RU2703533C1 - Agent for treating hormone-dependent tumors and a method for production thereof - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: group of inventions relates to medicine and concerns an agent for treating adenocarcinoma of mammary gland containing methyl ester N-tert-butyloxycarbonyl-S-tetrahydropyranylcysteinyl-phenylalanyl-D-tryptophyl-N-benzyl-oxycarbonyl-precyltreonine (P), where the agent is a lyophilizate for preparing an injection solution, additionally contains phosphatidyl choline (PCH), cholesterol (CH) and DSPE-PEG-2000 (PEG-lipid) and saccharose (S) in molar ratio of components: M:M:M:M:M=1.0:71.1:13.9:0.2:346.5. Group of inventions also relates to a method of producing said agent, involving dissolving P, PS, CH and PEG-lipid, taken in ratio of 1.0:71.1:13.9:0.2, evaporation of the formed chloroform solution on a rotary evaporator under vacuum, dispersion of the film formed during evaporation, filtration and extrusion of the obtained liposomal dispersion, addition of cryoprotector solution, dosing and stabilization by lyophilisation.EFFECT: group of inventions provides higher bioavailability and anticancer activity of the drug.2 cl, 1 tbl, 3 ex

Description

The invention relates to medicine and relates to the creation of agents for the treatment of endocrine and hormone-dependent tumors.

The number of patients with hormone-dependent tumors is constantly growing. So, for example, about 60 thousand people get cancer of the stomach per year, 40 thousand intestines, and 15 thousand people get pancreas. The effectiveness of existing drugs in the treatment of endocrine tumors is only from 15 to 20% [1. Davydov M.I., Axel E.M. Malignant neoplasms in Russia and the CIS countries. M .: - 2005]. Therefore, the creation of fundamentally new domestic antitumor drugs that differ significantly in selectivity, toxicity and spectrum of action from cytostatics used in medical practice is very relevant today.

In recent years, fundamental biomedical science has achieved considerable success in studying the mechanism of malignant transformation of cells and the process of metastasis of neoplasms. This allowed us to identify new targets for exposure to potential antitumor agents [2. Lichinitser M.R., Stepanova E.V. Drug therapy of tumors in the XXI century. Gorbunova V.A. (ed.), Artamonova E.V., Bazin S.G. et al. Etudes of chemotherapy - M: Litterra, 2006. - S. 28-30 .; 2. Lichinscher M.R., Stepanova E.V. Targeted therapy is a new direction in the treatment of malignant tumors. Gorbunova V.A. (ed.), Artamonova E.V., Bazin S.G. et al. Studies of chemotherapy - M: Litterra, 2006. - S. 31-38.].

In the light of modern ideas about the importance of hormones in the development of hormone-dependent tumors, the search for fundamentally new antitumor substances among the peptide hormones of the hypothalamus that can selectively affect the processes of receptor-mediated interaction and participate in the transmission of intracellular signals [A. Schally AV, Comary-Schally AM, Nagy A. et. al. Hypotalamic hormones and cancer // Front. Neuroendocrinol. - 2001. - No. 22. - P. 248-291; b.Weckhecker G., Lewis I., Albert R., Schmidt H.A. et. al. Opportunities in somatostatin research: biological, chemical and therapeutic aspects // Nature Rev. Drug Discovery. - 2003. - Vol. 2. - P. 999-1017.].

Particular attention is drawn to the hypothalamic hormone somatostatin. One of the main functions of somatostatin in the body is to inhibit the secretion of growth hormone, along with somatostatin has a wide range of biological effects - inhibits the release of prolactin, insulin, glucagon, pancreatic hormones and the gastrointestinal tract, stimulating proliferative processes in the cell [6. Buscail L., Vernejoul K, Fame P., Torrisani J., Susini C. Regulation of cell proliferation by somatostatin // Ann Endocrinol. - 2002. - Vol. 63. - P. 2S13-8.]. The mechanism of inhibition of secretory and proliferative processes of somatostatin and its analogues is realized through specific receptors, which are widely represented on the cells of target tissues: in the central nervous system (hypothalamus, pituitary, spinal cord) and the gastrointestinal tract (mainly in the pancreas, stomach, upper parts of the thin intestines). High expression of somatostatin receptors on malignant tumor cells has been shown: gastrinoma, glucagon, carcinoid tumors, small cell lung cancer, and others [7. Nagy A., Schally A.V. Targeting cytotoxic conjugates of somatostatin, luteinizing hormone-releasing hormone and bombensin to cancer expressing their receptors: a "smarter" chemotherapy // Current Pharmaceutical Design. - 2005. - Vol. 11, No. 9 - P. 1167-1180.]. A distinctive feature of compounds of this class is the absence of toxicity and high selectivity of action. The disadvantage is that they metabolize rapidly under the action of proteinases. A short half-life of somatostatin in the blood (three minutes) is undesirable in its clinical use. Enzymatic stability of peptide hormones can be achieved by chemical modification of the amino acid chain of a somatostatin molecule, in particular, by replacing amino acids in the native peptide with non-natural amino acids [8. Schally A.V. Oncological applications of somatostatin analogs // Cancer Research. - 1988. - Vol. 48. - P. 6977-6985.].

The foreign drug currently used in clinical practice, the structural analogue of somatostatin, Sandostatin or octreotide, consists of 8 amino acids and has a cyclic structure. Indications for the use of sandostatin are tumors of the islet apparatus of the pancreas, pancreatic cancer, carcinoid tumors, medial thyroid cancer, pituitary adenomas [9. Comary-Schally A.M., Schally A.V. A clinical overview of carcinoid tumors: respectives for improvement in treatment using peptide analogs (review) // International Journal Of Oncology. - 2005. - Vol. 26 (2). - P. 301-309 .; 10. Schally A.V., Szepeshazi K., Nagy A. et al. New approaches to therapy of cancers of the stomach, colon and pancreas based on peptide analogs // Cellular and Molecular Life Sciences. - 2004. - Vol. 61. - P. 1042-1048.]. Taken as the base object.

In FSBI “RONTs im. N.N. Blokhin’s Ministry of Health of Russia, over the years, studies have been conducted to search for potential antitumor compounds in the range of analogues of hypothalamic hormones, in particular, somatostatin analogues.

Known agent with antitumor activity, which is a linear pentapeptide of the General formula:

Where

R is tert-butyl carbonyl or H,

R ₁ is tetrahydropyranyl or H,

R ₂ - N ³ - benzyloxycarbonyl or H,

R ₃ -O-methyl or S-tetrahydropyranylcysteine methyl ester. [eleven. RF patent RU 2254139 C1, published on June 20, 2005]. This compound has great activity and bioavailability when administered intraperitoneally, but has insufficient solubility in injection solvents (the suspension was administered intraperitoneally), which makes it difficult to administer them intramuscularly for therapy.

была создана пероральная лекарственная форма в виде таблеток, дополнительно содержащих в составе лактозу, микрокристаллическую целлюлозу, крахмал, поливилпирролидон (повидон), тальк и стеарат магния [12. Михаевич Е.И., Яворская Н.П., Голубева И.С., Орлова О.Л., Полозкова А.П., Партолина С.А., Оборотова Н.А. Вопросы биологической, медицинской и фармацевтической химии. - 2011. - №10. - С. 68-72.], которая в настоящий момент проходит первую фазу клинических исследований. При проведении клинических исследований выявлено, что биодоступность пероральной лекарственной формы слишком низкая, для больных необходима системная доставка препарата или значительного увеличения дозы активного вещества -Using the above compound

an oral dosage form was created in the form of tablets, additionally containing lactose, microcrystalline cellulose, starch, polyvylpyrrolidone (povidone), talc and magnesium stearate [12. Mikhailovich E.I., Yavorskaya N.P., Golubeva I.S., Orlova O.L., Polozkova A.P., Partolina S.A., Oborotova N.A. Questions of biological, medical and pharmaceutical chemistry. - 2011. - No. 10. - S. 68-72.], Which is currently undergoing the first phase of clinical research. When conducting clinical studies, it was found that the bioavailability of the oral dosage form is too low, for patients, systemic delivery of the drug or a significant increase in the dose of the active substance is necessary -

This tool is taken as a prototype.

The disadvantages of the prototype are:

1. The impossibility of systemic drug delivery to the patient.

2. Inadequate bioavailability.

3. A large dose for the patient (a large number of tablets at a time).

4. Lack of antitumor activity.

The objective of the invention is to provide an injectable agent for the treatment of hormone-dependent tumors.

дополнительно содержащее фосфатидилхолин, холестерин, DSPE-PEG-2000 и сахарозу при следующем молярном соотношении компонентов: 1,0: 71,1: 13,9: 0,2: 346,5. Фосфатидилхолин для формирования нановезикул, холестерин - прочность липосом, пегилирование липосом (DSPE-PEG-2000) - для предотвращения захвата липосомы ретикулоэндотелиальной системой (РЭС), сахароза - криопротектор для избежания «слипания» липосом при сублимации. Данное молярное соотношение компонентов обеспечивает получение липосом приемлемого размера (180±6 нм) с минимальными потерями активного вещества на стадии фильтрации и экструзии, а соответственно с самым высоким уровнем его включения в везикулы - (97±1) %. Изменение доли указанных вспомогательных веществ в соотношении как в сторону их уменьшения, так и в сторону увеличения способствовало снижению количества включенного активного вещества и укрупнению липосом.The problem is solved by the fact that the proposed tool for the treatment of hormone-dependent tumors, which is a lyophilizate for the preparation of a solution for injection, including

additionally containing phosphatidylcholine, cholesterol, DSPE-PEG-2000 and sucrose in the following molar ratio of components: 1.0: 71.1: 13.9: 0.2: 346.5. Phosphatidylcholine for the formation of nanovesicles, cholesterol - liposome strength, liposome pegylation (DSPE-PEG-2000) - to prevent liposome uptake by the reticuloendothelial system (RES), sucrose - a cryoprotectant to avoid liposome “sticking” during sublimation. This molar ratio of components provides liposomes of an acceptable size (180 ± 6 nm) with minimal loss of active substance at the stage of filtration and extrusion, and, accordingly, with the highest level of its incorporation into vesicles - (97 ± 1)%. A change in the proportion of these auxiliary substances in a ratio both to the side of their decrease and to the side of increase contributed to a decrease in the amount of active substance included and enlargement of liposomes.

The proposed tool was obtained using the Bengem method (Bangham) [13. Liposomes: a practical approach. In D. Rickwood and B.D. Hames (eds.). Practical Approach Series, oil Press at Oxford University Press, 1990; G. Gregoriadis (ed.), Liposome technology, Vol. 1, CRC Press, Boca Raton, Fl, 1984], adapted for insoluble substances.

, фосфатидилхолин, холестерин и DSPE-PEG-2000 в хлороформе. Далее упаривали образовавшейся хлороформный раствор на роторном испарителе под вакуумом до полного удаления хлороформа. Затем, диспергировали образованную при упаривании пленку водой для инъекций встряхиванием. Затем проводили фильтрацию полученной липосомальной дисперсии и экструзию (для получения более мелких липосом). Средний размер липосом после экструзии, измеренный на приборе Submicron Particle Sizer NICOMP-380, составлял 160-190 нм. После экструзии в липосомальную дисперсию дополнительно вводили раствор сахарозы, после чего дисперсию стерилизовали фильтрацией и дозировали. Добавление раствора сахарозы после экструзии обеспечивает высокое (более 90%) включение

в липосомы.The inventive tool was obtained as follows: dissolved

, phosphatidylcholine, cholesterol and DSPE-PEG-2000 in chloroform. Then the resulting chloroform solution was evaporated on a rotary evaporator under vacuum until complete removal of chloroform. Then, the film formed upon evaporation was dispersed by shaking water for injection. Then, the resulting liposomal dispersion was filtered and extruded (to obtain smaller liposomes). The average size of the liposomes after extrusion, measured on a Submicron Particle Sizer NICOMP-380, was 160-190 nm. After extrusion, a sucrose solution was additionally introduced into the liposomal dispersion, after which the dispersion was sterilized by filtration and dosed. The addition of sucrose solution after extrusion provides a high (over 90%) inclusion

into liposomes.

The liposomal dispersion obtained by this method, provided that it is stored at a temperature of + 2 ... + 8 ° C, is stable for a short period of time - for 5 days. During longer storage, a gradual fusion and the formation of larger vesicles and a decrease in the pH of the liposome dispersion were observed, which is due to the lipid peroxidation processes that make up the dosage form. This drawback prevents the implementation of mass production of the claimed funds. To increase the stability during storage and extend the shelf life, stabilization of the liposomal dispersion by freeze-drying is proposed, which allowed to increase the shelf life of the claimed drug up to 2 years. Freeze-drying is as follows:

0. Start, room temperature + 20 ... + 25 ° С

1. -15-17 ° C. Cooling from room temperature to solidification (occurred at the temperature of the products according to the sensors -2 ... -4 ° C), bringing the temperature to -7 ... -9 ° C, keeping for 50 minutes. - 2.5-3 hours.

2. -25 ... -27 ° C. Cooling from -8 ... -10 ° С to -18 ... -20 ° С (according to temperature sensors of products), keeping for 50 minutes. - 1.5-1.8 hours.

3. -35 ... -37 ° C. Cooling from -18 ... -20 ° С to -28 ... -30 ° С, keeping for 40 minutes. -1.3-1.6 hours.

4. -45 ... -47 ° C. - Cooling from -28 ... -30 ° С to -38 ... -40 ° С, keeping for 40 minutes. Upon reaching a shelf temperature of -40 ° C, the cooling condenser of the vacuum trap is turned on for 1.3-1.6 hours.

5. -50 ° C. Product cooling from -38 ... -40 ° С to the minimum attainable temperature -42 ... -45 ° С, balancing the temperature -1.0-1.2 hours.

After reaching and balancing the minimum temperature of the product, the vials with the preparation were kept for 3 hours, after which air pumping started. After turning on the vacuum pump, equalizing the vacuum and drying the surface layer of the frozen dispersion was carried out:

1. Heating the shelves to a temperature of -20 ° C at a speed of + 5 ° C / hour.

2. Holding the shelves at a temperature of -20 ° C for 2 hours

3. Heating the shelves to a temperature of -10 ° C at a speed of + 3 ° C / hour.

4. Heating the shelves to room temperature + 20 ... + 22 ° С with a speed of + 5 ° С / hour. After this, the preparation was dried up for a duration of about 3 hours according to the criterion of the invariability of the residual vapor pressure in the sublimation chamber when the vacuum line was closed. The total time of freezing and drying the drug was 36-38 hours.

The following are specific examples of the proposed injection tool (The inventive tool) for the treatment of hormone-dependent tumors (mg).

Example 1 88; Phosphatidylcholine - 4599; Cholesterol - 462; DSPE-PEG - 200058; Sucrose 10220; Deionized water up to 100 ml. Example 2 103; Phosphatidylcholine 5410; Cholesterol 543; DSPE-PEG-2000 68; Sucrose 12023; Deionized water up to 100 ml. Example 3 118; Phosphatidylcholine 6222; Cholesterol 624; DSPE-PEG-2000 78; Sucrose 13826; Deionized water up to 100 ml.

Comparative studies of the biological activity of the claimed drug for the treatment of hormone-dependent tumors and prototype

To study the antitumor efficacy of the inventive tool, female F ₁ hybrid mice (C ₅₇ Bl / ₆ × DBA / 2) were used. The studies were carried out on transplantable mammary adenocarcinoma Ca-755 of mice from the Bank of tumor strains of the Federal State Budget Scientific Center named after N.N. Blokhin »Ministry of Health of Russia. Strain Ca-755 was maintained on female C ₅₇ Bl / ₆ linear mice. For the experiment, Ca-755 was transferred to mice subcutaneously (s / c) in the right axillary region with 0.5 ml (50 mg) of a suspension of tumor cells at a dilution of 1:10 in medium 199 [14. Experimental evaluation of anticancer drugs in the USSR and the USA. Edited by Z.P. Sof'ina, A.B. Syrkina (USSR), A. Golge, A. Klein (USA) M .: "Medicine". - 1980. - S. 71-112.]. The control group consisted of 10 mice, the experimental group of 8 animals. Treatment was started 48 hours after tumor transplantation [15. Translators N.I., Gorbunova V.A. (Ed.) Guidelines for Chemotherapy of Tumor Diseases. M .: Practical medicine. - 2015 .-- 686 p .; 16. Guidelines for preclinical studies. Part one, (edited by A.N. Mironov, Federal State Budgetary Institution NTsESMP of the Ministry of Health of Russia.) - M., 2012 - 944 p .; 17. Bolshakov O.P., Neznanov N.G., Babakhanyan R.V. Didactic and ethical aspects of research on biomodels and laboratory animals. WHO, 2000. Recommendations to ethics committees examining biomedical research // Qualitative clinical practice. - 2002. - No. 9. - S. 1-15.].

In order to determine the optimal route of administration, at which the greatest efficiency is achieved, sc and intravenous (iv) methods of administration of the claimed drug were used.

A comparative study of the antitumor activity of the experimental model of the proposed drug, depending on the method of administration on Ca-755, showed the advantage of sc administration of 5-fold administration. So, a statistically significant high antitumor effect at doses of 10 and 20 mg / kg persisted for 14-18 days after the end of treatment: inhibition of tumor growth (TPO) = 83-65% and TPO = 82-62%, respectively. Moreover, the increase in life expectancy (LLL) of mice with Ca-755 when s / to the introduction of the inventive tool at a dose of 10 and 20 mg / kg was 28 and 45%, respectively, with a minimum activity criterion ≥25%.

With iv administration at doses of 5 and 10 mg / kg, a short-term antitumor effect of the claimed drug was observed immediately after treatment: TPO = 71% and TPO = 85%, respectively. With the on / in the introduction of a dose of 20 mg / kg, moderate antitumor activity persisted for 7 days: TPO = 70-57%. A comparative evaluation of the effectiveness of the claimed drug for iv administration and the Prototype for oral (per os) administration to mice with Ca-755 revealed a similar antitumor effect at a dose of 20 mg / kg for 7 days after the end of treatment: TPO = 10-51% and SRW - 59-53%, respectively.

The invention is illustrated in the table, which shows a comparative study of the antitumor activity of the claimed drug, depending on the method of administration on Ca-755.

It was shown that the antitumor activity of the claimed drug when sc is administered in comparison with the prototype when per os applied to Ca-755 mice is more effective. So at doses of 10 and 20 mg / kg, the duration of the antitumor effect of the claimed drug with sc administration was observed for 14-18 days and amounted to TPO = 87-65% and TPO = 87-62%, respectively. Whereas, per os the introduction of similar doses of the prototype inhibited the growth of Ca-755: TPO = 79-50% and TPO = 59-53%, respectively. The antitumor effect was maintained for 7 days after the end of treatment.

Claims (3)

1. An agent for treating breast adenocarcinoma, including N ^α -t-butyloxycarbonyl-S-tetrahydropyranylcysteinyl-phenylalanyl-D-tryptophil-N ^ε -benzyl-hydroxycarbonyllysyltreonine (P) methyl ester, characterized in that it is a lyophilisate for solution for injection, additionally contains phosphatidylcholine (PF), cholesterol (X) and DSPE-PEG-2000 (PEG-lipid) and sucrose (C) in a molar ratio of components: M _P : M _PX : M _X : M _PEG-lipid : M _C = 1.0: 71.1: 13.9: 0.2: 346.5. 2. A method of obtaining a means for treating breast adenocarcinoma according to claim 1, comprising dissolving methyl ester N ^α -t-butyloxycarbonyl-S-tetrahydropyranylcysteinyl-phenylalanyl-D-tryptophil-N ^ε- benzyl-hydroxycarbonyllysyltreonine (P), phosphatidylcholine , cholesterol (X) and DSPE-PEG-2000, taken in a proportion of 1.0: 71.1: 13.9: 0.2, evaporation of the resulting chloroform solution on a rotary evaporator under vacuum until the chloroform is completely removed, dispersion of the film formed by evaporation water for injection, filtration and extrusion liposomal dispersion, the addition of a solution of cryoprotectant - sucrose to a molar ratio M _P : M _C = 1.0: 346.5, dosing and stabilization by lyophilization for 36-38 hours.