[The mechanisms of anti-inflammatory action of enisamium iodide].

AIM: The role of cyclooxygenases (COX-1 and/or COX-2), transcription nuclear factor NF-κB, anti-inflammatory cytokines - TGF1b, IL-4, IL-10 and pro-inflammatory cytokines IL-1, IL-6 were studied to substantiate the expediency of antiviral agent enisamium iodide (Nobazit) using to regulate key inflammatory components in acute respiratory infections, IL-8, TNF-alpha in the realization of the pharmacological activity of this drug. MATERIALS AND METHODS: Gene expression was determined by real-time RT-PCR, the concentration of interleukins was determined by ELISA, and the viability of peripheral blood mononuclear cells (PBMC) was assessed by the MTT spectrophotometric method. The chemiluminescence method was used to assess PBMC oxidant activity. RESULTS: Enisamium iodide (10 µM) reduced mRNA levels of COX-1, COX-2, NF-κB, TGF1b, IL-1, IL-6 in stimulated PBMC of healthy donors by an average of 48% (p≤0.05). At 5 times higher concentration, 50 µM, enisamium iodide suppressed the expression of these genes by an average of 43% (p≤0.05). At a concentration of 100 µM, enisamium iodide reduced the expression of COX-2, TGF1b, IL-1, IL-6 by an average of 47% (p≤0.05). At a concentration of 10 µM, enisamium iodide stimulated the secretion of IL-10 by mononuclear cells by 1.2 times, p≤0.05. The tested drug at a concentration of 50 µM did not affected on the concentration of IL-1, IL-4, IL-8 and TNF-alpha, but significantly stimulated the production of IL-10 by 1.5 times, p≤0.05. The chemiluminescence method revealed that enisamium iodide in the entire concentration range (10-100 µM) does not reduce the viability of macrophages, but inhibits their oxidative activity (maximum value of CL intensity) by an average of 55% (p≤0.05). CONCLUSION: The anti-inflammatory effect of enisamium iodide at a concentration of 10 µM may be associated with inhibition of the expression of COX-1, 2, NF-κB, IL-1, IL-6, TGF1b and an increase in the expression and production of IL-10. An additional contribution to the anti-inflammatory activity of enisamium iodide is made by its antioxidant and antiradical activity. The absence of the effect of enisamium iodide (10-100 µM) on the viability of PBMC indicates its safety for the cells of the immune system and the expediency of using it to suppress inflammatory reactions in acute respiratory infections, restore the quality of life of patients and the possibility of using Nobazit as an effective agent for treatment of these infections of various etiologies.

[INFLUENCE OF PROGESTERONE DERIVATIVES ON THE VIABILITY AND EXPRESSION OF ESTROGEN RECEPTOR-ALPHA MRNA IN HELA CELLS.]

We have studied the effect of new ligands of progesterone receptors, including pregna-D'-pentaran 6-methoxyimino-16a,17a-cyclohexanopregn-4-en-3,20-dio-ne (K1047), 17a-acetoxy-3b-butanoyloxy-6-methylpregna-4,6-dien-20-one (buterol), progesterone (P4), and medroxyprogesterone acetate on the viability of HeLa cells and expression of estrogen receptor-alpha (Era) mRNA gene in these cells. K1I047 and buterol exhibited high cytostatic activity, which exceeded the activity of reference compounds on the average by 15% (p < 0.05). Both buterol and K-1047 (at 10(-6)M) effectively suppressed ERa mRNA gene expression in HeLa cell culture by 83.4 - 9 8.6%.

[Antitumor and antiproliferative action of the steroidal cytostatic antiestrogen cytestrol acetate on hormone-dependent tumor models].

Cytestrole acetate (CA), in the structure of which the steroidal antiestrogen component is associated with bis-ß-cloroethylamino group, exhibits a strong cytotoxic activity against hormone-dependent cancer cell lines (CaOV, HeLa, MCF-7). In doxorubicin-resistant MCF-7 cells, CA potentiates the cytotoxic effect of etoposide and doxorubicin, and the IC50 for CA in these cells is 40 times lower than that for tamoxifen (TAM). In transplantable mice breast adenocarcinoma Ca-755, the therapeutic CA dose is 25 mg/kg when administered subcutaneously in oil solution for 5 days. On the DMBA-induced mammary tumors in rats, CA injected subcutaneously led to partial regressions 4 weeks after treatment in 75% of test rats, whereas TAM produced this effect in 43% of rats. Among various drug forms of CA, the most active were oil solution of CA in gelatin capsules for oral use and liposomal emulsion for intravenous administration, since these forms exhibited the highest values of Ca-755 tumor growth inhibition index (TGI = 97 - 98%).

[Antiproliferative and antioxidant activity of new dihydroquercetin derivatives].

Effect of nine new derivatives of dihydroquercetin (taxifolin) on the viability of cultivated normal and tumor cells, their antioxidant activity, and interconnection of the antioxidant activity with the chemical structure have been studied. Among these dihydroquercetin derivatives, the maximum antiproliferative activity on the model of rat fibroblast culture exhibited KN-2, KN-4, KN-7, and KN-8 compounds, while KN-7 and KN-8 compounds also showed maximum activity on the model of MCF-7 tumor cell culture (human breast cancer). The maximum general antioxidant activity was observed for the native dihydroquercetin and KN-8 compound. There is a strong correlation (with a correlation coefficient of 0.93) between the antiproliferative effects of dihydroquercetin derivatives on murine skin fibroblasts and MCF-7 cells (human breast cancer).

[Pharmacological properties of complex iron oxide nanoparticles entering in magnetic resonance tomography contrast agent].

A colloidal solution of iron oxide nanoparticles has been obtained that ensures an increase in the accuracy of diagnostic information from magnetic resonance tomography (MRT) due to the acceleration of proton relaxation in tissues, which improves the contrast of T1- and T2-weighed images. Improved visualization of small vessels in rat brain has been observed after intravenous injection of 0.1 ml solution containing 5.0 mg of contrasting iron nanoparticles. The paramagnetic properties of iron oxide nanoparticles were studied by the method of proton relaxation, and their size was determined by the method of transmission electron microscopy. The toxic properties ofnanoparticles were determined by their effect on cultured HeLa cells (MTT test). It is recommended to use a colloidal solution of superparamagnetic nanoparticles (size, 8.2 nm) for obtaining a pharmaceutical form of the new magnetic-resonance contrast medium Ferotrast.

[Interaction of the new gestagen compound 17alpha-acetoxy-3beta-butanoyloxy-6-methylpregna-4,6-dien-20-one with human blood proteins and calf embryo serum].

Molecular mechanisms of sex hormones (progesterone, medroxyprogesterone acetate (MPA), and new synthetic gestagen 17alpha-acetoxy-3beta-butanoyloxy-6-methylpregna-4,6-dien-20-on (ABMP) with human serum albumin, globulins, and calf embryo serum were studied. The binding of ABMP to albumin and progesterone-binding proteins were investigated using spectroscopic techniques (with the use of 1-anilino-8-naphthalenesulfonate as fluorescent probe) and radiolabeled progesterone, (either progesteron or MPA as comparative progestines). There is no difference between the non-specific binding of ABMP, progesterone, and MPA, but the ABMP binding is much smaller as compared to the binding of progestines, so that the new progestine ABMP will produce a more effective action on the target tissue than comparative progestines.

[A new Russian gestagen with anticancer activity].

The authors present results obtained in a complex study of 17alpha-acetoxy-3beta-butanoyloxy-6-methyl-pregna-4,6-dien-20-on (ABMP). ABMP was shown to differ from existing analogues by high gestagen activity and prolonged action. The substance does not possess androgenic or mineralocorticoid activity, is not toxic when used in high doses, and possesses significant cytostatic and chemiosensitizing activity. These properties of ABMP demonstrate that the substance should be studied in clinical setting as a gestagen with anticancer and chemiosensitizing activity for further application to therapy of hyperplastic processes in the female genital system and tumors that are sensitive to female sex hormones.

Combined effect of bis-beta-chloroethylamine estrogen derivatives and doxorubicin on proliferation of sensitive and resistant MCF-7 cells.

The sensitivity of normal (MCF-7/WT) and doxorubicin-resistant (MCF-7/R) breast cancer cells to the antiproliferative effect of ethynylestradiol 11alpha-derivatives with the cytostatic residue in the 3-position of the steroid ring (antiestrogen cytostatics) was studied by evaluating cell viability using methylthiazole tetrazolium staining. The antiproliferative effects of these agents on cell lines in the presence of doxorubicin were compared. Antiestrogen cytostatics produced weaker cytostatic effect on MCF-7/WT cells, but more potent cytostatic effect on MCF-7/WT cells compared to those of doxorubicin. Moreover, administration of these agents in combination with doxorubicin more significantly suppressed proliferation of tumor cells. Accumulation and efflux of cytostatic doxorubicin in MCF-7/R cells were studied in the presence and absence of antiestrogen cytostatic Po716. Confocal laser microscopy showed that doxorubicin accumulation in MCF-7/R cells in the absence of Po716 took 20 min, while in the presence of antiestrogen cytostatic this process took 5 min. The rate of doxorubicin transport from tumor cells was much lower in the presence of the test antiestrogen cytostatic. Our results suggest that antiestrogen cytostatics increase the sensitivity of resistant MCF-7/R cells to doxorubicin by modulating the mechanisms of multidrug resistance of tumor cells.

[Genomic and extragenomic mechanisms of antiglucocorticoid action of gestagens on thymocytes].

Effects of a new synthetic progesterone derivative 17a-acetoxy-3b-butanoyloxy-6-methyl-pregna-4,6-dien-20-one (ABMP) and the reference gestagen preparations on the rat thymus were evaluated by the degree of variation of the intracellular levels of calcium and cAMP, 3H-uridine inclusion into RNA, thymocyte viability, and thymus mass. It is shown that gestagens can produce antiglucocorticoid action on thymocytes, this activity being most pronounced in the case of ABMP.

[Antitumor activity of the new gestagen 17alpha-acetoxy-3beta-butanoyloxy-6-methyl-pregna-4,6-dien-20-one]. / Izuchenie protivoopukholevoi aktivnosti novogo gestagena 17 al'fa-atsetoksi-3 beta-butanoiloksi-6-metil-pregna-4,6-dien-20-ona.

Antitumor activity of a new highly active promising gestagen 17alpha-acetoxy-3beta-butanoyloxy-6-methyl-pregna-4,6-dien-20-one (butagest) was studied in mice with model cervical carcinoma (RShM-5). The reference drug was medroxyprogesteron acetate (MPA, Depo Provera) used in clinics. The new preparation introduced perorally in a dose of 1 mg per mice inhibited the model tumor growth by 73%, which was 18% (p < 0.01) more effective than the action of the reference drug MPA. The effect of the new gestagen was also studied in vitro with respect to human breast carcinoma of the MCF-7 line and human cervical carcinoma HeLa. The viability of the tumor cells was studied during a 6-day incubation with the drug at a concentration of 10(-7)-10(-5) M (MTT test). The reference compounds were progesterone and MPA. These drugs suppressed the growth of both MCF-7 and, in higher concentrations, of HeLa. Butagest inhibited the growth of HeLa in all concentrations. Thus, the new gestagen 17alpha-acetoxy-3beta-butanoyloxy-6-methyl-pregna-4,6-dien-20-one is capable of suppressing the viability of human breast carcinoma and human cervical carcinoma, being comparable or even more effective than the reference drugs.

Combined action of doxorubicin and gestagens on doxorubicin-sensitive and doxorubicin-resistant MCF-7 cells.

The combined cytostatic effect of doxorubicin and gestagens progesterone, medroxyprogesterone acetate, mecigestone, and butagest on doxorubicin-resistant and doxorubicin-sensitive human breast cancer MCF-7 cells was studied by the MTT assay. On the 6th day of incubation progesterone, medroxyprogesterone acetate, mecigestone, and butagest in high concentrations (10(-5) M) potentiated the cytostatic action of doxorubicin in sensitive and resistant cells by 30-50%. Potentiation of the cytostatic effect produced by doxorubicin in sensitive cells is related to intrinsic cytotoxic activity of gestagens. In resistant cells these changes are associated with potentiation of the effect of doxorubicin.

Effect of richlocaine on proliferative activity of osteoblasts and intracellular calcium content in rats.

We studied the effect of local anesthetic richlocaine on proliferation and intracellular calcium content in cultured osteoblasts from rat parietal bone. In a concentration of 1 mg/ml this drug produced a cytotoxic effect on osteoblasts. In concentrations of 0.01 and 0.001 mg/ml richlocaine in the absence and presence of subtoxic dose of sodium cyanide (0.2 mM) increased the number of osteoblasts by 15.4 and 36.6 or 13.8 and 38.6%, respectively. In a concentration of 1 mg/ml, richlocaine increased the content of cytosolic calcium in osteoblasts by 105%. These effects of richlocaine in low concentrations (0.01 and 0.001 mg/ml) can be related to stimulation of metabolic processes in osteoblasts.

Influence of estrogen cytostatics on activity of plasma membrane enzymes 5'-nucleotidase and N+-K+-ATPase.

We studied the effect of four conjugated synthetic derivatives of estrone and ethynylestradiol and bis-beta-chloroethylamine-containing substance on activity of plasma membrane enzymes 5'-nucleotidase and N+-K+-ATPase. As differentiated from precursors, estrogen cytostatics decreased activity of plasma membrane enzymes. Reference preparations chlorophenacyl and estradiol had little effect on activity of 5'-nucleotidase and N+-K+-ATPase. These data suggest that damage to plasma membrane enzymes is related to the effect of estrogen cytostatic molecules. Test compounds produced an antiproliferative effect on estrogen-independent tumor cells, which strongly correlated with a decrease in activity of plasma membrane enzymes 5'-nucleotidase and N+-K+-ATPase. The derivative of ethynylestradiol with the cytostatic residue in the 3-position of the steroid nucleus (Po-714-11alpha) most significantly modulated enzyme activity.

Binding of bis-beta-chloroethylamine derivatives of synthetic estrogens to proteins: dependence on the chemical structure.

We studied binding of 10 new bis-beta-chloroethylamine derivatives of synthetic estrogens of different chemical structure to estradiol receptors in cytosolic fraction of breast carcinoma tissue and to blood plasma proteins. 11alpha-derivatives of estrone and ethynylestradiol with bis-beta-chloroethylamine radical at the 3-position were most potent, while 11beta-substances with the cytostatic residue in this position less effectively competed with labeled estradiol for estradiol receptors. Estrone derivatives with cytostatic residue at the 3-position bound primarily to serum albumin. Ethynylestradiol derivatives with cytostatic residue at the 3-position of the steroid nucleus bound to plasma globulins. Cytostatic radical at the 11-position changed spatial conformation of estrogen cytostatics and they lost their ability to interact with estradiol receptors and blood plasma proteins.

6alpha-Methyl-16alpha,17alpha-cyclohexane progesterone and progesterone inhibit growth of doxorubicin-sensitive MCF-7 and HeLa tumor cells.

Temporal and concentration dependencies of the effects of gestagens (6alpha-methylpentarane and progesterone) and cytostatic doxorubicin on proliferation of MCF-7 and HeLa tumor cells was studied using(3)H-thymidine test. Gestagens produced the maximum inhibitory effect of on cell proliferation in a concentration of 10(-5)M; the effect developed on day 6 of incubation. 6 alpha-Methylpentarane in a concentration of 10(-8)inhibited proliferation of HeLa cells more effectively than progesterone (p<0.05). In experiments with combined treatment of doxorubicin-sensitive MCF-7 and HeLa cells, progesterone in a concentration of 10(-7)M attenuated the cytostatic effect of doxorubicin (p<0.05), while 6alpha-methylpentarane in the studied concentrations did not modulate it.

Effects of synthetic bis-beta-chloroethylamine estrogen derivatives on proliferation of skin sarcoma cells.

The effects of four new synthetic bis-beta-chloroethylamine-containing estrogens and known cytostatic agents chlorophenacyl and estradiol mustard were compared on monolayer cultures of transformed L-929 fibroblasts (from murine skin sarcoma). The drugs within the concentration range of 10(-5)-5 10(-7)M inhibited proliferation of cultured cells by 67%. Chlorophenacyl displayed the least antiproliferative activity (15% inhibition at 10(-5) M). Steroid nucleus introduced into the molecule enhanced antiproliferative activity of test drug in comparison with chlorophenacyl, probably due to accumulation of the hormone-cytostatic molecules in cells. Estradiol had no effect on proliferative activity of L-929 cells, and no specific estrogen-binding sites were found in cultured transformed fibroblasts. The antiproliferative effect of hormone-cytostatics on this culture is not mediated via specific interactions with estrogen receptors.

[The use of the DiaMorf (Russia) computer-programmed unit for evaluating the effect of steroids and cytostatic agents on cultured cells]. / Primenenie apparatno-programmnogo kompleksa "DiaMorf" (Rossiia) dlia otsenki vliianiia steroidov i tsitostaticheskikh sredstv na kletki kul'tury.

The effect of some steroid hormones and their synthetic analogs--dexamethasone, estradiol, progesterone, testosterone, and cytostatics--on the fibroblast nuclei in a monolayer culture was studied. Their proliferation was studied at the same time by the radioindicator method. The method of computerized morphodensitometry with the use of the apparatus-programmer complex DiaMorf (Russia) showed that the compounds under study induce reorganization of interphase chromatin of fibroblast nuclei over the molecules, the amount of which may be recorded by means of the computer system of analysis of cell and tissue images.