WO1998004250A1 - Pharmaceutical compositions for the reversion of multiple resistance to drugs - Google Patents

Abstract

The invention concerns the inhibition, or treatment by reversion, of multiple resistance to drugs, in particular the reversion of Plasmodium falciparum chemoresistance to chloroquine and the reversion of multiple resistance to drugs in the treatment of cancer. The active principle acts on the membrane potentials linked to the transmembrane movements of Ca<2+>/Na<+> inhibiting the membrane potassium canal and the membrane sodium canal, with or without a parallel action on the membrane calcium canal. It must be a class III antiarythmic agent with a positive inotropic action. It may be a bis-phenethyl-amine, in particular N-methyl-N-bis(3,4-dimethoxy-phenylethyl)-amine.

Description

 PHARMACEUTICAL COMPOSITIONS USEFUL FOR THE REVERSION OF MULTIPLE DRUG RESISTANCE

The present invention relates to the reversal of multiple resistance to drugs or drugs. It relates more particularly by way of illustration, but without this constituting any limitation, the reversion of the chemoresistance of Plasmodium falciparum to chloroquine and the reversion of the multiple resistance to drugs in oncology, in particular in the case of an anticancer treatment with anthracycine type products.

The invention relates more particularly to a pharmaceutical composition for the inhibition, or the treatment by reversion, of multiple resistance to drugs.

Multiple resistance to drugs is a hindrance in many treatments or in the prevention of pathological conditions and the means proposed so far to remedy them have proved in all cases to be too toxic to allow their use. In addition, it was not known until now on what basis to seek an effective therapeutic agent for the reversal of multiple drug resistance and work in this direction undertaken in the past has even been interrupted.

There was therefore a need to be able to effectively treat states of multiple drug resistance in animals, including humans, under conditions of non-toxicity or acceptable toxicity.

It has now been unexpectedly found that a bis-phenethylamine known otherwise for its efficacy in the treatment of cardiovascular diseases and disturbances of the cerebral circulation can be used for therapeutic purposes in the fight against multiple resistance to drugs. . Moreover, the action mechanisms detected in this regard make it possible to propose a general effective means in this regard and to provide the person skilled in the art with elements enabling him to determine by simple tests whether a therapeutic agent is capable of being effective in above therapeutic application.

ou ses sels d'addition avec des acides phar aceutiquement acceptables .The first object of the invention is thus a pharmaceutical composition useful for the inhibition, or treatment by reversion, of multiple resistance to drugs, said composition comprising an effective amount of at least one active principle or therapeutic agent capable of acting on membrane potentials linked to Ca ²⁺ / Na ⁺ transmembrane movements and those linked to potassium current, in particular by inhibiting the membrane potassium channel and the membrane sodium channel, with or without parallel action on the membrane calcium channel, said agent being in in addition to a class III antiarrhythmic agent having a positive inotropic effect. It has indeed been unexpectedly found according to the invention that a compound acting on the membrane potentials linked to the movements of Ca ²⁺ / Na ⁺ transmembrane by inhibiting them, in particular by inhibiting the potassium potassium channel and the sodium sodium channel, with or without parallel action on the membrane calcium channel, can give a composition particularly effective in the fight against multiple drug resistance, in the absence of notable clinically toxic side effects. The pharmaceutical composition according to the present invention advantageously, but without limitation, as active ingredient at least one compound chosen from bis-phenethylamines, in particular N-methyl-N-bis (3,4-dimethoxyphenylethyl) amine of general formula I

or its addition salts with aceutically acceptable acid phar.

The compound of formula I above will, for the sake of simplification, be designated hereinafter most often by the designation "YS 035".

For the sake of convenience, this latter compound will be referred to in the remainder of this description, but everything that will be explained about it can be transposed by a person skilled in the art, mutatis mutans, to other therapeutic agents which prove to be or prove to be equivalent thereof, without thereby departing from the scope and scope of the present invention.

Rosemund, Kϋlz and Buth, in US Patent 2006,114, in Ber. 72 B 18-28, 1939 and in patent DE 617647, described the synthesis of a series of bis-phenethylamines having properties analogous to those of papaverine and spasmolytic properties. Although they cited N-methyl-N-bis (3,4-dimethoxyphenylethyl) amine in this connection, they did not actually have this product. Indeed, the compound mentioned under this name, without indication of structure, by these authors was actually another molecule and its hydrochloride in no way corresponded to that of N-methyl-N-bis (3, 4-dimethoxyphenylethyl) amine. The latter compound was presented for the first time in document FR-A-2 541 113 with a method for obtaining it and indications for application in the treatment of cardiovascular diseases and disturbances of the cerebral circulation. The representative of the aforementioned bisphenethylamines, designated in the remainder of this description by YS 035, has structure indicated above by formula I, confirmed by spectroscopy:

H ¹ NMR spectrum (recorded in CDCI3, with TMS as internal reference). The values of the chemical shifts of the protons, expressed in δ, were: 2.35 (s,

3H, N-CH3); 2.7 (S, 8H, N- (CH2-CH2) 2) 3.8 (s, 12H, 4

(OCH3)); 6.7 s, 6H aromatic).

The hydrochloride of compound YS 035 has a m.p. of 180-185 ° C. Elementary analysis for C21H30NO41CI (PM = 395.93):

Calculated (%): C = 63.70; H = 7.63; N = 3.54; Found (%): C = 63.42; H = 7.47; N = 3.39. The present invention also relates to a pharmaceutical composition presented and / or packaged for inhibition or treatment by reversion, of multiple resistance to drugs, comprising as active principle at least one compound as defined above and / or a salt or other pharmacologically acceptable derivative thereof. Said composition is advantageously in the form of tablets, tablets or lozenges, possibly effervescent, or also of solutions or oral suspensions. The dosage forms of this composition comprise in practice per unit dose approximately 120 mg to 240 mg of active principle or a weight equivalent, on the basis of the pharmacological effect targeted, of any equivalent of this active principle or of their different salts. Those skilled in the art are able to determine such a weight equivalent based on their own knowledge and on routine tests. According to a preferred embodiment, the present invention comprises the use of an effective amount of at least one active compound as defined above.

More advantageously, said effective amount consists of daily doses of approximately 240 mg to 360 mg of the active principle or mixture of active principles or a dose of pharmacologically equivalent anti-reversion effect of an equivalent compound. These doses may, depending on the route of administration used, the condition of the patient and the product used vary over time and be administered according to a dosage comprising administration in a single administration per day, per week or per month. Thus a daily dose as mentioned above can itself be replaced by any other dose of administration of the active ingredient, provided that the pharmacological effect obtained is substantially the same as that obtained by daily administration. Oral administration is preferred. The active ingredient or therapeutic agent can be administered together with a physiologically acceptable carrier or carrier or diluent to thereby form a complete pharmaceutical composition ready for use. Such compositions advantageously contain more than 80% by weight of at least one therapeutic agent according to the invention and can be prepared by conventional techniques allowing their formulation in usual dosage forms, for example tablets, capsules, capsules, dragees, dispersible powders, syrups, elixirs, suspensions or solutions, drops, nebulizations. Suitable diluents or pharmaceutical fillers include, for example, water, alcohols, oils or waxes or gums, natural or hardened, calcium or sodium carbonates, calcium phosphate, kaolin, talc and lactose, as well as suitable preservatives, such as for example methyl hydroxybenzoate, suspending agents, such as methyl cellulose, gum tragacanth and sodium alginate, wetting agents, such as lecithin, polyoxyethylene stearate and polyoxyethylene sorbitan maleate, granulating and / or disintegrating agents such as starch and acid algenic, cohesion agents, such as starch, gelatin and gum arabic, and lubricating agents such as magnesium stearate, stearic acid and talc. The compositions in the form of tablet can be coated by known techniques, so that the disintegration of the tablet and the absorption of the active ingredient in the gastrointestinal tract are delayed and so that a delayed and / or prolonged action is obtained over a long period. period. A person skilled in the art is able to determine in each species whether or not a compound is an equivalent as above falling within the scope of the present invention by resorting to routine tests and by proceeding if necessary iteratively, preferably based on the indications and proposed methods provided below.

The invention is described in more detail in the examples, given below for purely illustrative purposes, which in no way limit it and in which, unless otherwise stated, the dosages of the active ingredient are related to body weight.

The pharmacological tests carried out and described below are, at least in part, illustrated by the appended figures, in which: - FIG. 1 represents a graph showing the current voltage curve in a stable state under control conditions (C) and in the presence of 100 μmol / 1 of compound YS 035 (100).

Fig. 2 represents in graph form the in vitro activity of YS 035 on a strain of P. falciparum resistant to chloroquine.

Fig. 3 shows in graph form the in vitro activity of YS 035 on resistance to chloroquine. - Fig. 4 represents in graph form the evolution of the activity of compound YS 035, as a function of its concentration in an in vitro combination of YS 035 and chloroquine, on a strain of P. falciparum resistant to chloroquine.

- Fig. 5 shows in graph form the effect of YS 035, at concentrations lower than any activity of its own, on the IC50 of chloroquine of a strain of chloroquine resistant P. falciparum.

The above active ingredient has been subjected to toxicological studies and to a series of pharmacotoxicological tests by comparing it with known drugs bearing the international nonproprietary names Verapamil, D 600,

Nifedipine and Diltiazem.

Toxicological studies

I - Acute toxicity: a) YS 035 was administered, in the form of hydrochloride in 1% solution, intravenously to 40 male and female rats. The LD50s were as follows, 14 days after a single injection:

- in male rats: 19.0 mg / kg (16.77-21.52)

- in female rats: 24.4 mg / kg (21.15-28.12) b) in 60 male and female mice, YS 035 was administered in the form of hydrochloride by the venous route. The LD50s were as follows, 14 days after a single injection:

- in male mice: 36.1 mg / kg (32.7-39.8)

- in female mice: 42.3 mg / kg (39.0-45.9) c) YS 035 was administered orally as a base in 60 male rats and 60 female rats. The LD50 values were as follows, 14 days after a single administration:

- in male rats: 217 mg / kg (179.6-264.4)

- in female rats: 313 mg / kg (277.0-354.9) d) In 60 male mice and 60 female mice, YS 035 was administered orally as based. The LD50 values were as follows, 14 days after a single administration:

- in male mice: 253 mg / kg (218.0-295.2)

- in female mice: 250 mg / kg (214.8-290.9)

II - Subacute toxicity:

This toxicity was investigated in 20 male SD strain rats CD, orally. These rats received orally in 5 days increasing doses of YS 035 (base), which corresponded respectively to 44-66-99-148 and 220 mg / kg of body weight. The animals were sacrificed 12 days after the last administration.

There were no spontaneous deaths. The highest dose (222.75 mg / kg) thus administered for 5 days induced between 30 and 90 minutes after each administration of clonic discharges and a pseudocoma (prone position). No effect on body weight or on food and water consumption was observed. 12 days after the last treatment, the number of clumped and white blood cells and platelets had increased. Biologically and histologically, no change in hepatocellular functions or cytolysis has been observed.

III - Subchronic toxicity (4 weeks of oral administration 7 days a week):

This study was carried out on 96 male and SD female rats of CD strain, aged 5 to 6 weeks and with an average weight of 169 ± 1.1 g for males and 158 ± 1.4 g for females .

Chrez the males and the females 4 batches were constituted by distribution in groups constituted at random: 1 control batch and 3 batches receiving respectively 22.5 - 45 and 90 percent of the LD50 previously determined on subjects of the same origin and of the same sex, The results were: no mortality; no change in behavior; no significant change in body weight; increased food intake in males; no haematological modification; increased GP transaminase for both sexes at the higher dose; impaired renal function in the bypassed tubes at the highest dose for both sexes. Histological examination in the male showed the existence, at the highest dose, of a focused fatty degeneration of the liver, an enlarged epithelium of the cells of the tubes bypassed in one case and a slight hyperchromatophilia of CA3 neurons in 3 cases, and in females the existence, at the highest dose, the existence of picnosis of CA3 neurons in only one case.

It should be noted that the active doses in the treatment of cerebral or cardiac ischemic attacks were in the same animal species respectively 3 and 1.5 mg / kg orally.

IV. Pharmacokinetic study in rats:

This study was done in the fasting rat intraperitoneally (5 groups of 3 rats, receiving 30 mg / kg of YS 035 in suspension at 1% in gum arabic) and orally (5 groups of 3 rats receiving 30 mg / kg of YS 035 in 1% solution in gum arabic). The animal sacrifices took place 0.25 - 0.50 - 1 - 2 and 4 hours after I.P. administration and 0.5 - 1 - 2 - 4 and 8 hours after oral administration, respectively.

From a group of 3 rats kept in a metabolic cage, 24-hour urine was collected and analyzed. By using digital data processing techniques (SAA - 27 parameters), the following bi-exponential kinetics were obtained for administration I. P. : X (t) = 1.55e ^{~ 3} + 0.97e ^~ ° ' ³ , where X is the concentration in μg / ml and t the time in hours.

The half-life was 2, 3 hours. The area under the curve (AUC) was 3.75 μg / ml / h. The total clearance (dose / AUC) was 8 1 / h / kg. X (O) being 2.52 μg / ml, the volume of distribution was 11.9

1 / kg.

After oral administration of 30 mg / kg the plasma kinetics followed a triexponential equation: X (t) = -8.03e ^-1 ' ^8t + 7.93e ^-2 ' ⁰ + 0, the ^_0 ' ² .

The plasma peak appeared at 30 minutes (0.343 μg / ml). The AUC value was 0.920 μg / ml / h. The bioavailability of the product calculated using the ASCperos / ASCip ratio was 24.5%.

Activity of the YS 035 on the transmembrane movements of Ca ²⁺ and comparison with Nifedipine and D 600

It is generally assumed and accepted that Ca ²⁺ antagonists exert their action by reducing the entry of Ca ²⁺ through the plasma membrane (Fleckenstein et al., 1972, Arzneim. Forsch. 22: 22-33). compound YS 035 was therefore tested on the transport of Ca ²⁺ by different cells. It has been possible to show an accumulation as a function of time of Ca ²⁺ in the muscle cells of a chicken embryo in 1 ^• absence and in the presence of the compound YS 035 and a significant reduction of this accumulation under the effect of a addition of 30 μM of YS 035 in the incubation medium (R. Deana et al., Biochem. J., vol. 2: 8-1984).

This inhibitory effect is observed for doses greater than 1 μM and is dose-dependent between 10 and 30 μM and is not altered at concentrations greater than 50 μM. Other Ca ²⁺ antagonists, such as Nifedipine (10 μM) and D 600, have been shown to produce similar inhibition.

Mitochondria are generally considered to have two different specific Ca ²⁺ release systems which are respectively sensitive and insensitive to the inhibition produced by Ruthenium Red. Ruthenium Red is known to ingest Ca ²⁺ uptake by mitochondria and that the addition of this dye to a mitochondria suspension causes Ca ^{2 + to be released} by them (Moore CL., 1971, Biochem Biophys. Res. Comm. 42: 298-305; Rossi et al., 1973, Biochem. Biophys. Res. Comm. 50: 846-852; Stucki and Inneichen, 1974, Eur. J. Biochem. 48: 365- 375). The mechanism of action of Ruthenium Red has been the subject of research by various laboratories. The release of Ca ²⁺ from the mitochondria when Ruthenium Red is added to inhibit active uptake can be divided into its three origins: a) non-specific passive permeability due to membrane damage and sensitive to albumin and l 'ADP (Harris et al., 1979, Biochem. J. 182: 455-464); b) a rapid modification of Na ⁺ / Ca ^+, particularly in excitable tissues (Crompton et al., 1977, Europ. J. Biochem. 79: 549-558); c) an electroneural modification of H ⁺ / Ca ²⁺ (Fiskum and Lehninger, 1979, J. Biol. Che. 254: 6236-6239).

Anyway, Ca ⁺ leakage from mitochnodria caused by Ruthenium Red is the result of a blockage of the Ca ²⁺ cycle through the inner membrane.

According to the methodology known to those skilled in the art and described in the abovementioned publications, it has been possible to establish that the compound YS 035 radically inhibits the slow release of ca ²⁺ induced by ruthenium red at the level of the cardiac mitochondria and of way partial the rapid leakage of Ca ²⁺ due to the addition of NaCl.

This effect was found to be dose-dependent for YS 035. The inhibition by compound YS 035 of the release of Ca ²⁺ dependent on Na ⁺ is of the non-competitive type with an apparent Ki of 28 μM.

It is not currently possible to establish whether the inhibition exerted by YS 035 with respect to the Ca ²⁺ leakage induced by Ruthenium Red is a specific effect on Ca ²⁺ / H ⁺ or a non-specific prevention of damage to the mitochondrial membrane. The specific inhibition of Ca ²⁺ leak regulated by Na ⁺ / Ca ²⁺ seems to be relevant for cardiac mitochondria. The leakage of Ca ²⁺ from the mitochondria of the liver caused by Ruthenium Red is completely blocked by the compound YS 035, whereas the latter, in the absence of Ruthenium Red, does not cause any leakage of Ca ²⁺ .

Conversely, Nifedipine is capable by itself of causing Ca ²⁺ leakage, probably by decoupling effect.

As for compound D 600 (another Ca ²⁺ antagonist and derived from Verapamil), it alone does not cause any mitochondrial leakage of Ca ²⁺ and exerts no inhibition on the action of Ruthenium red (Deana et al., Biochem. J., supra).

The same author also established that the compound YS 035 does not affect the mitochondrial release of Ca ²⁺ obtained after addition to the suspension medium of the mitochondria of the chelating agent EGDTA or by a decoupling (carbonyl-cyanide of p-trifluoro -methoxyphenyl- hydrazone) or a breathing inhibitor (Antimycin A)

Compound YS 035 at concentrations below 100 μM produces a slight modification of the common parameters for the evaluation of mitochondrial function, such as the respiratory index, the degree of ATP synthesis or the membrane potential. Nifedipine produces a drop in all these parameters from the concentration of 100 μM, which is correlated with its liberation action.

Ca ⁺ linked to a damage which results in a collapse of the membrane potential.

The two mechanisms which contribute to increasing the cytoplasmic concentration of Ca ²⁺ , the capture of Ca ²⁺ and the release of this by the mitochondria, being inhibited by YS 035, this compound can be considered as the ideal type of calcium antagonist.

One of the remarkable properties of the compound YS 035 is thus to be a membrane stabilizer, by its action both on mechanisms of capture and of extrusion of Ca ²⁺ , in particular by the mitochondria.

Action of YS 035 on the slow action potentials of the papillary muscle of the guinea pig

The action of YS 035 was then analyzed by research on the slow action potentials of the papillary muscle isolated from the guinea pig after depolarization, with 27 μM / 1 of KC1 at approximately -45 mV. The maximum increase in the slope of the slow potential, which can be used as a measure of slow input of calcium current, remained virtually unchanged by YS 035 at concentrations up to 30 μM / 1 and a marked reduction did not was observed only from 100 μM / 1.

In addition, the amplitude of the slow action potentials and the isometric contractility were only reduced at the highest concentrations. The EC ₅₀ for reducing _my V χ was calculated at a value of 1.6 x 10 ^"4 M / 1. By comparison, the values of EC ₅ 0 of Diltiazem and Nifedipine were 2.0 x 10 "^ M / 1 and 2.9 x 10 ^-8 M / 1. The inhibitory action of YS 035 is therefore only discernible at relatively high concentrations, but is associated with a reduction in the duration of the slow action potential which is characteristic of Ca ²⁺ antagonists. This same compound YS 035, at concentrations of 1 to 10 μM / 1, induces a marked prolongation of the slow action potential, associated with an increase in isometric contractility. Compound YS 035 shows a dependence in use at 100 μM / 1, which resembles that of Nifedipine, in particular with regard to the reduction of V _ma χ and of contractility.

Action of compound YS 035 on the action potentials of the papillary muscle of the guinea pig

On the papillary muscle with normal resting action potential, the compound YS 035 was found to increase, up to a concentration of 100 μM / 1, the duration of the action potential, respectively at 30 and 90% repolarization.

For an aumgentation of approximately 30%, the highest values were obtained for concentrations of 2 to 10 μM.

As with a slow response, as described above, in this case the prolongation of the action potential is associated with an increase in contractility. YS 035 behaves like the blocker β, d-sotalol, a class III antiarrhythmic drug.

From 100 μM / 1 and beyond the YS 035 reduced the Vmax to. because of its inhibitory effect on the sodium channel. These results show that up to 30 μM / 1, YS 035 markedly delays depolarization which, at the concentration of 100 μM / 1, is marked by an inhibitory effect on the calcium and sodium channels.

Action of compound YS 035 on the action potentials of the left atrium of the guinea pig

For concentrations of 1 μM / 1 and above, YS 035 has been shown to prolong the atrial action potential. At the concentration of 10 μM / 1 the durations of the action potentials respectively at 30 and 90% of repolarization were increased by approximately 50% and the contractility increased in parallel. At 100 μM / 1 the variations in the duration of the action potentials respectively at 30 and 90% of repolarization increased by 274 and 235% respectively, compared to the control values, but on the other hand the contractility was lowered to its value basic.

Action of compound YS 035 on the action potentials of the papillary muscle of the guinea pig. partially depolarized

On papillary muscle depolarized at -72.6 mV in the presence of 9 μM / 1 of KC1, YS 035 exerts an inhibitory effect on the sodium channel, starting at 1 μM and reaching 42.8% of the control values at the concentration 100 μM / 1. It has been established that the repolarization time reaches its peak at 10 μM / 1, even with a reduction in the potential of the resting membrane. The sodium flow inhibition was increased by depolarization. The action of YS 035 on the action potentials became stronger at 10 μM / 1 and weaker at 100 μM / 1.

Action of compound YS 035 on ionic currents of Purkinπe fibers in sheep

This action was sought using a clamp program. Voltage curves were drawn, in a stable state, in controls and in the presence of 100 μM / 1 of YS 035 (see FIG. 1). Figure 1 attached shows such curves, the voltage being plotted on the abscissa in mV, while the intensity ikl is plotted on the ordinate in nA. The intersection of the voltage curves in the controls and in the presence of YS 035 at -80 mV suggests that the compound YS 035 inhibits the current i] ςl and reduces the external current (iout) measured under stable conditions. The YS 035 mainly reduces instantaneous components of the studied potentials from -5 to +45 mV. This inhibition is due to potassium ions, which explains the delay in repolarization.

Compound YS 035 thus proved to be a powerful inhibitor of external potassium current in cardiac preparations, instantaneous current being first affected. This effect begins at the concentration of approximately 1 μM / 1 and delays repolarization. This corresponds to the extension of the duration of the action potentials of the left atrium, the papillary muscle and the Purkinje fibers. The effects of this compound on the sodium and calcium channels are only observed at concentrations equal to or greater than 100 μM / 1. The inhibition of the sodium channel can also be increased by an increase in the pulse speed or depolarization of the cell membranes.

With regard to its comparison with Verapamil and Nifedipine, it should be emphasized that YS 035 has an extremely weak calcium antagonist effect compared to that of Verapamil as well as that of Nifedipine.

At best, YS 035, which is a derivative of Verapamil, should be considered to have significant anti-calcium properties due to its weak side effects.

In addition, Verapamil is a class IV antiarrhythmic drug depressing atrioventricular conduction, while compound YS 035 is a class III antiarrhythmic drug, responding in this respect to the mechanism of action of myodarone. This same mechanism is implemented with a neuroleptic, such as for example Melperone or Trioruoroperazine. As regards the compound YS 035, it is therefore not the anticalcic property as with Verapamil which is the property concerned in the therapeutic effect sought, but the membrane effect Ca ²⁺ / Na ⁺ dependent and especially K ⁺ dependent. Phenomenon of multiple drug resistance and activity of YS 035

In 1989, T.J. Lampidis et al., J. Cell. Pharmacol. 16-22, have shown that heart muscle, a number of cancer cells, and Friend's leukemia cells can accumulate relatively large amounts of anthracyclines and positively charged dyes. The intracellular accumulation of these positively charged compounds was correlated with the existence of high electronegative potentials of the plasma membrane found in these cells and with a reduction in the level of P-glycoprotein. Conversely, for multiple drug resistance (RMD) one must find less accumulation of positively charged compounds and lower membrane potentials.

This suggests that chemical load plays an important role in the selection of intrinsically sensitive heart fibers, cancer cells, Friend's leukemia cells and their intrinsic or acquired RMD. T.J. Lampidis et al. have been postulated that adriamycin can accumulate as well in cancer cells with high membrane potential, as are heart muscle cells. In other words, they defined the importance of the relation existing between electronegative membrane potentials and the increase in the attraction, then of the accumulation of compounds with positive electric charge in sensitive cells and by comparison in cells exhibiting RMD by the fact that they acquired resistance to Adriamycin. The action of Verapamil in this regard is to restrict efflux by modifying the membrane permeability.

In a publication (Proceedings of the American Association for Cancer Research, 1990, vol. 31: 373, In vitro cardiac potencies of multidrug resistance modulators, TJ Lampidis, D. Kolanias, H. Tapiero, JN Savaraj and J. Cahn reported that, using a video system described by TJ Lampidis et al. (1989, J. of Cell. Pharmacol. 16-22) and taking as reference substances Verapamil (Vpl) and a neuroleptic, Calmodulin inhibitor, Trifluoperazine (TFP) capable of reversing RMD in leukemia cells of Friend, with study of their action on the spontaneous contractility of myocytes in culture, they obtained the following results by seeking the comparative action of YS 035:

- the dose of YS 035 necessary (50,000 ng / ml) to stop the beating of myocytes was 200 times higher than that of Vpl (250 ng / ml) and 10 times higher than that of TFP;

- a positive chronotropic effect was always observed at 10 ng / ml of YS 035, while with doses as high as 20,000 ng / ml in continuous treatment the beating of myocytes was always observed 24 hours later;

- the dose producing a detectable effect on the myocytic cardiac function was respectively 5 ng / ml for Vpl and 750 ng / ml for TFP and that necessary to stop the contraction of myocytes was respectively 250 ng / ml for Vpl and 5000 ng / ml for TFP.

This clearly shows the fundamental differences between the compounds thus compared.

In this regard, it should be emphasized that the method developed by TJ Lampidis et al. was only a sorting method, allowing only comparative analysis of chemical compounds by an in vitro method not allowing to make a significant difference between - Verapamil which is a powerful calcium antagonist, anti-arrhythmic class IV of certain toxicity,

- Trifluoperazine, which is a neuroleptic whose property is a class antiarrhythmic effect

III, like Melperone, but with manifest toxicity, and

- Compound YS 035, slightly anticalcic, practically devoid of cytotoxicity, but which is in reality a class III antiarrhythmic.

It has been established according to the present invention that the compound YS 035 is on the contrary particularly advantageous, because it has been found:

. have a membrane effect mainly exerted on the potassium channel,

. be class III antiarrhythmic, and. exert a positive inotropic cardiac effect. Such a compound has membrane activity capable of modifying the membrane permeability, of increasing the membrane potentials when they are depressed, and this in a remarkable manner without the appearance of cytotoxicity.

therapeutic uses of RMD reversion This reversion is of particular interest in two areas of human pathology, malaria and cancer.

It had been practically abandoned with regard to attempts to combat malaria, because the products used (Verapamil, Imipramine and

Cyproheptadine) have all demonstrated the possibility of a reversal of chloroquine resistance, but at toxic concentrations in vitro or at doses inducing toxic effects in vivo. The same was true for the reversal of resistance to anti-cancer substances. There was therefore currently no available means capable of exerting in these types of human pathology a reversal of resistance to drugs without clinically toxic side effects.

YS 035 action on multiple drug resistance in oncology

Appropriate tests using techniques published by H. Tapiero et al. (Molecular Basis for Selectivity and Effects of Anthracyclines in Drug Résistance Mechanisms and Reversai, Enrico Mihich éd., John Libbey - CIC Publishers, June 19-21, 1989, pages 233-243) have enabled studies of cell survival with Doxorubicme to be verified. (hereinafter abbreviated to Dox) with respect to a cell strain of human myelogenic leukemia, and to measure the intracellular concentration of Dox compared to the membrane potentials for normal cells and cells exhibiting multiple resistance.

According to these authors, a certain number of anti-cancer products derived from anthracyclines of which Doxorubicin is a part, such as for example Pirarubicin, Fluoro-Doxorubicin, MEJ 2303 and its metabolite Ml, Morpholino-anthracycline and 1 'Aclarubicine, which respond to The formulas below show different behavior towards the aforementioned human myelogenous leukemia cells depending on whether these cells are insensitive or sensitive to Doxorubicin (see Table 1 below). DOXORUBICIN (DOX) PIRARUBICIN (THP-DOX

FLUORO -DOXORUBICIN (MEJ 2303) MEJ 2303 -M1

MORPHOLINO-ANTHRACYCLINE (KRN 8602) ACLARUBICINE (ACR)

Tableau 1 : Accumulation d'anthracyclines dans les cellules de la leucémie myélogène humaine respectivement sensibles et résistantes à la Doxorubicine.

Table 1: Accumulation of anthracyclines in human myelogenous leukemia cells respectively sensitive and resistant to Doxorubicin.

The cells were exposed for the time and to the drug concentration necessary to induce an IC50. They were then washed, the drug was extracted with DMSO and analyzed by HPLC. [] *: Intracellular metabolism of MEJ 2303 to Ml. This confirms a fact well known to the oncologist who prescribes compounds from the anthracycline group as anti-cancer agents, namely that the first treatment triggers an RMD phenomenon and that thus the use of another product of the same origin can result in marked cardiac toxicity, also resulting from the necessary increase in the dosage for the anticancer effect to be maintained. By the way, the effects of class antiarrhythmia

III demonstrated with YS 035 are the basis of the present invention, while T. Lampidis et al. (Association for Cancer Research, vol. 31, p. 373, 1990) had put on the same level Verapamil (a class IV anti arrhythmic, which is a calcium antagonist), Trif luoperazine (a neuroleptic) and YS 035 (considered to be derived from Verapamil). These authors could not explain, on the basis of knowledge at the time, the different activity of YS 035, which maintains a concentration of 200 ng / ml, or even 20,000 ng / ml for 24 h in continuous, its effects on myocytic beats for 24 hours without toxicity, whereas we cannot exceed 5 ng / ml with Verapamil, due to its negative chronotropic and inotropic effects. Although one does not wish to be bound by any theory, it is believed that it is because YS 035 is a class III antiarrhythmic agent acting on membrane potentials and potassium channels that it exerts an inotropic action. positive. The invention therefore also relates to a pharmaceutical composition comprising a combination with an anticancer agent of anthracyclinic origin of a compound of formula I, or of its addition salts with acceptable pharmaceutical acids, with an anticancer agent of anthracyclinic origin , in proportions that a person skilled in the art is able to define suitability in each case, useful for inhibiting or avoiding the development of multiple drug resistance and induced cardiac toxicity.

Action of the YS 035 on the reversion of the. resistance to Doxorubicin

Appropriate tests have been carried out to find the dose necessary to obtain the maximum RMD reversion effect. For doxorubicin (Dox) 20 μg / ml of YS 035 were necessary, compared to 10 μg / ml of Vpl and 1 μg / ml of TFP. Moreover, this dose of 20 mg / ml of YS 035 reduced 40-fold resistance to Dox, while the 10 mcg / ml VPL had the major disadvantage of the form l / ^40th of the dose able to stop the heart, while TFP only reduced Dox resistance by 4 times.

The results obtained are all the more important since Doxorubicin is the prototype of anticancer agents which are of the anthracycline type, capable of generating RMD and of causing cardiac disorders, just like aclarubicin, epirubicin, zorubicin, pirarubicin and idarubicin as examples.

It can be estimated that the efficacy and therefore the advantage of the compound YS 035 and its equivalents, in particular for the reversion of multiple resistance to drugs in vivo with regard to anti-cancer products, have as essential bases the low level of cardiac side effects and very marked RMD modulator activity.

Action of YS 035 as a reversal of the chemo-resistance of Plasmodium falciparum to chlorocruine

Chloroquine sulfate, sold by the Specia Laboratory, having a molecular mass of 418 and belonging to the class of amino-4-quinolines, was used. YS 035, on the other hand, is a synthetic derivative of the Verapamil class. Its chemical formula is C21H29NO4, corresponding to N, N-bis- (3,4-dimethoxyphen-ethyl) -N-methylamine, of molecular mass 359.442. YS 035 is a weak calcium pump inhibitor, which has been tested on several cell types.

The plasmodial strains tested were strains of P. falciparum, adapted in continuous in vitro culture and coming from malarious patients, managed in the Department of Infectious, Parasitic, Tropical and Public Health Diseases of the Pitié-Salpêtrière Hospital Group.

One strain, isolated in 1988 from a patient who contracted malaria in Niger, remained sensitive to the four antimalarials that are chloroquine, quinine, mefloquine and halofantrine (P. Mirovsky et al.,

Trans. R. Soc. Too much. Med. Hyg., 84: 511-515, 1989).

Another strain, isolated in 1989 from a patient suffering from malaria on his return from Benin, was adapted and maintained in culture in the laboratory. The strain concerned was resistant to chloroquine and sensitive to quinine, mefloquine and halofantrine. She has kept this profile steadily since then.

Reversers of chloro-resistance

After the emergence of resistance to chloroquine (the most widely used antimalarial agent) in the 1960s, several strategies were adopted to work around this problem. A chloroquine-resistant strain is characterized by the increase in the efflux of chloroquine outside the resistant parasite, as well as by the reversibility of this phenomenon under the action of certain molecules. This is how the synergistic properties of calcium pump blockers have been widely studied on different strains of Plasmodium falciparum (mainly Verapamil) (Martin, SK, Oduola, AMJ and Milhous, .K., 1987, Reversion of chloroquine resistance in Plasmodium falciparum by Verapamil, Science 235: 889-901), tricyclic antidepressants (Imipramine) and antihistamines (Cyproheptadine) (Bitonti, AJ, Sjoerdsma, A., McCann, PD et al., 1988, Reversion of chloroquine resistance in P. falciparum by desipramine. Science 242: 1301-1303; Peters, W., Ekong, R, Robinson, BL, arhurst, DC and Pan XQ, 1989, Antihistamine drugs reversing chloroquinesisance in P. falciparum, Lancet 11: 334-335).

The in vivo application of these proposals has shown that all these substances would only be active in animals, including humans, at toxic concentrations. As a result, work on the chloroquino-resistance reversers had been virtually abandoned.

It was then unexpectedly found, as explained above, that the compound YS 035 and any functional equivalent thereof, such as for example a compound meeting the definition of activity indicated above with reference to the subject of the present invention constitutes an active principle or therapeutic agent, on the contrary, exceptionally effective.

Methods

In vitro chemosensitivity test

In this study, the isotopic microtest described by RE Desjardins et al., Antimicrob, was used. Agents Che other., 16: 710-718, 1979. This isotopic microtest, which is a major application of the method of in vitro culture of P. falciparum developed by W. Trager and JB Jensen, Sciences, 193: 673-675 , 1976. It is based on the incorporation by parasites of tritium-labeled hypoxanthine in the presence of increasing concentrations of antimalarial agents in 96-well microtiter plates.

After 48 hours of incubation in a CO 2 bell, the culture was stopped. The radioactivity was then measured using a liquid scintillation beta radiation counter (Beckman LS 1701 model). The representation of the number of pulses per minute as a function of the concentration of the antimalarial agent resulted in a sigmoid curve. The concentration inhibiting by half the parasite growth (IC50) was calculated by extrapolation on the linear portion of this curve.

Determination of the antimalarial activity specific to YS 035: Several ranges of concentrations of YS 035 were used in the presence of the two strains of P. falciparum mentioned above and tested according to the isotopic method previously described.

The results shown in FIG. (2) give the percentage of parasitic inhibition as a function of the concentration of 1ΥS035.

This study revealed a specific antimalarial activity of YS 035 from 600 ng / ml. 40% inhibition of parasite growth was obtained with a YS 035 concentration of 4000 ng / ml (see Fig. 2).

A significant reversion of in vitro resistance to chloroquine was obtained precisely from a concentration of 4000 ng / ml of YS 035, which is, as we have just seen, the concentration of the same compound resulting in an inhibition of 40 % of parasite growth (see Fig. 3).

Also, when compound YS 035 is added in vitro to chloroquine to study its effects on a strain of P. falciparum resistant to chloroquine

(Fig. 4), it is observed that the inhibitory concentration 50 (IC50 in nmol / 1) of the compound YS 035 is of the order of 3 μg / ml.

In addition, examination of FIG. 5 shows the effect of YS 035, at concentrations (in μg / ml) lower than any specific activity, on the IC ₅ 0 (in nmol / 1) of chloroquine from a strain of P. falciparum chloroquino- resistant. It seems likely that YS 035 has its own antimalarial activity for a concentration of less than 1 μg / ml.

Determination of the activity of YS 035 in combination with chloroquine

The abovementioned strains were brought into contact with increasing concentrations of chloroquine. Fixed concentrations of YS 035 were then added. The results obtained show a considerable lowering of the IC ₅₀ of chloroquine in the various cases studied.

Claims

1. Pharmaceutical composition useful for the inhibition, or treatment by reversion, of multiple resistance to drugs, characterized in that it comprises an effective amount of at least one active principle or therapeutic agent capable of acting on the potentials of membrane linked to Ca ²⁺ / Na ⁺ transmembrane movements and those linked to the potassium current by inhibiting the membrane potassium channel and the membrane sodium channel, with or without parallel action on the membrane calcium channel, said therapeutic agent also being an antiarrhythmic agent class III and having a positive inotropic effect. 2. Composition according to claim 1, characterized in that the therapeutic agent is a class III antiarrhythmic compound with positive inotropic action. 3. Composition according to one of claims 1 or 2, characterized in that said active principle or therapeutic agent comprises at least one compound chosen from bis-phenethylamines, in particular N-methyl-N-bis (3,4- dimethoxyphenylethyl) amine of general formula I

or its addition salts with pharmaceutically acceptable acids. 4. Composition according to any one of claims 1 to 3, characterized in that it comprises a combination of said active principle or therapeutic agent and of an anticancer agent of the anthracycline type. 5. Composition according to any one of claims 1 to 3, characterized in that it is in a form for oral administration and comprises per unit dose of 120 to 240 mg of at least one active principle or therapeutic agent as defined above. 6. Use of a pharmaceutical composition according to any one of claims 1 to 5 for obtaining a medicament intended for the inhibition and / or the treatment of multiple resistance to drugs. 7. Use according to claim 6 for obtaining a medicament intended for the reversion of the chemoresistance of Plasmodium falciparum to chloroquine. 8. Use according to claim 6 for obtaining a medicament intended for the reversion of multiple resistance to drugs in oncology. 9. Use according to claim 8, characterized in that said therapeutic composition further comprises an anticancer agent of the anthracycline type.