RU2067870C1 - Antitumor agent - Google Patents

Abstract

FIELD: medicine, oncology. SUBSTANCE: proposed agent is hexapeptide of the formula Leu-Val-Val-Tyr-Pro-Trp that is used as antitumor agent. Indicated hexapeptide shows expressed cytostatic effect, inhibits the proliferation of different tumors (lympholeukosis P-388, melanoma B-16). This hexapeptide is able to recover T-lymphocyte function depressed by tumor action that are important the main role in antitumor protection. Hexapeptide is not toxic and can be used for treatment of broad spectrum oncologic diseases. EFFECT: high effectiveness of agent. 3 tbl

Description

 The invention relates to medicine, namely to Oncology, and consists in the use of a hexapeptide of the formula Leu-Val-Val-Tyr-Pro-Trp as an antitumor agent.

 Known use in cancer practice of polypeptides (cytokines), such as interferon, colony stimulating factors, interleukin-2. They have a molecular weight of 15–70 kDa, they are produced in a biological, genetic engineering way. The introduction into the patient's body of these relatively large substances in therapeutic doses causes, as a rule, severe side effects. It is much preferable to use low molecular weight peptide compounds, especially of endogenous nature, as therapeutic agents. They are effective in extremely small doses and produce, as a rule, a regulatory, corrective effect, without exerting harmful effects on the body.

 The hexapeptide of formula I was found in the supernatant of a pig bone marrow cell culture. It is known that, in a mixture with a hexapeptide of formula II Phe-Leu-Gly-Phe-Pro-Thr, it produces a modulating effect on pain sensitivity: with a low initial threshold of pain sensitivity, peptides increase it, and with an increased threshold of pain sensitivity it is lowered. The synthesis of these endogenous peptides made it possible to obtain them in a separate state. Other biological properties have not been studied.

 The purpose of the invention to apply the peptide Leu-Val-Val-Tyr-Pro-Trp in oncology.

 This goal is achieved in that the hexapeptide of formula I Leu-Val-Val-Tyr-Pro-Trp cancels the inhibitory effect of tumor cells on the functional activity of T-lymphocytes and inhibits the growth of a number of mouse transplantable tumors (lymphocytic leukemia P-388, melanoma B-16).

 The hexapeptide is prepared by the solid phase method by expanding the peptide chain at the N-terminus. The amino group protected Fmoc amino acids are attached using the carbodiimide method.

 Example 1. The hexapeptide of formula I restores the activity of T-lymphocytes suppressed by human tumor cells.

 It is known that tumor cells of patients with AML, as well as cells of the HL-60 line, originating from the leukemia cells of the bone marrow of these patients, produce proteins that compress the functions of T-lymphocytes, which is expressed in a sharp decrease in their ability to respond to proliferation upon mitogen exposure (phytohemagglutinin, PHA).

Freshly isolated peripheral blood T-lymphocytes from healthy donors at a concentration of 1 × 10 ⁶ cells / ml stimulated PHA proliferation (3 μg / ml). At the end of 3 days of incubation of T-lymphocytes with mitogen, N-thymidine (2 μCi / mmol) was introduced into the incubation mixture, by the inclusion of which proliferative activity was judged in the T-lymphocytes DNA, and held for 4 hours. If at the beginning of incubation, add to this incubation mixture 10% of the conditioned medium (CS) from HL-60 leukemia cells, the level of T-lymphocyte proliferation in response to PHA decreases by an average of 50% compared with the control (100% incubation without HL CS -60) (table 1).

 It was found that the hexapeptide of formula I, in contrast to the hexapeptide of formula II, which is closely related to the physicochemical properties, restores the response of T-lymphocytes reduced by PHA leukemia cells to a normal level. The ability of the hexapeptide of formula II to restore the suppressed proliferative response of T-lymphocytes exhibits a clear dose dependence. The hexapeptide of formula II, closely related in physicochemical properties to the hexapeptide of formula I, but having a different amino acid sequence, not only does not restore the reduced PHA response of T-lymphocytes, but even enhances the toxic effect of HL-60 leukemia cells (table 1).

 Thus, of the two hexapeptides of endogenous nature, previously described as affecting pain sensitivity (3), one (the hexapeptide of formula I) also has antitumor activity.

 Example 2. The introduction of a hexapeptide of formula I to mice inhibits the growth of a grafted tumor of lymphocytic leukemia P-388.

 In experiments, hybrid female mice BDF-1 were used. Lymphocytic leukemia P-388 was inoculated subcutaneously by a conventional technique by suspension of leukemia cells in 199 nutrient medium in an amount of 1 million cells per mouse. Hexapeptide was administered intraperitoneally 48 hours after transplantation of the tumor once, twice and five times at intervals of 96 and 24 hours, respectively. A single dose range of 0.5 to 4.0 mg / kg was used.

 The effectiveness of the hexapeptide was judged by the inhibition of tumor growth (TPO,), which was calculated as the difference in the average volumes of tumors in the control and experimental groups, expressed as a percentage. Table 2 presents the average data from two series of experiments. The number of mice in the control groups without treatment was 29. The number of mice in each experimental group was 8. The toxicity of hexapeptide was estimated by the number of dead mice in the experimental groups before the death of animals in the control groups.

 The data presented show that the hexapeptide of formula I has a significant and reliable antitumor effect in mice against subcutaneous lymphocytic leukemia P-388.

 This hexapeptide is non-toxic in the doses used.

 Example 3. The introduction of the hexapeptide of formula I to mice inhibits the growth of a graft of a solid tumor of melanoma B-16 grafted by it.

 In experiments, linear C 57 BL / 6 mice were used. Melanoma B-16 was inoculated with 50 mg of crushed tumor tissue per mouse in nutrient medium 199. The hexapeptide was administered 1- or 2-fold with an interval of 96 hours. Treatment began 72 hours after tumor inoculation. Doses of the hexapeptide, as well as a method for evaluating its effectiveness were the same as in example 2. The results of the experiments are presented in table 3.

 From the data of table 3 it follows that the antitumor effect of the hexapeptide of formula I was significantly manifested on this solid tumor. He was most pronounced on the 7th day after the end of the course of treatment.

 As in example 2, the peptide is non-toxic at the used doses of administration.

 Example 4. Synthesis of a hexapeptide of formula I Leu-Val-Val-Tyr-Pro-Trp.

 The peptide was synthesized on an automated Biosearch 9600 peptide synthesizer (USA) on a PAC resin using the Fmoc / DIPCDI method according to the standard c: FDI program supplied with the device. Fmoc amino acids were condensed using the carbodiimide method; equimolar amounts of 1-hydroxybenzotriazole were added to suppress racemization. The tyrosine residue was introduced as its o-tert-butyl ether. The starting amino acid tryptophan was attached to 5 g of the PAC polymer in an amount of 0.76 mmol of amino acid per 1 g of polymer. Next, the Trp polymer was treated with 0.4 M solutions of the corresponding protected amino acids containing approximately a 7-fold excess of the corresponding amino acid and diisopropylcarbodiimide. Cleavage of the Fmoc group after condensation was carried out with a mixture of piperidine-toluene-dimethylformamide (30:35:35). After completion of the synthesis, the peptide was cleaved from the resin with trifluoroacetic acid containing 2.5% ethanediol and 2.5% water while cooling with ice for 1.5 hours. The resulting peptide was isolated and purified by high-performance chromatography on a Diasorb-130 C-16 T column, 10 μm, size 26x250 mm in an acetonitrile gradient in 0.05 M phosphate buffer pH 3.0 and desalted on the same column. After lyophilization, 2 g of a white amorphous powder homogeneous according to HPLC was obtained. Analytical chromatography was performed on an ODS-3 Ultrasphere column in an acetonitrile gradient (20–80%) in 0.05 M phosphate buffer pH 3.0, and detection was carried out at 220 nm. Amino acid analysis of an acid hydrolyzate (6N HCl, 20 hours) showed the presence of the following amino acids: Leu 1 (0.95), Val 2 (1.87), Tyr 1 (1.03), Pro 1 (0.96).

Next, 1 mg of the obtained peptide was dissolved in 0.5 ml of 98% formic acid, 0.25 ml of acetic anhydride was added and kept for 30 min at 18 ^o С. Then it was evaporated at room temperature and purified by high-performance chromatography on an Ultrasphere C-18 ODS column (4.6 x 250 mm) in a gradient of acetonitrile in 0.1% trifluoroacetic acid. Received 0.5 mg of the peptide. TTT1 TTT2

Claims (1)

 Use of a hexapeptide of the formula Ley-Val-Val-Tyr-Pro-Trp as an antitumor agent.

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