WO1997033575A1 - Use of tiamulin for preparing a drug for use as a cancer multidrug resistance reversion agent - Google Patents

Abstract

The use of the antibiotic tiamulin for inducing drug-sensitivity in chemotherapy-resistant human or animal tumour cells as soon as the tumour appears or during tumour development and/or cancer treatment, with a view to preparing a drug for treating malignant blood diseases and solid cancerous tumours in mammals, is disclosed. The cytotoxic activity caused by increased intracellular cancer drug retention may be studied by producing highly resistant tumour lines, i.e. mouse lymphoid leukaemia line P388 resistant to 25 νg/ml of adriamycin, rat hepatocellular carcinoma line AS30-D resistant to 5 and 10 νg/ml of colchicine, and human lymphoblastic leukaemia line CEM resistant to 0.45, 3.6 and 5 νg/ml of vinblastine.

Description

T π TIAMULIN SOAP FOR THE PREPARATION OF A MEDICINAL PRODUCT AS A REVERSION AGENT FOR CANCER MULΗCHEMIC RESISTANCE

Technical field indication

The present invention relates to the use of tiamulin, a semi-synthetic derivative of the natural antibiotic pleuromutilin, for the reversion of the multichimioresistance phenotype (known as MDR phenotype for Multidrug Resistance) developed by certain tumor cells either immediately or after chemotherapy anticancer.

Indication of art was

The clinical resistance that cancer cells oppose to chemotherapeutic agents is currently a major obstacle to their curative potential in the treatment of human or animal cancers. The multichimioresistance phenotype, also known as MDR (for multidrug resistance) is a very studied mechanism by which cancer cells manage to resist the cytotoxic effects of antineoplastic agents. The tumor cells selected in vitro for their MDR phenotype overexpress the mdή gene, which results in the overproduction of a plasma membrane gtycoprotein, called P-glycoprotein, or Pgp, or Pgp170, of molecular weight 170 kDa; it acts like a pump expelling the drugs towards the extracellular medium what confers the resistance with respect to a broad range of chemotherapeutic drugs commonly used. Numerous compounds having the capacity to inhibit the activity of expulsion of drugs by Pgp have been identified: they reverse the cellular resistance towards cytotoxic agents used in experimental systems [1]. This observation led to the idea that the clinical resistance of human and animal tumors to chemotherapy drugs, which often results in the overproduction of Pgp, could be potentially overcome by administering inhibitors to patients. Pgp along with chemotherapy drugs. This strategy has already started in clinical trials [2]. Since the discovery, 10 years ago, of the possibility of pharmacological blockage of Pgp, a large number of these inhibitors, also called chemosensitizers or modulators-MDR, have been able to be identified from a wide variety of chemical classes ( reviews: 3,4). Many of these products have been found to be toxic (particularly significant cardiotoxicity for channel blockers) or ineffective in vivo. This has led to a redoubling of interest in the identification of new classes of chemosensitizers for potential clinical use.

History of the invention During our research work, a resistant tumor cell line was infected with Mycopiasma hyohnis. By treating this line with tiamulin to disinfect it, we found that at the concentration required to ensure the antimycoplasmic effect, the resistant cells would die while the sensitive cells were not affected. The difference was in that to maintain the resistance phenotype, it was necessary to cultivate resistant cells in the presence of the anticancer drug against which these cells were resistant. We have found that the fact of temporarily removing this drug during the treatment of cells with tiamulin no longer causes cell death and allows tiamulin to act against the mycoplasma. We have tested this product on all our resistant tumor lines: the joint use of tiamulin and anticancer drug quickly leads to the death of resistant tumor cells. These findings led us to propose tiamulin as a chemosensitizing agent reversing the MDR phenotype of resistant tumor cells from different species including man, thanks on the one hand to its strongly and rapidly cytotoxic effect on these cells. in conjunction with the use of an anticancer drug, and on the other hand, its non-cyctotoxic effect on healthy cells, in particular on the calcium channels of myocardial cells.

Disclosure of the Invention This invention relates to the use of a semi-synthetic antibiotic derived from the pleuromutilin family, tiamulin or (2- (Diethylamino) ethyl) thio] aœtic acid 6-ethenyldecahydro-5-hydroxy- 4, 6 _r 9, 1 Metramethyl-1-oxo-3a, 9-propano-3aH-cyclopentacycloocten-8-yl ester, as a chemosensitizing agent in the reversal of the multichimioresistance phenotype (MDR) of animal and human cancer cells, an original use in this meaning that tiamulin is currently used in veterinary therapy against bacterial and mycoptamic infections of farm animals intended for human consumption (6-8] [For this use, tiamulin is covered by a patent: Geπnan Patent 2,248,237 corresponding to US Patent 3,919, 290 (1973 and 1975 resp., Both to Sandoz).] This product also has an inhibitory action on cytochrome P450 [9,10]. The structure of this molecule is presented in Figure 1.

Figure 1.

We tested its cytotoxic effect on three highly resistant tumor cell lines. Since lines with a very high level of resistance are not available on the scientific market, we had to produce them from sensitive or already weakly resistant parental strains by gradually increasing the amount of anticancer drug in their culture medium. , and this over a period of two years, followed by a period of eight-month phenotypic stabilization These are the following lines: a line of P388 lymphoid leukemia resistant to 25 μg / ml of adπamycme (FIG. 2A), a line of rat hepatocarcinoma AS30-D resistant to 5 and 10 μg / ml of colchicine (Figures 2B and 2C respectively) and a line of human lymphoblastic leukemia CEM resistant to 0.45 and 3.6 μg / ml of vinblastine (Figures 2D and 2E respectively) These cells express the MDR phenotype by overproduction glycoprotein-P which can represent 36 to 40% of membrane proteins [11] It is then possible to characterize the level of resistance to the anticancer drug that these cells have reached by measuring the CI ^ of each drug used , i.e. the quantity of drug necessary to induce 50% of cytotoxicity For a given drug, the ratio of the ICso obtained with a resistant line to that obtained with the sensitive parental line gives the index of r resistance (or IR) which is a characterization of the level of resistance that the cells have reached The IR obtained for each resistant line is represented in Table I

Table I: Induced drug IC ₅₀ of the inducing drug Daunorubicin (μg / ml) IC∞ (μg / ml) IR le. " (μg / m () IR

AS30-D / S - 0 17 ± 008 - - 0 055 ± 0.028 -

AS30-D / COL10 colchicine (10) 1748 ± 235 col 103 20.35 ± 7.52 370

P388 / S - 0 0054 + 0003 - - 0.0037 ± 0.0023 -

P388 / ADR25 adπamycin (25) 42.28 ± 8 8 adr 7830 7.68 ± 2.01 2075

CEM / S - 000058 ± 000034 - - 0.0030 ± 0.0027 -

CEM / VLB3.6 vinblastine (3,6) 844 + 37 vlb 14552 1.80 ± 1.03 600

CEM / VLB5 vinblastine (5) 10 9 + 2 11 vlb 18793 3.25 ± 1.65 1083

Similarly, in order to know the cytotoxic effect in vitro of tiamulin used in conjunction with an anticancer drug, on resistant tumor cells, dose-response curves are established for each of the resistant lines. These are presented in the Figure 2. We deduce the IC 50 of tiamulin for each of the lines tested. The mean ICso values (in μg / ml) from 4 experiments are. P388 / ADR25 0.31 ± 0.26, AS30-D / COL5: 1.01 ± 0.61, AS30-D / COL10: 0.85 ± 1.0, CEM / VLB045: 1.8 ± 0.39 , CEM / VLB3.6 0.83 ± 0.45, CEMΛ / LB0.45: 1.8 ± 0.39.

The addition of tiamulin at a concentration of 1 μM in a culture of resistant cells in the presence of the anticancer drug at the indicated concentrations, leads rapidly (24 to 48 hours depending on the cell line) to cell death. Adding tiamulin alone to resistant or sensitive tumor cell cultures has no effect, suggesting that tiamulin exerts a chemosensitizing effect on resistant cells under these conditions.

We have shown, by flow cytometry experiments that tiamulin could induce the intracellular retention of the anticancer drug, which leads to its cytotoxic effect A typical result is shown in Figure 3 where, for each line tested, we clearly see that the addition of tiamulin to cells pretreated with daunorubicin instantly leads to a significant increase in the intracellular retention of the anticancer drug Since most of the chemosensitizers tested proved to be toxic, in particular by their calciblocking effect, we tested the effect of tiamulin on the calcium channels of rat myocardiac cells. Figure 4 shows that it is necessary to reach 10 μM of tiamulin to obtain 25% inhibition of the calcium channels. No effect is measurable at lower concentrations, and consequently at the concentrations which have been found to be cytotoxic in the assays. chemosensitization (0.01 to 2 μg / ml).

Description of the figures and table Figure 1: chemical formula of tiamulin. Figure 2: dose-response curves to determine the 50% tiamulin cytotoxicity index (ICS _Q ). The percentage of viability is increased according to the concentration of tiamulin used. Viability is determined by the exclusion of trypan blue. P388 murine lymphoid leukemia and AS30-D rat hepatoma cells are cultured in DMEM medium and human lymphoblastic leukemia cells are cultured in RPMI1640 medium. The culture media are enriched with 10% fetal calf serum, 100 U / ml of penicillin, 100 μg / ml of streptomycin, 0.25 μg / ml of amphotericin B. The cells are incubated at 37 ° C. in a atmosphere saturated with humidity in the presence of 5% of C0 ₂ . The culture medium of the AS30-D cells is enriched with 0.5% glucose as described previously [12]. The highly resistant cells P388 / ADR25 (Figure 2A), AS30- D / COL5 (Figure 2B), AS30-D / COL10 (Figure 2C), CEMΛ / LB0.45 (Figure 2D) and CEM / VLB3.6 (Figure 2E) ) were obtained by incubation of sensitive parental lines in the presence of increasing amounts of the corresponding inducing anticancer drug adriamycin, colchicine and vinblastine, over a period of 24 months; their phenotype was then stabilized for an additional 8 months. The cells thus selected can grow in a medium containing the greatest quantity of drug possible, that is to say 25 μg / ml of adriamycin for P388 / ADR25, 5 and 10 μg / ml of colchicine for AS30-D / COL5 and AS30-D / COL10 and 0.45 and 3.6 μg / ml of vinblastine for CEMΛ LB0.45 and CEM / VLB3.6. The determination of the 50% cytotoxicity index (ICg,) for tiamulin is carried out as follows: the cells are passed into fresh medium at a concentration of 0.2 × 10 ⁶ / ml 24 hours before the test. The viability of each line in the presence of the anticancer drug (for resistant cells) or in its absence (for sensitive cells) is taken as 100%. Tiamulin is then added in increasing concentrations as indicated on the curves, over a few decades depending on the lines concerned. The IC 50 for tiamulin is calculated from dose-response curves representing the viability measured as a function of the concentration of tiamulin used. Figure 3: Cytograms showing the effect of tiamulin on the retention of daunorubicin by the lines AS30-D (cytograms 1, 2), CEM (cytograms 3, 4) and P388 (cytograms 5, 6). The cytograms represent the cell population as a function of the intensity of fluorescence emitted in red by the cells. For each line, the odd cytograms represent the sensitive parental line while the even cytograms represent the resistant derivative line. The name complete of each line is indicated at the top of each frame. The measurement was taken 6 hours after the addition of tiamulin. In each cytogram there are three curves: T = autofluorescence control (cells incubated in the absence of daunorubicin), D = cells incubated in the presence of 2 μg / ml of daunorubicin, D + TM = cells incubated in the presence of 2 μg / ml of daunorubicin and 1 μg / ml tiamulin. The retention tests for tiamulin by flow cytometry are carried out as follows: after a viability test with Trypan blue, the resistant or sensitive cells, in the exponential phase, are washed twice in PBS buffer and incubated for 1 hour fresh medium at a concentration of 0.3 10 ⁶ / ml, at 37 ° C., before the addition of the daunorubicin marker at 1 μg / ml. Tiamulin is added to a final concentration of 2.5 μg / ml. The cells are then incubated for 5 hours at 37 "C. Aliquots of 300 μf containing 10 ⁵ cells are immediately removed (for time zero), then at the time indicated, centrifuged for 1 minute at 100 g, rinsed twice in PBS , taken up in 100 μl of PBS and finally analyzed by flow cytometry. Cell viability is tested extemporaneously with propidium iodide (5 ng / ml). The source of excitation for daunorubicin and propidium iodide is a laser. ionic with argon emitting a beam of 488 nm at 15 mW. The red fluorescence of daunorubicin (A * ^, max: 484 nm; λ ^ max 560 nm) and of propidium iodide (λ _"x max: 540 nm; λ ^ max: 625 nm) is collected through a 585/42 nm bandwidth filter (FL2) and is measured on a 4-decade logarithmic scale in which 10,000 events are taken into account Figure 4: effect of 10 μM of tiamulin on the intensity of calcium currents of type L of cellul es of the rat myocardium. The variations in the intensity of the current L are plotted as a function of time.

Susceptibility of industrial application

→ Given the non-toxicity of tiamulin used alone on sensitive or resistant cells,

→ in view of its low impact at high concentration on the calcium channels, → in view of its very large and rapid selective cytotoxic effect on resistant tumor cells by increasing the intracellular retention of the anticancer drug or drugs used jointly, tiamulin - and probably its derivatives and its history - may be likely to be used in animal and human oncology in order to reverse the multi-drug resistance phenotype which manifested itself either immediately at the time of diagnosis, or following tumor progression and / or anticancer treatment .

References cited 1- Ford JM, Hait WN, Pharmacot. Rev. 42: 156-199, 1990 "Pharmacology of drugs that alter multidrug resistance in cancer") 2- Lum BL, Fisher GA, Brophy NA, et al., Cancer, 72: 3502-3514, 1993 "Clinical trials of modulation of multidrug resistance" 3- Ford JM, Hait WN, Cytotechnology, 12: 171-212, 1993 "Pharmacology circumvention of multidrug resistance

4- Lum B.L., Gosland M.P., Kaubish S. et al., Pharmacotherapy, 13: 88-109, 1993 "implications of the multidrug resistance gene" 5- McOrist S .; Mackie R.A .; Lawson G.H., J. Clin. Microbiol. 33: 1314-7, 1995 "Antimicrobial susceptibility of ileal symbiont intracellularis isolated from pigs with proliferative enteropathy." 6- Laak E.A .; Noordergraaf J.H .; Verschure M.H., Antimicrob. Chemothβr agents. 37: 317-21, 1993 "Susceptibilities of Mycoplasma bovis, Mycoplasm dispar, and Ureaplasma diversum strains to antimicrobial agents in vitro." 7- McOrist S .; Mackie R.A .; Lawson G. H., J. Clin. Microbiol. 33: 1314-7, 1995 "Antimicrobial susceptibility of ileal symbiont intracellularis isolated from pigs with proliferative enteropathy."

8- Uphoff C.C .; Gignac S.M .; Drexler H.G., J. Immunol. Methods 149: 55-62, 1992 "Mycoplasma contamination in human leukemia ce" lines. II. Elimination with various antibiotics. "

9- Witkamp R.F., Nijmeijer S.M., Monshouwer M. and Van Miert A.S., Drug. Metab. Dispos. 23: 542-547, 1995 The antibiotic tiamulin is a cotent inducer and inhibitor of cytochrome P450A via the formation of a stable metabolic intermediate complex. Studies in primary hepatocyte cultures and liver microsomes of the pig "

10- De Groene E.M., Nijmeijer S.M., Horbach G.J. and Witkamp R.F. Biochem. Pharmacol. 50: 771- 773, 1995 Tiamulin inhibits human CYP3A4 activity in an NIH / 3T3 cell line stably expressing CYP3A4 cDNA "

11- Dong M., Penin F. and Baggetto L.G., "Complet purification of P-giycoprotein for functionai and structural studies" submitted for publication, 1996

12- Baggetto L.G. and Lehninger A.L., "Formation and utilization of acetoin, an unusual product of pyruvate metabolism by Ehrlich and AS30-D tumor mitochondria". J. Biol. Chem. 262: 9535-9541, 1987

Claims

1) Use of tiamulin, a semi-synthetic antibiotic (Fig. 1) from the pleuromutilin family for the preparation of a drug for the treatment of cancer. 2) Use according to claim 1 characterized in that the antibiotic reverses the multichimioresistance phenotype of tumor cells (Fig. 2, 3) resistant to chemotherapeutic treatments. 3) Use according to claims 1 and 2 characterized in that the antibiotic is active at low concentrations (0.5 to 50 μg / ml) in vitro on highly resistant tumor lines. 4) Use according to claims 1, 2 and 3 characterized in that the antibiotic has no ionotoxic effect at these doses. 6) Use according to claim 1 in cells from malignant hemopathies and cancerous tumors. 7) Use according to claims 1 to 6 in mammals. 8) Use according to claims 1 to 7 with known anticancer chemotherapy drugs. 9) Extension of the use according to claims 1 to 8 to the active derivatives of pleuromutilins, tiamulin and its derivatives.