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WO1996018652A1 - Hexapeptides presentant une activite antitumorale - Google Patents

Hexapeptides presentant une activite antitumorale Download PDF

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Publication number
WO1996018652A1
WO1996018652A1 PCT/IB1994/000430 IB9400430W WO9618652A1 WO 1996018652 A1 WO1996018652 A1 WO 1996018652A1 IB 9400430 W IB9400430 W IB 9400430W WO 9618652 A1 WO9618652 A1 WO 9618652A1
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WO
WIPO (PCT)
Prior art keywords
val
leu
tyr
pro
trp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB1994/000430
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English (en)
Inventor
Augusta A. Mikhailova
Rem V. Petrov
Larissa A. Fonina
Leonid A. Strelkov
Sergei A. Guriyanov
Galina K. Gerassimova
Elena M. Treshchalina
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Individual
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Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Individual filed Critical Individual
Priority to PCT/IB1994/000430 priority Critical patent/WO1996018652A1/fr
Priority to AU11175/95A priority patent/AU1117595A/en
Publication of WO1996018652A1 publication Critical patent/WO1996018652A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Tumor cells of various origin produce substances inhibiting or disrupting the normal functions of the immune system (Miescer et al.. Functional properties of tumor-infiltrating and blood lymphocytes in patients with solid tumors, J. Im ⁇ munol. 136, 1899-1907 (1986)).
  • the dysfunc ⁇ tion of T-lymphocytes characteristic of acute myeloid leuke ⁇ mia is connected with the suppressive effect of leukemic cell products (Chiao et al.. Suppression of lymphocyte acti ⁇ vation and functions by a leukemia cell-derived inhibitor, Proc. Natl. Acad. Sci. USA 83, 3432-3436 (1986)).
  • Cytokines are polypeptides with a molecular weight of 15 - 70 kDa. Admi ⁇ nistration of such comparatively large polypeptide substan ⁇ ces at effective doses to the patients induces as a rule harmful side effects. It would be more preferable to use clinically low molecular weight peptide compounds, particu ⁇ larly ones of endogenic origin (Fridman & Michon, Pathophy- siology of cytokines, Leukemia Res. 14, 675-677 (1990)).
  • cytokines are based on an enhancement of their reduced activity in the body of the tumor carrier.
  • positive effects of cytokines are short-termed and require supportive injections. It is therefore necessary to administer these substances many times and at rather high doses.
  • Bone marrow cells produce a group of bioregulatory molecules - myelopeptides (MPs) .
  • MPs display various biological acti ⁇ vities: immunoregulatory, cell differentiating, neurotropic activity etc.
  • a mixture of un ⁇ identified MPs isolated from the supernatant of porcine bone marrow cell culture has been used in Russia in both human and veterinary applications for a variety of effects.
  • this mixture inhibited the suppressive effect of leukemic cells HL-60 on T-lymphocyte functional activity (Strelkov & Mikhailova, Myelopeptides abolish T-lymphocyte suppression induced by human leukemia cells, Immunologia 6 (in Russian), 32-35 (1990)).
  • the hexapeptide Leu-Val-Val-Tyr-Pro-Trp together with the hexapeptide Phe-Leu-Gly-Phe-Pro-Thr were isolated from the supernatant of porcine bone marrow cell culture as substance modulating pain sensitivity. It induced hypoalgesia in high pain sensitivity threshold and hyperalgesia in low pain sen ⁇ sitivity threshold (Fonina et al.. Structures of two myelo ⁇ peptides affecting pain sensitivity, Doklady Academy Nauk 319 (in Russian), 755-757 (1991)). No further biological activities of these peptides are known.
  • the present invention provides new hexapeptides possessing antitumor activity as well as the use of known hexapeptides for the preparation of pharmaceutical compositions having antitumor activity.
  • the present invention is directed to a hexapeptide of the formula (1) X 1 -Y 1 -Y 2 -Y 3 -Tyr-Pro-Trp-X 2 in which
  • X 1 is H, Form, Ac or any amino acid, or an acid addition salt or complex thereof,
  • X 2 is H, any salt, amide, substituted amide or any amino acid
  • Y 1 is Leu, lie, Val, Nle, Nva, Ala, Gly,
  • Y 2 is Leu, lie, Val, Nva, Ala,
  • Y 3 is Leu, He, Val, Nva, Ala, except the compound Leu-Val-Val-Tyr-Pro-Trp.
  • Bivalfor is the new compound OHC-Leu-Val-Val- Tyr-Pro-Trp which is named Bivalfor.
  • Bivalfor has a low molecular weight (927 Da) . It is able to stimu ⁇ late IL-2 production in T-lymphocytes.
  • the presence of the N-terminal formyl group of the peptide molecule increases its stability to aminopeptidases and provides a prolongation of its action in the body.
  • the present invention is further directed to pharmaceutical compositions comprising the hexapeptides and to a method of reducing tumor growth in a mammal and to inhibit the sup ⁇ pressive effect of tumor cells on T-lymphocyte functional activity.
  • the present invention is directed to the use of the hexapeptide Leu-Val-Val-Tyr-Pro-Trp for the preparation of a pharmaceutical composition for reducing tumor growth in a mammal and to inhibit the suppressive ef ⁇ fect of tumor cells on T-lymphocyte functional activity.
  • the hexapeptide comprises at least 10%, 25%, 50% or 90% of the pharmaceutical composition of the invention.
  • the peptides of the invention are synthesized according to usual methods, e.g. the solid phase method (see, e.g. "Aminosauren, Peptide, Proteine", Eds. H.D. Jakubke and H. Jeschkeit, Akade y Verlag, Berlin, 1982) .
  • the peptide was synthesized by the solid phase method on Biosearch 9600 (USA) .
  • the hexapeptide was prepared by car- bodiimide method using poly(4-hydroxymethyl) phenylacet- amidomethyl resin.
  • the free peptide was purified by HPLC using Diasorb-130 C-16 T column, 10 ⁇ (250x26 mm) with an acetonitrile gradient (10 - 100%) in 0.05 M phosphate buffer, pH 3.0. Its homogeneity was checked by HPLC on an Ultrasphere ODS-3 column (500x4.6 mm) with an acetonitrile gradient (20 - 80%) in 0.05 M phos ⁇ phate buffer, pH 3.0. Amino acid analysis: Leu 0.95 (1), Val 1.87 (2), Tyr 1.03 (1), Pro 0.96 (l). 1 mg of this pep ⁇ tide was dissolved in 0.5 ml 98% HCOOH and treated with 0.25 ml (CH 3 CO)2 ⁇ .
  • Bivalfor was isolated by HPLC on Ultrasphere C-18 ODS (4.6x250 mm) with an acetonitrile gradient (5-60%) in 0.1% trifluoroacetic acid. The yield of Bivalfor was 47%.
  • PHA mitogen- phytohe agglutinin
  • T-lymphocytes from peripheral blood of healthy donors were cultured at lxlO 6 cells per ml and stimulated with PHA (3 ⁇ g/ml) during 3 days.
  • PHA-stimulated proliferation was de ⁇ termined by the 3 H-thymidine incorporation procedure (Chiao et al., supra).
  • HL-60 cells conditioned media (DM) with in ⁇ hibitory activity collected from cells during logarithmic growth phase (on the 3rd - 4th day of growth) and various concentrations of Bivalfor were added to the cultures at the start of cultivation.
  • J H-thym ⁇ d ⁇ ne pulsing was for 4 hours using 2 ⁇ Ci/ml on the 3rd day of PHA stimulation.
  • the cells were harvested and their radioactivity was measured in a li ⁇ quid scintillation counter. The typical results of one of four experiments are presented in Table 1. Table 1.
  • Each figure is an average value of three parallel tests (in the range of +/- 200 cpm) .
  • HL-60 CM decreased 3 H-thymidine incorporation to 40.6% of the control value.
  • the addition of Bivalfor to the T-lym ⁇ phocyte culture increased the proliferative response in a dose dependent manner.
  • the best restoration of T-lymphocyte proliferative response (at least 80% of control) took place at a dose of Bivalfor of 100 ⁇ g/ml. It was shown that the inhibitory effect of HL-60 CM on T-lymphocyte proliferative response is accompanied by a decrease of IL-2 production in T-lymphocytes (Chiao et al., supra) .
  • Spleen cells were isolated from (CBAxC57BL)Fl mice and incu ⁇ bated in RPMI 1640 medium supplemented with 10% fetal calf serum in 5% C0 at 37°C. The cell concentration was 5xl0 6 cells/ml. Con A (5 ⁇ g/ml) and Bivalfor (1 ⁇ g/ml) were added at the start of cell cultivation. IL-2 production in T-lym ⁇ phocytes was determined by the ability of Con A-activated spleen cell supernatant to support the proliferation of the IL-2 dependent cytotoxic T-cell line CTLL-2.
  • CTLL-2 cells were incubated in 96-well plates (lxio 4 cells/well) and the tested supernatant was added to each well at final dilutions of 1/2, 1/4, 1/8, 1/32, 1/64 and 1/128.
  • Supernatant of Con A-activated mouse spleen cells cultivated without Bivalfor was added at the same dilutions to the control wells.
  • the proliferation of CTLL-2 cells was measured by rapid quantity colorimetric method based on the reduction of tetrazolium salt MTT to a blue farmason product (Mosmann, Rapid colori ⁇ metric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65, 55-63 (1986)) .
  • Bivalfor was administered to mice in therapeutic doses (0.5 - 4 mg/kg) and in 30-fold increased doses as compared to therapeutic doses (120 mg/kg) .
  • the peptide was administered 1, 2 or 5 times with 0, 96 and 24 hour intervals, respectively.
  • the animal survival was registered on the 10th day after the last Bivalfor injec ⁇ tion.
  • the results presented in the Table 3 show the full absence of Bivalfor toxicity.
  • Table 3 Survival of (CBAxC57BL)Fl mice after Bivalfor in ⁇ jections.
  • Example 5 Ability of the hexapeptide Leu-Val-Val-Tyr-Pro- Trp to reduce the tumor size in mice injected with lympho- leukemia P-388 cells.
  • Leukemic P-388 cells were inoculated in hybrid mice BDF ⁇ subcutaneously at lxlO 6 cells per mouse.
  • the tested peptide was injected intraperitoneally once, twice (with a 96 hour interval) or 5 times (with 24 hour intervals) .
  • the start of the peptide treatment was 48 hours after leukemic cell in ⁇ oculation.
  • the antitumor effect of the peptides was examined by the in ⁇ hibition of tumor growth (ITG) which was calculated as the difference between the average volume of tumors in experi ⁇ mental and control groups, expressed in percent.
  • the con ⁇ trol group without peptide treatment contained 29 mice. Each experimental group contained from 7 to 10 mice. The results of one of three such experiments are presented in Table 4. We can see the suppressive effect of hexapeptide on growth of tumor. This scheme of peptide treatment had no effect on the life span of the animals (data not shown) .
  • Example 6 Ability of the hexapeptide L ⁇ u-Val-Val-Tyr-Pro- Trp to decrease the tumor size in mice injected with mela ⁇ noma B-16 solid tumor.
  • Melanoma B-16 was inoculated in C57BL mice subcutaneously at 50 mg tumor tissue per mouse (cell suspension in Pasher's media) .
  • the peptide treatment was started at 72 hours after tumor inoculation. The interval between two peptide injections was 96 hours. The results of one of two experiments are presented in Table 5. 7-8 mice were used in each group. Table 5. Influence of Leu-Val-Val-Tyr-Pro-Trp on melanoma fi ⁇ le spreading.
  • the injected peptide in the doses used had no toxic effect.
  • There were special animal groups consisting of 8 to 10 healthy mice for each peptide dose. Not one mouse died after peptide treatment.

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

Abstract

La présente invention se rapporte à des hexapeptides de la formule (1): X?1-Y1-Y2-Y3¿-Tyr-Pro-Trp-X2, dans laquelle X1 représente H, Form, Ac ou un aminoacide quelconque, ou un sel d'addition d'acide ou complexe de ceux-ci, X2 représente H, un sel, amide, amide substitué ou aminoacide quelconque, Y1 représente Leu, Ile, Val, Nle, Nva, Ala, Gly, Y2 représente Leu, Ile, Val, Nva, Ala et Y3 représente Leu, Ile, Val, Nva, Ala. Ces hexapeptides présentent une activité antitumorale.
PCT/IB1994/000430 1994-12-16 1994-12-16 Hexapeptides presentant une activite antitumorale Ceased WO1996018652A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/IB1994/000430 WO1996018652A1 (fr) 1994-12-16 1994-12-16 Hexapeptides presentant une activite antitumorale
AU11175/95A AU1117595A (en) 1994-12-16 1994-12-16 Hexapeptides possessing antitumor activity

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB1994/000430 WO1996018652A1 (fr) 1994-12-16 1994-12-16 Hexapeptides presentant une activite antitumorale

Publications (1)

Publication Number Publication Date
WO1996018652A1 true WO1996018652A1 (fr) 1996-06-20

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB1994/000430 Ceased WO1996018652A1 (fr) 1994-12-16 1994-12-16 Hexapeptides presentant une activite antitumorale

Country Status (2)

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AU (1) AU1117595A (fr)
WO (1) WO1996018652A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998030581A1 (fr) * 1997-01-07 1998-07-16 Primamedic Ltd. Myelopeptides et leur utilisation therapeutique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 116, no. 1, 6 January 1992, Columbus, Ohio, US; abstract no. 4237u, I A FONINA ET AL.: "Structure of two myelopeptides affecting pain perception" page 419; *
CHEMICAL ABSTRACTS, vol. 122, no. 15, 10 April 1995, Columbus, Ohio, US; abstract no. 185289e, L A STRELKOV ET AL.: "Myelopeptide abolishing the tumor cell toxic effect on T-lymphocyte functional activity" page 850; *
DOKL. AKAD. NAUK, vol. 319, no. 3, 1991, pages 755 - 757 *
DOKL. AKAD. NAUK, vol. 338, no. 1, 1994, pages 125 - 126 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998030581A1 (fr) * 1997-01-07 1998-07-16 Primamedic Ltd. Myelopeptides et leur utilisation therapeutique
US6469137B1 (en) 1997-01-07 2002-10-22 Primamedic, Ltd Myelopeptides and their therapeutic use

Also Published As

Publication number Publication date
AU1117595A (en) 1996-07-03

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