RU2308966C1 - Antimetastatic composition - Google Patents

Abstract

FIELD: medicine, oncology, pharmacy.

SUBSTANCE: invention proposes a composition comprising serotonin salts with pharmaceutically acceptable acids and mellitine in the ratio 0.5-25 moles of serotonin salt per 1 mole of mellitine, 10 moles of serotonin salt per 1 mole of mellitine. Invention provides preparing the composition possessing broad range of antimetastatic effect.

EFFECT: valuable medicinal property of composition.

2 cl, 5 tbl, 2 ex

Description

This invention relates to medicine, new pharmacologically active compositions based on the natural peptide of melittin and serotonin, in aggregate possessing new pharmacological activity, namely, activity against metastatic malignant neoplasms.

Oncological diseases of various localization are widespread and are one of the main causes of mortality of the population of all ages.

Tumor growth is a complex multi-stage process. The appearance and progressive development of a tumor depends both on the properties of the cancer cells and on the state of the immunological reactivity of the body (Batchelor JR, Immunol. Today, 1989, 10, N 2, p. 37-40; Pollock RE, Sem. Surg. Oncol., 1989, 5, p. 414-419). This determines the variety of approaches to cancer exposure.

In the treatment of malignant neoplasms, one or several basic methods are used: surgical intervention, radiation therapy, chemotherapy and immunocorrection. The goal of most treatment methods is to minimize the mass of tumor cells. With solid tumors, this is usually achieved by surgical removal of the tumor. In leukemia and other generalized diseases, this goal is achieved with the help of massive radiation or chemotherapy.

However, no single method is able to destroy all tumor cells and achieve a radical result. Therefore, modern oncology almost always uses the combined effect of several treatment methods to eliminate the remaining tumor cells. The final fate of these cells, and therefore the fate of the patient, depends on the specific activity of the mechanisms of antitumor immunity (Takeda Y., J. Cancer Res. Clin. Oncol., 1989, 115, p. 211-228; Ehrke M., Minich E. , Reticuloendothel. Syst. Compr. Treatise, 1985, v. 8, p.309-347).

In accordance with the “Guidelines for the preclinical study of drugs with the ability to inhibit the process of metastasis and increase the effectiveness of the cytostatic therapy of malignant tumors” G.K. Gerasimova, V.E. Goldberg, AMKozlov, et al., Moscow, 1992, p. 2 -5; antimetastatic drugs should take into account the following features.

Malignant tumor metastasis is a cascade of selective biological processes, including the specific interaction of tumor cells with a high metastatic potential with cells and components of the extracellular matrix of the microenvironment, invasion of adjacent normal tissues, the penetration of tumor cells into lymph and blood, their transportation with the flow of biological fluids, adhesion to cellular and subcellular substrates in target organs and secondary proliferation in areas of secondary growth. Antimetastatic activity should be understood as the ability of a pharmacological agent, acting on certain biological processes that play a significant role in the mechanism of metastasis of a malignant tumor, to impede the implementation of the signs of a metastatic phenotype of tumor cells.

The selection of drugs is carried out on animals with transplantable tumors, while the effect of the test substance on both the severity of the metastatic process and the effectiveness of the cytostatic treatment is evaluated. Compounds characterized as possessing antimetastatic activity and promising for clinical testing should be able to effectively inhibit the process of spontaneous metastasis of transplanted tumors (35-75% inhibition).

Currently, in the treatment of malignant neoplasms, cyclophosphamide (S. Haga, T. Shimizu, H. Imamura, O. Watanabe, J. Kinoshita, M. Fukushima and T. Kajiwara, Antitumor efficacy of combination chemotherapy with UFT and cyclophosphamide against human breast cancer xenografts in nude mice. Anticancer Res, May 1, 1999; 19 (3A): 1791-6).

However, he is most active in combination chemotherapy.

The closest clinical product in terms of its properties, nature and direction of action is the only antimetastatic drug widely used to date - razoxan (JPBraybrooke, KJO'Byrne, DJPropper, A.Blann, M. Saunders, N.Dobbs, C. Han, J. Woodhull, K. Mitchell, J. Crew, K.Smith, R. Stephens, TS Ganesan, DC Talbot, AL Harris. A Phase II Study of Razoxane. December 2000 Clinical Cancer Research, Vol.6, 4697-4704)

The use of razoxan as an antimetastatic drug is based on the following features. Razoxane is able to inhibit the catalytic activity of topoisomerase 2 in micromolar concentrations. Razoxane is an active inhibitor of angiogenesis; enhances the effect of cisplatin based on the ability to enhance the antiproliferative effect of cisplatin. Razoxane has been studied in various animal tumor models. The dose range is 25-80 mg / kg (Gilbert JM, Thompson EM, Slavin G., Kark AE Inhibition of experimental calorectal cancer by razoxane (ICRF-159). Br.J.Surg., 1984, vol. 71, pp. .600-603; Lazo JS, Ingber DE, Sartorelli AC Enhancement of experimental lung metastases by cultured B16 melanoma cells treated with ICRF-159. Cancer Res., 1978, 38, pp. 2263-2270; Giraldi T. et. Al. Selectivity of the antimetastatic and cytotoxic effects of 1-p- (3,3-dimethyl-1-triazeno) benzoic acid potassium salt, 1,2-di (3,5-dioxopiperazin-1-yl) propane in mice bearing Lewis Lung Carcinoma. Cancer Res., 1981, 41, pp. 2524-2528; Bellet RE et. Al. ICRF-159: current status and clinical prospects. Europ. J. Cancer, 1977, vol. 13, pp. 1293-1298) . Higher concentrations are toxic, lower ones are inactive. It should be noted that in the entire range of active concentrations, razoxan works with approximately the same efficiency - the inhibition of metastasis practically does not differ at a concentration of 30 mg / kg from the inhibition at a concentration of 50-60 mg / kg (Giraldi T. et. Al. Selectivity of the antimetastatic and cytotoxic effects of 1-p- (3,3-dimethyl-1-triazeno) benzoic acid potassium salt, 1,2-di (3,5-dioxopiperazin-1-yl) propane in mice bearing Lewis Lung Carcinoma. Cancer Res. 1981, 41, pp. 2524-2528).

для лечения злокачественных опухолей (М.Д.Машковский «Лекарственные средства», М. «Медицина» 1993, ч.1, стр.366). Однако известен и отрицательный эффект от его применения - стимуляция роста опухолей (Е. Vicaut, E. Laemmel and О. Stucker. Impact of serotonin on tumour growth. Ann Med, April 1, 2000; 32 (3): 187-94). Имеются данные о том, что при некоторых формах опухолей увеличивается концентрация серотонина в тромбоцитах и, вероятно, в плазме крови и экскреция с мочой его метаболитов (I.P. Kema, E.G. de Vries, M.J. Slooff, В. Biesma, and F.A. Muskiet. Serotonin, catecholamines, histamine, and their metabolites in urine, platelets, and tumor tissue of patients with carcinoid tumors. Clin. Chem., Jan 1994; 40: 86-95).The use of serotonin is known

for the treatment of malignant tumors (MD Mashkovsky “Medicines”, M. “Medicine” 1993, part 1, p. 366). However, the negative effect of its use is also known - stimulation of tumor growth (E. Vicaut, E. Laemmel and O. Stucker. Impact of serotonin on tumor growth. Ann Med, April 1, 2000; 32 (3): 187-94). There is evidence that for some forms of tumors, the concentration of serotonin in platelets and, probably, in blood plasma and urinary excretion of its metabolites increases (IP Kema, EG de Vries, MJ Slooff, B. Biesma, and FA Muskiet. Serotonin, catecholamines , histamine, and their metabolites in urine, platelets, and tumor tissue of patients with carcinoid tumors. Clin. Chem., Jan 1994; 40: 86-95).

However, the use of serotonin in the treatment of malignant tumors requires a rather high concentration of it in the drug. At the same time, no evidence of the use of serotonin as a preparation of antimetastatic action of antitumor therapy was found.

The task to which the invention is directed is to expand the arsenal of agents that can be used as drugs that are active against metastatic malignant neoplasms.

The technical result that can be obtained by carrying out the invention is to obtain a new antimetastatic preparation based on a melittin peptide and a salt of serotonin with pharmaceutically acceptable acids.

The objective is achieved by creating a biologically active composition containing salts of serotonin with pharmaceutically acceptable acids, the novelty of which is that it additionally contains melittin in the ratio of 0.5-25 moles of serotonin salt per 1 mol of melittin, which has a wide range of antimetastatic action.

Melittin structure - Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg -Gln-Gln-NH2 is described in (Habermann E., Jentsch J. Sequence analysis of melittin from tryptic and peptic degradation products. Hoppe Seylers Z Physiol Chem 1967 Jan 348: 1 37-50). Some experimental studies have shown the antitumor activity of melittin against a number of tumor cell cultures (Bechinger B. J Membr Biol 156: 197-211, 1997, Kourie JI and Shorthouse AA Am J Physiol Cell Physiol, 2000, 278: C 1063 - C 1087) . There is experimental evidence that melittin is specifically selective against cells with high levels of ras oncogen expression (Sharma S.V. Melittin resistance: A counterselection for ras transformation., Oncogene, 1992 Vol. 7 No.2 pp. 193-201).

However, to date, there is no data indicating the presence of antimetastatic activity.

The proposed composition, consisting of a peptide of melittin and salts of serotonin with pharmaceutically acceptable acids, in combination have a new pharmacological activity, namely activity against metastatic malignant neoplasms.

The biologically active composition containing 10 mol of serotonin salt per 1 mol of melittin has the highest antimetastatic activity and a higher therapeutic index.

As salts of serotonin with pharmaceutically acceptable acids, any of the salts currently available commercially available, for example, creatinine sulfate, adipinate, hydrochloride, maleate, oxalate, is taken. The use of salt in the composition of serotonin is associated with the instability of its base.

The composition is active against metastatic tumors in a concentration range of 0.001 to 2.5 mg / kg. Most preferred is a concentration range of 0.001 to 0.125 mg / kg.

The above examples confirm, but do not exhaust, the invention.

Example 1. A method of obtaining a composition.

The melittin peptide is prepared using a known peptide preparation (Chromatography. Practical application of the method. Edited by E. Heftman, M., Mir, 1986, part 1, p. 43-68) single-stage high-performance preparative reverse phase chromatography on preparative columns large diameter. Columns of 50 × 250 mm and larger are used, filled with Diasorb 130 C16T or similar media. Isolation of the peptide takes place in a gradient mode. The advantage of this method is the ability to obtain a peptide of high purity (up to 99%) in a single chromatography without the use of complex equipment or multi-stage methods. The peptide is then lyophilized in freeze drying.

The antimetastatic composition is prepared by a standard method for preparing compositions with biological activity by mixing in the required proportion with any of the currently available commercially available salts of serotonin, for example, serotonin creatinine sulfate, dissolving the mixture in bidistilled water, followed by sonication in ultrasound in for 5-20 minutes and removing water in a freeze dryer. The lyophilized preparation is stored at room temperature without loss of activity. Before using it, the drug is dissolved in physiological saline, bringing to the required concentration of the drug.

Experiment

Studies were performed using the following types of tumors:

leukemia P 388,

melanoma B16,

metastatic Lewis lung tumor,

adenocarcinoma Ca 755.

Lewis lung tumor, B16 melanoma, Ca 755 adenocarcinoma are supported on C57BL / 6 or DBA / 2 or BDF ₁ mice (DBA / 2 × C57BL / 6) (females are used in all cases). For experiments, 10 ⁵ cells are transplanted - in the case of a Lewis lung tumor intramuscularly, for melanoma B16 - subcutaneously. Adenocarcinoma Ca 755 is transplanted in 0.3 ml of tumor tissue homogenate at a dilution of 1: 2 sc. Experimental animals sit on cells and are used in experiments starting from the first day after tumor inoculation. The studied compounds are administered intraperitoneally in physiological saline with a volume of not more than 0.2 ml per animal. To assess the effect of the studied compounds on various stages of tumor growth, it is administered at different times from the moment of tumor cell transplantation. Animals with a Lewis lung tumor and B16 melanoma are killed on 23-25 days after transplantation.

Example 2. Comparative antileukemic activity of serotonin, melittin and a composition consisting of a natural peptide of melittin and serotonin creatinine sulfate in a molar ratio of 1:10.

Conducting an experiment. The experiments were carried out on female hybrid mice of the first generation BDF ₁ (DBA / 2 × C57BL / 6), weighing 18-22 g. Leukemia P388 was inoculated intraperitoneally with 1 × 10 ⁶ cells in a volume of 0.2 ml. The age of the inoculum in all experiments was 7 days. All drugs were studied with two main regimens: once, only 24 hours after leukemia transplantation, and repeatedly, daily from 1 to 9 days after transplantation. The compositions were used in aqueous solutions; the volume of the injected solution did not exceed 0.5 ml per mouse. The injection site was treated with 96% ethanol.

As controls were used animals with leukemia P388, which was not administered drugs. The average life expectancy of untreated control was 10.5-11.5 days. In all experiments in control animals, in 100% of cases, death from the development of the leukemia process was observed.

Each dose of drugs was tested on a group of 6 animals, all experiments were repeated twice.

Evaluation of the results was carried out 60 days after transplantation of experimental leukemia. Animals that survived to 60 days without signs of tumor growth were considered survivors. Additionally, the increase in life expectancy / ILS / was determined as the ratio:

Table 1 Comparative antileukemic activity of serotonin, melittin and antimetastatic compositions A drug Life expectancy increase (ILS,%) The number of surviving animals melittin 61 1/12 composition 145 2/12 serotonin 47 0/6

Example 3. The activity of the composition consisting of the natural peptide of melittin and serotonin creatinine sulfate in a molar ratio of 1:10, in relation to a Lewis lung tumor.

table 2 The activity of the composition against Lewis lung tumor. A Lewis lung tumor is maintained on C57BL / 6 mice; for the experiments, 10 ⁵ cells are transplanted. As reference drugs use the antimetastatic drug razoxan and cyclophosphamide. Compound Dose (mg / kg) The mode of administration, day The average number of metastases The percentage of inhibition of metastasis Composition 0.125 1-9 4.2 ± 0.2 86 Composition 0.06 1-9 5.7 ± 0.3 81 Composition 0.018 1-9 6.2 ± 0.2 79 Composition 0.125 5-14 5.0 ± 0.2 80 Composition 0.06 5-14 5.2 ± 0.2 82 Composition 0.018 5-14 2.8 ± 0.1 91 Composition + 0.06 1-9 5.6 ± 0.1 77 cyclophosphamide 200 Composition + 0.125 1-9 3.8 ± 0.3 84 cyclophosphamide 200 Cyclophosphamide 200 5-14 7.6 ± 0.1 69 Razoxan 25 5-14 6.2 ± 0.3 75 Razoxan thirty 1-7 2.7 ± 0.2 82 * Razoxan thirty 2-7 3.7 ± 0.1 85 * Razoxan thirty 3-7 14.4 ± 0.2 42 *

* (K. Hellmann, K. Burrage, Control of malignant metastases by ICRF159, Nature, 1969, 224, pp. 273-275).

A characteristic feature of razoxan is its effectiveness only in certain periods of metastasis. Razoxan is most effective when used starting from the first day after tumor inoculation. With the use of razoxan at a later date, its effectiveness is markedly reduced (Giraldi T. et. Al. Selectivity of the antimetastatic and cytotoxic effects of 1-p- (3,3-dimethyl-1-triazeno) benzoic acid potassium salt, 1,2 -di (3,5-dioxopiperazin-1-yl) propane in mice bearing Lewis Lung Carcinoma. Cancer Res., 1981, 41, pp. 2524-2528, K. Hellmann, K. Burrage, Control of malignant metastases by ICRF159, Nature, 1969, 224, pp. 273-275), while the effectiveness of the proposed composition does not depend on the time of use and has a wider time range of effectiveness.

Example 4. The activity of the composition consisting of the natural peptide of melittin and serotonin creatinine sulfate in a molar ratio of 1:10, in relation to metastatic melanoma B16.

Razoxane antimetastatic drug and standard combined chemotherapy in the form of cyclophosphamide and nitrosomethylurea (HMM) are used as comparison preparations.

Table 3 The activity of the composition against metastatic melanoma B 16. Melanoma B16 is maintained on C57BL / 6 mice; for the experiments, 10 ⁵ cells are transplanted. Substance Dose mg / kg Mode (days) Tumor weight, mg Tumor growth inhibition,% Inhibition of metastasis,% Composition 0.006 1-9 5.4 12 73 Composition 0.125 one 5.28 fourteen 76 Composition + 0.006 1-9 3.9 39 68 cyclophosphamide + one hundred NNM 10 Composition + 0.125 one 6.4 0 71 cyclophosphamide + one hundred NNM 10 Razoxan thirty 1-9 3.9 39 41 Cyclophosphamide + one hundred 1-9 5.4 12 43 NNM 10

Example 5. The activity of the composition consisting of the natural peptide of melittin and serotonin creatinine sulfate in a molar ratio of 1:10, in relation to recurrent adenocarcinoma 755.

Ca 755 adenocarcinoma is maintained on C57BC / 6 mice; for experiments, 0.3 ml of tumor tissue homogenate is transplanted at a dilution of 1: 2 s.c.

As comparison drugs use cyclophosphamide.

Table 4 The activity of the composition consisting of the natural peptide of melittin and serotonin creatinine sulfate in a molar ratio of 1:10, in relation to recurrent adenocarcinoma 755 Substance Dose mg / kg Mode (days) ILS,% Surviving animals Composition 0.38 one 59 1/10 Composition 0.125 one 75 1/6 Composition 0.018 1-9 52 1/7 Composition + 0.38 one 57 1/9 cyclophosphamide one hundred Composition + 0.125 one 49 2/9 cyclophosphamide one hundred Cyclophosphamide one hundred 10 26 0/9

Example 6. The activity of the composition consisting of the natural peptide of melittin and serotonin creatinine sulfate in various molar combinations, against a tumor of the Lewis lung.

Table 5 The activity of the composition against Lewis lung tumor. A Lewis lung tumor is maintained on C57BL / 6 mice; for the experiments, 10 ⁵ cells are transplanted. The compound, the ratio of melittin: serotonin, mol Dose (mg / kg) The mode of administration, day The average number of metastases The percentage of inhibition of metastasis Composition, 1:10 0.125 1-9 4.2 ± 0.2 86 Composition, 1:25 0.125 1-9 6.7 ± 0.3 46 Composition, 1:15 0.125 1-9 5.4 ± 0.2 79 Composition 1: 0.5 0.125 1-9 6.8 ± 0.2 62 Composition 1: 0.1 0.125 1-9 7.8 ± 0.2 27 Composition, 1:30 0.125 1-9 8.1 ± 0.2 29th

From an analysis of the experimental data of examples of the effects of the compositions, it follows that a composition consisting of a peptide of melitin and serotonin in a molar ratio of 1:10 shows high efficiency in a large time range and in a large concentration range not only in relation to antitumor activity (see tables 1, 3 , 4), but also as antimetastatic (see tables 2, 3, 5). The most optimal results are obtained with a molar ratio of the peptide of melitin and serotonin equal to 1:10. A distinctive and important feature of the action of the composition is the low effective concentration: the concentration necessary to achieve the effect is more than 1,500 times lower than that of the well-known antimetastatic drug razoxan or more than 800 times lower than that of the well-known antitumor drug cyclophosphamide (Table 1-5).

Claims (2)

1. A biologically active composition containing salts of serotonin with pharmaceutically acceptable acids, characterized in that it further comprises melittin in a ratio of from 0.5 to 25 mol of serotonin salt per 1 mol of melittin, having a wide range of antimetastatic action. 2. The biologically active composition according to claim 1, characterized in that it contains 10 mol of serotonin per 1 mol of melittin.