RU2494742C1 - Agent inhibiting multiple drug resistance of tumour cells - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, veterinary science and pharmaceutical industry. The invention provides using triterpene glycosides from holothuria that is frondoside A or a complex of frondoside A and cholesterol as an agent for inhibiting the multiple drug resistance of tumour cells, and for preparing a pharmaceutical composition inhibiting the multiple drug resistance of tumour cells.

EFFECT: using the invention enables extending the range of products inhibiting the multiple drug resistance of tumour cells.

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Description

The invention relates to medicine and veterinary medicine and relates to agents that inhibit the multidrug resistance of tumor cells.

Resistance of tumor cells to many toxic antitumor drugs simultaneously, which can be used for chemotherapy of oncological diseases that are dissimilar in chemical structure and mechanism of action, has long been a known phenomenon called multidrug resistance (MDR) [Endicott JA, Ling V. The biochemistry of P -glycoprotein-mediated multidrug resistance // Annu. Rev. Biochem. 1989. V. 58. P.137-171;

Stavrovskaya A.A. Tumor cell in defense // Soros Educational Journal. 2001. V.7, No. 7. S.17-23; Stavrovskaya A.A. Cellular mechanisms of multidrug resistance of tumor cells // Biochemistry. 2000.V.65, Issue 1. S.112-126]. Cells that have MDR or acquire it during chemotherapy (by artificial selection under the influence of poisons) become resistant to the action of drugs, and their destruction or inhibition of their proliferation requires the use of chemotherapeutic drugs-cytostatics, such as cyclophosphamide, doxorubicin, vinblastine, etoposide, fluorouracil, in such large doses, which are often incompatible with the survival of patients.

The most common MDR mechanism is the activation of transmembrane transport proteins that remove various substances from the cell. The main protein of this type is P-glycoprotein [Endicott J.A., Ling V. The biochemistry of P-glycoprotein-mediated multidrug resistance // Annu. Rev. Biochem. 1989. V. 58. P. 137-171]. R-glycoprotein acts as a pump, which, using the energy of ATP, can pump a wide variety of substances from the cell, including fluorescent dyes that freely penetrate through cell membranes by diffusion. Using such dyes, the functional activity of P-glycoprotein can be determined by comparing the fluorescence of resistant cells and cells treated with a P-glycoprotein inhibitor. Fluorescence of cells in which P-glycoprotein is inhibited will be significantly higher due to the lack of active transport of dye from the cells.

Known drugs that help overcome the multidrug resistance of tumors. Such substances include verapamil, cyclosporin A, nifedipine and its derivatives, amiodarone, trifluorkerazine, quinine and others [Ford J.M., Halt W.N. Pharmacology of drugs that alter multidrug resistance in cancer. // Pharmacol. Res. 1990. V. 42. P.155-199]. The disadvantage of these compounds is the presence of serious side effects in the doses necessary to enhance the action of antitumor drugs. In particular, verapamil, an effective inhibitor of P-glycoprotein, causes heart failure and brain damage, and cyclosporin A has immunosuppressive activity.

In this regard, the search for MDR inhibitors among natural compounds with less pronounced side effects seems relevant. So, among natural substances that inhibit MDR, mention is made of avermectins - substances of a macrolide nature, obtained by microbial biosynthesis [RU 2250775 C2, 2002.11.06].

Also known are synthetically modified natural triterpenoids that inhibit the proliferation of cells with MDR [RU 2393165 C2, 07.17.2008].

As substances possessing various types of biological activity, natural compounds of marine origin, including triterpene glycosides from holothurium, attract attention. Saponins, or glycosides, consisting of a carbohydrate chain and triterpene or steroid aglycone, are widespread in plants. However, the presence of triterpene glycosides is characteristic of most holothurians belonging to the class Holothurioidea (type Echinodermata) and for some sponges. Holoturia triterpene glycosides have lanostane type aglycones, most have 18 (20) -lactone aglycones and belong to the holostane series. Holothuria glycoside carbohydrate chains are composed of 2-6 monosaccharide residues, including xylose, quinose, glucose, 3-0-methylglucose, and (sometimes) 3-O-methylxylose, 3-O-methylquinose, 3-0-methylglucuronic acid, and 6-O-acetylglucose. They may contain one, two or three sulfate groups. [Stonik VA, Kalinin VI, Avilov SA, Toxins from sea cucumbers (Holothuroids): chemical structures, properties, taxonomic distribution, biosynthesis and evolution // J. Nat. Toxins. 1999. V.8. P.235-248; Kalinin VI, Silchenko AS, Avilov SA, Stonik VA, Smimov AV Sea cucumbers triterpene glycosides, the recent progress in structural elucidation and chemotaxonomy // Phytochemistry Reviews. 2005. V.4, No.2-3. P.221-236; Avilov SA, Silchenko AS, Antonov AS, Kalinin VI, Kalinovsky AI, Smimov AV, Dmitrenok PS, Evtushenko EV, Fedorov SN, Savina AS, Shubina LK, Stonik VA Synaptosides A and Ai, two triterpene glycosides from the sea cucumber Synapta maculata containing 3-O-methylglucuronic acid and their cytotoxic activity against tumor Cells // J. Natural Prod. 2008. V.71, No.4. P.525-531; Antonov AS, Avilov SA, Kalinovsky AI, Anastyuk SD, Dmitrenok PS, Evtushenko EV, Kalinin VI, Smirnov AV, Taboada S., Ballesteros M., Avila C., Stonik VA Triterpene glycosides from Antarctic sea cucumbers I. Structure of liouvillosides A ₁ , A ₂ , A ₃ , B ₁ and B ₂ from the sea cucumber Staurocucumis liouvillei, new procedure for separation of highly polar glycoside fractions and taxonomic revision // J. Nat. Prod. 2008. V.71. P.1677-1685].

Due to the ability to form a complex with 5 (6) -unsaturated sterols of cell membranes, glycosides have a wide spectrum of biological activity, including hemolytic, antifungal, cytotoxic, and many other types of membranotropic effects [Kalinin V.I., Aminin D.L., Avilov S.A., Silchenko A.S., Stonik V.A., Stonik V.A. Triterpene glycosides from sea cucucmbers (Holothurioidae, Echinodermata), biological activities and functions // Studies in Natural Product Chemistry (Bioactive Natural Products) / Ed. Atta-ur-Rahman. V.35. P.135-196. The Netherlands: Elsevier Science Publisher, 2008].

However, the action of holothuria glycosides in subcytotoxic doses is of particular interest. These substances are characterized by two binding constants with biomembranes in cytotoxic and subcytotoxic doses [Pislyagin E.A., Gladkikh R.V., Kapustina I.I., Kim N.Yu. Shevchenko V.P., Nagaev I.Yu. Interaction of holothurian triterpene glycoside with biomembranes of mouse immune cells // International Immunopharmacology. 2012. V.14. P.1-8.].

Possibly, these last subtle interactions are related to their stimulating effect on cellular immunity, as well as on the antitumor effect, in which glycosides can cause apoptosis, both in vivo and in vitro [Marzouqi N., Iratni R., Nemmar A ., Arafat K., Al Sultan MA, Yasin J., Collin P., Mester J., Adrian T.E., Attoub S. Frondoside A inhibits human breast cancer cell survival, migration, invation and the growth of brest tumor xenografts // Eur. J. Pharmacology. 2011. V.668, No.1-2. P.25-34; Jin J.-O., Shastina VV, Shin S.-W., Park J.-I., Rasskazov VA, Avilov SA, Fedorov SN, Stonik VA, Kwak J.-Y. Differential effects of triterpene glycosides, fromdoside A and cucumerioside A ₂ -2 isolated from sea cucumbers on caspase activation and apoptosis of human leukemia cells // FEBS Lett. 2009. V.583. P.697-702; Jin J.-O., Shastina VV, Shin S.-W., Park J.-I., Rasskazov VA, Avilov SA, Fedorov SN, Stonik VA, Kwak J.-Y. Differential effects of triterpene glycosides, frondoside A and cucumerioside A ₂ -2 isolated from sea cucumbers on caspase activation and apoptosis of human leukemia cells // FEBS Lett. 2009. V.583. P.697-702].

In order to reduce the cytotoxic effect of glycosides and preserve their immunomodulating and antitumor properties, a complex of monosulfated glycosides from holoturia Cucumaria japonica (mainly cucumarioside A ₂ -2) with cholesterol (preparation Kumazid) was obtained [Stonik V.A., Aminin D.L. ., Boguslavsky V.M., Avilov S.A., Agafonova I.G., Silchenko A.S., Ponomarenko L.P., Prokofieva N.G., Chaykina E.L. Immunomodulating agent "Kumazid" and a pharmaceutical composition based on it // RU 2271820 C1, 02.07.2004; Aminin DL, Pinegin BV, Pichugina LV, Zaporozhets TS, Agafonova IG, Boguslavsky VM, Silchenko AS, Avilov SA, Stonik VA Immunomodulatory properties of Cumaside // Int. Immunopharm. 2006. V.6. P.1070-1082; Aminin DL, Chaykina EL, Agafonova IG, Avilov SA, Kalinin VI, Stonik VA Antitumor activity of the immunomodulatory lead Cumaside // Intern. Immunopharm. 2010. V.10. P.648-654.]. The drug Kumazid has an in vivo antitumor effect on mouse Ehrlich carcinoma, and also enhances the antitumor effect of fluorouracil.

It is known that cucumarioside A ₂ -2 and its complex with cholesterol (drug Kumazid) block the multidrug resistance of tumor cells [D.L. Aminin and others. New immunomodulating drug Kumazid. // Materials of the VI scientific-practical conference Fundamental science - medicine. June 2, 2011 Vladivostok, C.11-12].

However, data on the inhibition of MDR by frondoside A and its complex with cholesterol and compositions based on them in the patent and other scientific and technical literature were not found.

The objective of the invention is the expansion of the arsenal of drugs that inhibit the multidrug resistance of tumor cells.

The problem was solved by the use of frondoside A from the Okhotsk Sea fishing holothuria Cucumaria okhotensis or its complex with cholesterol as an agent that inhibits the multidrug resistance of tumor cells.

The problem was also solved by the use of frondoside A or its complex with cholesterol for the preparation of a pharmaceutical composition that inhibits the multidrug resistance of tumor cells.

Glycosides are obtained by isolation and purification from natural raw materials. The process of isolation of triterpene glycosides from holothurians, in particular, frondoside A, is a standard procedure described, for example, by A. Silchenko. and co-authors [Silchenko A.S., Avilov S.A., Kalinin V.I., Stonik V.A., Kalinovsky A.I., Dmitrenok P.S., Stepanov V.G. Monosulfated triterpene glycosides of Cucumaria okhotensis, a new species of holothurians from the Sea of Okhotsk // Bioorg. chemistry. 2007. Vol. 33, No. 1. S.81-90; Silchenko AS, Avilov SA, Kalinin VI, Kalinovsky AI, Dmitrenok PS, Fedorov SN, Stepanov VG, Dong Z., Stonik VA Constituents of the sea cucumber Cucumaria okhotensis. Structures of okhotosides B ₁ -B ₃ and cytotoxic activities of some glycosides from this species // J. Nat. Prod. 2008. V.71, No.3. P.351-356].

The essence of the method is as follows: animals are crushed and extracted twice with hot ethanol; the combined extracts are evaporated to dryness on a rotary evaporator and the dry residue is chromatographed on a column of Teflon powder or other hydrophobic carrier, equilibrated in water. Inorganic salts and polar impurities are washed out of the column with water, and the fraction containing glycosides is washed with 50% ethanol in water. Then the fraction is applied to a column of silica gel; eluent - chloroform-ethanol-water system (100: 100: 17). As a result, two main fractions are distinguished: the first contains monosulfated glycosides, and the second contains more polar glycosides. The individual monosulfated glycoside (frondoside A) is then isolated by high performance column chromatography (HPLC) using reverse phase columns.

The essence of the method for preparing the complex is as follows: frondoside A is dissolved in butanol, then a solution of cholesterol in aqueous butanol is added to the resulting solution (1 g of cholesterol in 60 ml of butanol saturated with water). The mixture was left overnight, after which the precipitated white precipitate was separated by centrifugation, washed with a small amount of ethanol and diethyl ether, and dried.

Pharmaceutical compositions that inhibit the multidrug resistance of tumor cells are prepared by mixing an effective amount of frondoside A with traditional pharmaceutically or pharmacologically acceptable excipients. The expression "pharmaceutically or pharmacologically acceptable" means molecular components and compositions that do not cause negative, allergic or other undesirable reactions when administered to an animal or person. The nature of the filler depends on the route of administration. For example, if oral administration is desired, a solid excipient may be chosen, while liquid saline may be used for intravenous administration.

The technical result provided by the invention lies in the ability of frondoside A or its complex with cholesterol to cause inhibition of multidrug resistance in tumor cells, as well as in the possibility of using frondoside A and its complex with cholesterol to prepare pharmaceutical compositions that inhibit multidrug resistance of tumor cells.

The invention expands the arsenal of agents and pharmaceutical compositions that inhibit the multidrug resistance of tumor cells.

Figure 1 shows the structure of the frontdoside A.

Figure 2 shows the effect of verapamil on the multidrug resistance of Ehrlich carcinoma cells, determined using a Calcein AM fluorescence probe.

Figure 3 shows the effect of frondoside A on the multidrug resistance of Ehrlich carcinoma cells, determined using a Calcein AM fluorescence probe.

Figure 4 shows the effect of the complex of frondoside A with cholesterol on the multidrug resistance of Ehrlich carcinoma cells, determined using a Calcein AM fluorescence probe.

The preparation of pharmaceutical compositions is illustrated by the following examples:

Example 1. Frondoside A in an effective amount is mixed with an appropriate amount of a mixture of starch and gypsum and pressed into a tablet.

Example 2. The complex of frondoside-A with cholesterol in an effective amount is mixed with an appropriate amount of starch and placed in a gelatin capsule.

Example 3. Frondoside A in effective concentrations is dissolved in saline.

Example 4. The complex of frondoside A with cholesterol in effective concentrations is suspended in saline in an ultrasonic bath.

The study of biological activity

I. Materials and methods

1. Triterpene glycosides

The ability of frondoside A and its complex with cholesterol to inhibit the multidrug resistance of tumor cells was studied. According to ¹³ C NMR spectroscopy, frontside A is an individual compound. Verapamil, whose ability to inhibit MDR of tumor cells, and more specifically P-glycoprotein, was previously known was used as a control.

Individual compounds were dissolved in sterile distilled water at a concentration of 1 mg / ml (initial concentration). A series of appropriate dilutions were made from this initial concentration. Suspensions of the glycoside-cholesterol complex were prepared by dispersing the complexes in sterile distilled water using an ultrasonic bath at a concentration of 1 mg / ml. A series of appropriate dilutions were made from this initial concentration.

2. Determination of the inhibition of multidrug resistance of tumor cells.

A museum tetraploid strain of mouse Erlich ascites carcinoma cells from the All-Union Cancer Center of the Russian Academy of Medical Sciences (Moscow) was used in the experiments. Ehrlich ascites carcinoma cells were passaged in the abdominal cavity of white outbred mice of both sexes weighing 18-20 g. For the experiment, cells were selected on the 7-10th day after tumor inoculation. For this, the mice were sacrificed by the method of perivisceral dislocation and ascites fluid containing tumor cells was collected using a syringe. Cells were washed three times from exudate by centrifugation at 1500 rpm (450 g) for 5 min in Dulbecco's phosphate buffered saline (PBS) of the following composition: NaCl - 137 mM; KCl - 2.68 mmol; Na ₂ HPO ₄ × 7H ₂ O - 8.06 mmol; NaH ₂ PO ₄ - 1.47 mmol, pH 7.4. The final cell concentration in the incubation medium was 2-5 × 10 ⁶ cells / ml.

Ehrlich ascites carcinoma cells at a concentration of 5 × 10 ⁵ cells / ml, 100 μl each, were added to each well of a 96-well plate containing 50 μl of the test substances in concentrations from 0.04 μg / ml to 120 μg / ml and incubated for 15 min at 37 °. Then 50 μl of Calcein AM (final concentration 0.25 μM) was added and incubated for 15 min at 37 ° C. Then the plates were centrifuged for 5 min at 4 ° C, 1500 rpm 3 times, each time draining the supernatant and adding 200 μl of culture medium to each well. Fluorescence was measured using a Fluoroscan Accent flat-format spectrofluorimeter (Finland) at λEx = 494 nm, λem = 517 nm. The fluorescence intensity of randomly selected 100 cell images was estimated using the AQM Advance 6 software (Kinetic Imaging Ltd., UK) and calculated from the average fluorescence intensity of each cell, expressed in pixels. In addition, the geometric parameters of the cells (area, perimeter, maximum chord and shape) were evaluated.

The experiment was repeated three times. Mean values, standard error and standard deviations were calculated and plotted using the SigmaPlot 3.02 software (Jandel Scientific, USA).

II. The effect of frondoside A, as well as its complex with cholesterol on multidrug resistance.

An MDR study was performed on Ehrlich ascites carcinoma cells using a calcein AM fluorescence probe. In normal cells, the probe easily penetrates through the plasma membrane into the cytoplasm and, under the influence of esterases, it turns into an unesterified form of calcein, and begins to fluoresce in the green region of the spectrum. In MDR cells, super-expression of P-glycoprotein is observed, which prevents the probe from entering the cell. Known standard blockers of P-glycoprotein, such as verapamil, which eliminate the effect of MDR and thereby increase the sensitivity of tumor cells to cytostatics and cytotoxins and can play the role of a positive control.

The blocking of MDR by the studied glycoside and its complex with cholesterol was studied. The results are presented in figure 3, 4.

The experiments showed that frontside A, as well as its complex with cholesterol, effectively block P-glycoprotein in the studied concentration range from 0.001 to 1 μg / ml, as a result of which the number of probe in the cytoplasm increases. The highest fluorescence of the probe was observed under the action of frondoside A or its complex with cholesterol at a concentration of 0.001 μg / ml, and the increase in intensity was about 54% with respect to the control. As a comparison drug used verapamil in a concentration range from 0.012 to 120 μg / ml (figure 2). The maximum effective concentration of verapamil was shown to be 0.012 μg / ml. This corresponded to an increase in probe fluorescence of approximately 45-50% relative to the control.

Thus, frondoside A, as well as its complex with cholesterol, inhibits the multidrug resistance of tumor cells, which is comparable in action with the known effective blocker of P-glycoporotein verapamil, which, due to its high cardiotoxicity, has not been used in the treatment of tumor diseases.

The studied triterpene glycoside, its complex with cholesterol and pharmaceutical compositions based on them can be used as therapeutic agents that inhibit the multidrug resistance of tumor cells in very low concentrations.

Claims (2)

1. The use of frontside A or a complex of frontside A with cholesterol as an agent that inhibits the multidrug resistance of tumor cells. 2. The use of frontside A or a complex of frontside A with cholesterol for the preparation of a pharmaceutical composition that inhibits multidrug resistance of tumor cells.

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