotactic activity of the peptides of the invention with the chemotactic activity of peptide T (C.B. Pert et al., Proc. Natl. Acad. Sci., Vol. 83, pp. 9254-9258, 1986) was undertaken. The results of the study, which are summarized in Table II, demonstrate that the peptides of the invention exhibit significant chemotactic activity at concentrations of 10 -12M.
  • the chemotaxis assay was performed generally in accordance with the technique described by Ruff et al., FEBS
  • Human monocytes were obtained by the known technique of layering heparinized blood on Ficoll-Hypaque in a centrifuge tube and spinning to separate the red blood cells from plasma and the monolayered monocytes. The monocytes were pipetted off and then washed with physiological saline.
  • the agarose plates were also prepared in accordance with conventional methods. Agarose type II
  • each plate was used to assay two test samples in triplicate.
  • 8 ul of the test peptide (in 120 mM HEPES buffer) was placed on the outermost well, 8 ul of monocyte cell solution was placed in the center well and 8 ul of RPMI-Hanks buffer was placed in the innermost well.
  • the migration of monocytes was visually observed and the number of migrating cells was determined.
  • a chemotactic index was calculated as the number of cells which migrated toward the attractant, i.e., test peptide or control, less the number of cells that migrated towards the buffer divided by 10.
  • Formyl-Met-Leu-Phe (FMLP) and (d-Ala)Peptide-T were used as the positive control and HEPES buffer and Leu-Gly-Gly were used as negative controls.
  • a method of treating AIDS with the synthetic peptides described herein is provided.
  • a therapeutically effective dosage of the peptide which may be obtained according to the methods described hereinabove, is administered to a patient infected with HIV.
  • a therapeutically effective dosage may be from about 0.05-10 mg of peptide administered parenterally once per day.
  • the dosage level may, of course, be adjusted to provide optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionately reduced, as indicated by the exigencies of the therapeutic situation.
  • the peptide may suitably be administered orally or parenterally, generally, together with a pharmaceutically acceptable carrier.
  • Pharmaceutical forms suitable for injectable use include sterile solutions, or dispersions and sterile powders for extemporaneous preparation of sterile solutions.
  • the carrier can be a solvent or a dispersing medium and such carriers and excipients as are known to those skilled in the art are useful.
  • the advantages of the therapeutic method of the present method are apparent. AIDS has already reached epidemic proportions in the United States. Because of the long incubation period from infection to the manifestation of the disease, the number of persons already infected with HIV is not known, but the number of persons with AIDS is expected to dramatically increase in the next few years.
  • the present invention provides a treatment for AIDS, which unlike presently available therapies, is directed to blocking the attachment of HIV to the virus targeted human T-lymphocytes.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Peptides synthétiques linéaires ou cycliques de formule [A]x-[B]x-Ser-Ser-Ser-Asn-Tyr-[C]x, dans laquelle A est L-Ala, DL-Ala, D-Ala, Gly, ou Val; B est L-Ala, DL-Ala, D-Ala, Gly, ou Val; C est L-Met, L-Met (0), Cys, Thr ou Gly; x est un nombre entier valant 0 ou 1. Est également décrit un procédé de traitement du SIDA par l'administration de ces peptides synthétiques.
PCT/US1988/001674 1987-05-22 1988-05-20 Peptides synthetiques et procede de traitement du sida Ceased WO1988009338A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US5331487A 1987-05-22 1987-05-22
US053,314 1987-05-22

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992014751A1 (fr) * 1991-02-25 1992-09-03 Carlbiotech Ltd. A/S Peptides a usage therapeutique
EP0467699A3 (en) * 1990-07-19 1993-02-24 Merck & Co. Inc. Cyclic hiv principal neutralizing determinant peptides
EP0471453A3 (en) * 1990-07-19 1993-03-10 Merck & Co. Inc. Cyclic hiv principal neutralizing determinant peptides
EP0467701A3 (en) * 1990-07-19 1993-03-10 Merck & Co. Inc. Cyclic hiv principal neutralizing determinant peptides
WO1993020102A1 (fr) * 1992-03-27 1993-10-14 Peptide Technology Limited Peptide t et peptides associes destines au traitement des inflammations, notamment la sclerose en plaques
US5795858A (en) * 1993-09-24 1998-08-18 Peptide Technology Limited Treatment or prevention of Crohn's disease and/or ulcerative colitis
US5990172A (en) * 1996-02-28 1999-11-23 Innapharma, Inc. Peptidomimetics for the treatment of HIV infection
US6011014A (en) * 1992-03-27 2000-01-04 Advanced Immunit, Inc. Peptide T and related peptides in the treatment of inflammation, including multiple sclerosis

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, (California, USA), Volume 143, No. 1, issued 27 February 1987, (PINCUS et al.), "A strong homology exsists between the active T-cell binding gp 120 octapeptide of human immunodeficiency virus and the subtilisin cleavage peptide of bovine ribonuclease A". See pages 248 and 251 in particular. *
FEBS LETTERS, (Amsterdam, Netherlands), Volume 211, No. 1, issued January 1987, (RUFF et al.), "CD4 receptor binding peptides that block HIV infectivity cause human monocyte chemotaxis", pages 17-22. *
PROC. NATL. ACAD. SCI. USA, (Washington D.C., USA), Volume 83, issued December 1986, (FAUCI), "Current issues in developing a strategy for dealing with the acquired immunodeficiency syndrome", pages 9278-9283. *
PROC. NATL. ACAD. SCI. USA, (Washington D.C., USA), Volume 83, issued December 1986, (PERT et al.), "Octopeptides deduced from the neuropeptide receptor-like pattern of antigen T4 in brain potently inhibit human immunodeficiency virus receptor binding and T-cell infectivity", pages 9254-9258. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0467699A3 (en) * 1990-07-19 1993-02-24 Merck & Co. Inc. Cyclic hiv principal neutralizing determinant peptides
EP0471453A3 (en) * 1990-07-19 1993-03-10 Merck & Co. Inc. Cyclic hiv principal neutralizing determinant peptides
EP0467701A3 (en) * 1990-07-19 1993-03-10 Merck & Co. Inc. Cyclic hiv principal neutralizing determinant peptides
WO1992014751A1 (fr) * 1991-02-25 1992-09-03 Carlbiotech Ltd. A/S Peptides a usage therapeutique
US5763406A (en) * 1991-02-25 1998-06-09 Carlbiotech, Ltd. A/S Method for the treatment of conditions caused by herpes virus infections
WO1993020102A1 (fr) * 1992-03-27 1993-10-14 Peptide Technology Limited Peptide t et peptides associes destines au traitement des inflammations, notamment la sclerose en plaques
US6011014A (en) * 1992-03-27 2000-01-04 Advanced Immunit, Inc. Peptide T and related peptides in the treatment of inflammation, including multiple sclerosis
US5795858A (en) * 1993-09-24 1998-08-18 Peptide Technology Limited Treatment or prevention of Crohn's disease and/or ulcerative colitis
US5990172A (en) * 1996-02-28 1999-11-23 Innapharma, Inc. Peptidomimetics for the treatment of HIV infection

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Publication number Publication date
AU1808088A (en) 1988-12-21

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