EP0920310A1 - Use of aminothiolester derivatives in pharmaceutics - Google Patents

Abstract

The invention concerns the use of at least an aminothiolester derivative for preparing a pharmaceutical composition for lifting the inhibition of a characteristic resistant to apoptosis induction in transformed cells, said characteristic arising from the presence of bcl2 gene in said cells. The pharmaceutical composition is more particularly for treating pathologies including breast cancer, B cell lymphoma, leukaemia, neuroblastoma, prostate adenocarcinoma, prolactinoma and other pituitary adenomas.

Description

 USE OF AMINOTHIOLESTER DERIVATIVES IN THE PHARMACEUTICAL FIELD

The present invention relates to the use of ammothiolester derivatives in the preparation of a pharmaceutical composition with a view to removing the inhibition of apoptosis due to the presence of the bcl ₂ gene in transformed cells.

There are two types of mechanism involved in cell death The first of the classic type is called necrosis Morphologically, necrosis is characterized by swelling of the mitochondπes and the cytoplasm and by nuclear alteration, followed by the destruction of the cell and its autolysis, this being accompanied by an inflammation phenomenon Necrosis occurs passively and incidentally Tissue necrosis is generally due to a physical trauma of the cells or a chemical poison, for example

The other form of cell death is called apoptosis [Kerr, JFR and Wyllie, AH, Br J Cancer, 265, 239 (1972)], but unlike necrosis apoptosis does not cause inflammation. It is described that apoptosis can be carried out under different physiological conditions. It is a highly selective form of cell suicide which is characterized by easily observable morphological and biochemical phenomena. Thus, there is in particular a condensation of the chromatin associated or not with a endonucleasic activity, the formation of apoptic bodies and a fragmentation of deoxyπbonucleic acid (DNA) by the activation of endonucleases into DNA fragments of 180-200 base pairs (these fragments can be observed by electrophoresis on agarose gel)

Apoptosis can be considered as a programmed death of cells involved in the development of tissue differentiation and renewal. It is also considered that cell differentiation, growth and maturation are closely linked to apoptosis and that substances capable of play a role in the differentiation, growth and maturation of cells are also linked to the phenomenon of apoptosis

It has already been described in patent application WO 96-20701 filed by the Applicant a compound chosen from methional malondialdehyde and any factor increasing the intracellular level of methional or malondialdehyde for use as a medicament, this medicament being more particularly intended to increase the phenomenon of programmed cell death (apoptosis) and thus can allow the treatment of many diseases, more particularly diseases linked to cellular hyperproliferation, such as in the case of cancer, autoimmune diseases or allergies. However, the addition of exogenous methional to cultured cells inhibits the growth of transformed cells than that of normal cells

To try to increase the level of endogenous methional in transformed cells, but not in normal cells, its metabolism has been studied

Thus, in methional metabolism, it is known that 4-methyithιo-2-oxobutanoic acid can be metabolized in vivo by the branched chain oxo-acid dehydrogenase complex present in the mitochondπes of liver, heart and heart cells. skeletal muscle via the methional to give methylthiopropionylCoA [cf Wu, G & Yeaman SJ (1989) Biochem J 257, 281-284, Haussinger, D, Stehle T & Gerok, W (1985) J Biol Chem 366, 527-536 Jones , SMA & Yeaman, SJ (1986) Biochem J 237, 621-623] It is also described that 4-methylthιo-2-oxobutanoιque acid can be metabolized in vivo by transamination to methionine [cf Ogier, G, Chantepie J Deshayes , C, Chantegrel, B Chariot, C, Doutheau, A & Quash, G (1993) Biochem Pharmacol 45, 1631-1644] The methional can optionally also be reduced or oxidized to methionol respectively by an aldehyde reductase or methylthopropionic acid by an aldehyde dehydrogenase The methional in association with the radi cal HO ^' can finally give malondialdehyde and methane thiol by a β-hydroxylation reaction [Quash G Roch AM Chantepie J Michal Y Fournet G and Dumontet C (1995) Biochem J 305 1017-1025]

In the following, the following abbreviations can be used

- MTOB represents 4-methylthιo-2-oxobutanoιque acid,

- MTPA represents methylthiopropionic acid,

E1 represents the decarboxylase of the branched chain oxo-acid dehydrogenase complex whose co-factor is thiamine pyrophosphate (TPP),

- E2 represents the transacylase of the branched chain oxo-acid dehydrogenase complex whose co-factor is thioctic acid (TA),

- ALDR represents aldehyde reductase

- ALDH represents the aldehyde dehydrogenase

It has also been described that transaminase inhibitors involved in the transformation of MTOB into methionine which are compounds of L methionine esters and pyπdoxal selectively inhibit the growth of several types of transformed cells but not that of normal MRC5 cells and weakly induce apoptosis also in BAF3 lymphoid cells cultured in the presence of interleukin 3

In the case of pathologies which are characterized by an overexpression of the bcl2 gene, such as in particular breast cancers, B cell lymphomas, leukemias, neuroblastomas, adenocarcmomas of the prostate, prolactinomas and other pituitary adenomas, this overexpression of the bcl2 gene gives cells resistance to apoptosis and therefore to chemotherapy or antiandrogens (Miyashita, T and Reed, JC (1993) Blood 81, 151-157, Furuya.Y et al (1996) Clinical Cancer Research 2, 389-398)

Furthermore, it has been described that the addition of an inhibitor of the ALDH enzyme, disulfiram (at 50 μM), to BAF3-bo cells induces fragmentation in 30% of cellular DNA, typical of apoptosis, while in apoptosis-resistant BAF3-bcl2 cells, this increase did not exceed 5% (Roch A -M et al, (1996) Biochem J 313, 973-981) The increase in the concentration of disulfiram did not achieve a higher percentage of fragmentation due to the intrinsic toxicity of this product

One of the aims of the present invention is therefore to partially or even completely inhibit this character resistant to the induction of apoptosis due to the bcl2 gene present in the transformed cells.

This object and others are achieved by the present invention which relates to the use of at least one aminothiolester derivative of formula (I) for the preparation of a pharmaceutical composition intended to remove the inhibition of a character resistant to l induction of transformed cell apoptosis, this characteristic being due to the bcl2 gene present in these cells

The present invention also relates to the use of at least one aminothiolester derivative of formula (I) for the preparation of a pharmaceutical composition intended to remove the inhibition of the character resistant to chemotherapy or to antiandrogens of transformed cells.

More particularly, the resistance to chemotherapy or antiandrogens of transformed cells is due to the bc! 2 gene present in these cells. The aminothiolester derivatives have the following formula (I)

(I) in which R1, R2 and R3, independently, represent an alkyl radical, linear or branched, saturated or unsaturated, of C1-C6.

Mention may in particular be made, among the saturated linear alkyl radicals having from 1 to 6 carbon atoms, of the methyl, ethyl, propyl, butyl, pentyl, hexyl radicals

Among the saturated branched alkyl radicals having from 1 to 6 carbon atoms, mention may be made in particular of the 2-methylbutyl, 2-methylpentyl, isopropyl and tert-butyl radicals

Among the unsaturated alkyl radicals having from 1 to 6 carbon atoms, there may be mentioned in particular the allyl radical

Preferably, R1, R2 and R3, independently, represent an alkyl radical from C1 to C3

Preferably, R1 and R2 represent a methyl radical and R3 a radical chosen from the methyl, ethyl and propyl radical.

Advantageously, R1, R2 and R3 represent the methyl radical

A compound of formula (I) is advantageously used in which the amine is in the form of ammonium, preferably in the form of organic ammonium and advantageously in the form of ammonium formate or acetate In the form of ammonium, the compounds of formula (I) have the advantage of being water-soluble, and therefore easily usable

These compounds can be obtained by (1) reaction of a dialkylpropargylamine derivative, in which the amino function is protected (these compounds are described in patent application EP 0 133 407), with a very strong base to give the carbanion of the radical propargyl, (2) this carbanion is then reacted with a carbon oxysulfide, then (3) an alkylation reaction is carried out on the sulfur of the product obtained in (2) and finally (4) the amine is deprotected. To obtain the compound of formula (I) in the form of ammonium, any means known per se is used. Thus, to obtain the compound of formula (I) in the form of formate. or ammonium acetate, the compound obtained in step (4) is reacted with respectively formic acid or acetic acid

Other characteristics, aspects, objects and advantages of the invention will appear even more clearly on reading the description which follows, as well as various concrete examples, but in no way limiting, intended to illustrate it.

FIG. 1 represents the percentage of DNA fragments obtained in BAF-bcl2 cells cultured for 6 hours with different compounds as a function of the concentrations (expressed in μM) of these different compounds which are 4-amino methyl-4 pentyne-2 al-1 (represented by D), S-methyl 4-amιno-4-methylpent-2-yne thioate (represented by M) and the mixture of 4-amιno-4-methylpent-2-yne thioate S-methyl (at different concentrations) and methional at 200 μM (represents by •)

FIG. 2 represents the percentage of DNA fragments obtained in BAF-bO cells cultured for 6 hours with different compounds as a function of the concentrations (expressed in μM) of these different compounds which are 4-amino 4-methyl pentyne-2 al-1 (represented by D), S-methyl 4-amιno-4-methylpent-2-yne thioate (represented by B) and the mixture of 4-amιno-4-methylpent-2-yne thioate S-methyl (at different concentrations) and methional at 200 μM (represented by *)

FIG. 3 represents the percentage of DNA fragments obtained in LNCaP cells (ATCC CRL 1740) cultured for 6 days with 4-amιno-4-methylpent-2-yne S-methyl thioate (represented by M) as a function of the concentrations of this compound (expressed in μM)

These compounds of formula (I) which inhibit the activity of the ALDH enzyme have the advantage of being of the "suicide" type (irreversible covalent bond with the ALDH enzyme), of not being toxic and of inhibiting the growth of transformed (cancerous) cells, not normal cells

The pathologies which are characterized by an overexpression of the bcl2 gene are in particular breast cancers, B cell lymphomas, leukemias, neuroblastomas, adenocarcmomas of the prostate, prolactinomas and other pituitary adenomas The pharmaceutical composition according to the invention comprises a physiologically acceptable medium

The administration of the composition according to the invention can be carried out by enteral, parenteral, topical or ocular route. Preferably, the pharmaceutical composition is packaged in a form suitable for application by a systemic route (for injection or infusion).

By enteral route, the composition, more particularly the pharmaceutical composition, may be in the form of tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid or polymeric vesicles allowing controlled release By parenteral route, the composition may be in the form of solutions or suspensions for infusion or for injection

The compounds of formula (I) according to the invention are generally administered at a daily dose of approximately 0.001 mg / kg to 100 mg / kg in body weight in 1 to 3 doses

Topically, the pharmaceutical composition according to the invention is more particularly intended for the treatment of the skin and mucous membranes and may be in the form of ointments, creams, milks, ointments, powders, soaked tampons, solutions, gels, sprays, lotions or suspensions It can also be in the form of microspheres or nanospheres or lipid or polymeric vesicles or polymeric patches and hydrogels allowing controlled release This composition by topical route can be presented either in anhydrous form, either in aqueous form

By eye, these are mainly eye drops

The compounds of formula (I) are used topically or ocularly at a concentration generally between 0.0001% and 10% by weight, preferably between 0 01 and 1% by weight, relative to the total weight of the composition

The compositions as described above can of course also contain inert or even pharmacodynamically active additives or combinations of these additives, and in particular methional, numerous antineoplastic agents, such as for example dexamethasone, cyclophosphamide, cisplatin, etoposide and BCNU (N, N-Bιs (2-chloroethyl) -N-nιtrosourea), which are also capable of inducing apoptosis

Thus, the present invention also relates to a pharmaceutical composition, characterized in that it comprises, in a physiologically acceptable carrier, at least one compound of formula (I), as described above, and at least one selected compound among methional and an antineoplastic agent

This composition is therefore more particularly intended for treating, preventively or curatively, diseases linked to cellular hyperproliferation, such as cancers, autoimmune or allergic diseases

The pharmaceutical composition preferably comprises methional and at least one compound of formula (I), as described above

Preferably, the antineoplastic agent is capable of inducing apoptosis and can thus be chosen from those mentioned above.

The composition according to the invention may be in the form of a kit comprising at least one compound of formula (I), as described above, and at least one compound chosen from methional and an antineoplastic agent, the compounds of this kit being packaged separately

Of course, a person skilled in the art will take care to choose the possible compound (s) to be added to these compositions in such a way that the advantageous properties intrinsically attached to the present invention are not or substantially not altered by the envisaged addition.

We will now give, by way of non-limiting example, several examples intended to illustrate the present invention.

EXAMPLE 1 Process for the preparation of S-methyl 4-amιno-4-methylpent-2-yne thioate

To a solution of 4.51 g (20 mM) of N, N- (1, 2-bιs (dιmethylsιlyl) ethane) -1, 1- dimethylpropargylamine (described in patent application EP 0 133 407) in 100ml of tetrahydrofuran, 16 ml of a 1.5 M solution of n-butyl thium in hexane are added over 5 minutes at -70 ° C. The reaction medium is left to return to room temperature in 30 minutes and then stirred for another hour. at this temperature (18 ° C.) After returning to -70 ° C., 9 ml of carbon oxysulphide previously condensed are cannulated. After one hour of stirring at -70 ° C. and then 30 minutes at + 3 ° C., 1.31 are added. ml (21 mM) of methyl iodide and stirring is continued for 2 hours at this temperature After dilution in 400 ml of ether, washing with saturated sodium chloride solution, drying over sodium sulfate and evaporated in vacuo, the amine is released on silica gel as described in patent application EP 0 133 407, 2 g (63%) of the amine are thus isolated

EXAMPLE 2

Process for the preparation of S-methyl 4-amιno-4-methylpent-2-yne formate thioate

2.7 mM of formic acid dissolved in 2 ml of ether at 0 ° C. are added to 418 mg (2.7 mM) of the amine obtained in Example 1 in anhydrous ether. minutes, then the mixture is brought to ambient temperature and evaporated to dryness. After taking up in anhydrous ether, the solid is dispersed and the supernatant is removed, then it is dried under vacuum and 415 mg (77%) of the amine formate are isolated. longed for

EXAMPLE 3

Effect of different compounds on the induction of apoptosis in BAF3-bO and BAF3- bc! 2 cells

The cells used correspond to a BAF3 mouse lymphocyte cell line which requires interleukin 3 (IL3) to grow and which undergoes apoptosis (more than 80% of the cells) in the absence of IL3 in 16 hours [cf Collins, MKL, Marvel, J, Malde, P & Lopez-

Rivas, A (1992) J Exp Med 176, 1043-1051]

BAF3-bcl2 cells correspond to BAF3 cells transfected with the bcl2 gene, BAF3-bO cells correspond to BAF3 cells not transfected by the bcl2 gene As stated previously, BAF3-bO cells undergo apoptosis (more than 80% cells) in the absence of IL3 in 16 hours On the other hand, the BAF3-bcl2 cells which are therefore transfected with the bcl2 gene show no sign of apoptosis in the absence of IL3 The BAF3-bO or BAF3-bcl2 cells, cultured in the presence of IL3, are labeled by an adaptation of the method described in Wright, S et al (1992) J of Cell Biochem 48, 344-355, by incubating 2 , 5 10 ⁵ cells / ml with 0.5 μCi [3H] -thymιdιne for 40 hours at 37 ° C. After two washes with a culture medium, 2.5 10 ⁶ cells are cultured in the presence of different compounds to be tested After incubation for 6 hours, these cells are recovered by centπfugation at 400g for 5 minutes and washed 3 times in PBS buffer. The cells recovered in the pellet are lysed in 2 ml of 0.1% Triton X-100, 20mM EDTA, 5mM Tris pH8 and centrifuged at 30,000 g at 4 ° C for 30 minutes The supernatants are recovered and the pellets dissolved in 0.3 ml of 0.5 N NaOH Aliquots of the culture medium (1 ml), of the supernatant (0.3 ml) and solubilized pellet (0.1 ml) are placed in the scintillation counter. The percentage of DNA fragments is c calculated as follows

% of fragments = dpm of the culture medium + dpm of the DNA supernatant dpm of the culture medium + dpm of the supernatant + dpm of the pellet solubilized

The results are collated in FIGS. 1 and 2 The results of amιno-4 methyl-4 pentyne-2 al-1 are given by way of comparison Amιno-4 methyl-4 pentyne-2 al-1 (represented by Z) is also an ALDH inhibitor but does not correspond to the compound of formula (I) used in the present invention Its process for obtaining is described in patent application EP 0 133 407 4-amιno-4-methylpent-2 -yne S-methyl thioate is the compound obtained according to the process described in Example 1

These results clearly show that the presence of S-methyl 4-amιno-4-methylpent-2-yne thioate, preferably in combination with methional, makes it possible to lift the inhibition of apoptosis due to the bcl2 gene in BAF3 cells. -bcl2 The association methιonal-4-amιno- 4-methylpent-2-yne thioate of S-methyl is particularly interesting, because it presents a synergy of the lifting of the inhibition of apoptosis, in fact only at 200μM the methional shows no activity (0% DNA fragments) EXAMPLE 4

Effect of S-methyl 4-amιno-4-methylpent-2-yne thioate on the induction of apoptosis in LNCaP cells

The cells used correspond to a prostate adenocarcinoma cell line LNCaP (ATCC CRL 1740) The LNCaP cells are cultured in a medium containing RPMI 1640 medium (marketed by the company GIBCO) and 7.5% serum of Ellles fetal calf are marked by an adaptation of the method described in Wright, S et al (1992) J of Cell Biochem 48, 344-355, by incubating 2.5 10 ⁵ cells / ml with 0.5 μCi [3H] - thymedene for 5 days at 37 ° C. After two washes with PBS buffer, 2.5 10 ⁶ cells are reincubated in a fresh culture medium containing RPMI 1640 medium 7.5% of fetal calf serum and 4-amino- S-methyl 4-methylpent-2-yne thioate in doses indicated After incubation for 6 days, these cells are recovered by centrifugation at 400 g for 5 minutes and washed 3 times in PBS buffer The cells recovered in the pellet are lysed in 2ml 0.1% Triton X-100, 20mM EDTA 5mM Tris pH8 and centr ifugated at 30000 g at 4 ° C. for 30 minutes The supernatants are recovered and the pellets dissolved in 0.3 ml of 0.5 N NaOH Aliquots of the culture medium (1 ml), of the supernatant (0.3 ml) and of the pellet solubilized (0.1 ml) are placed in the scintillation counter. The percentage of DNA fragments is calculated as follows

% of fragments - dpm of the culture medium + dpm of the DNA supernatant dpm of the culture medium + dpm of the supernatant + dpm of the pellet solubilized

The results are collated in Figure 3

These results clearly show that the presence of S-methyl 4-amιno-4-methylpent-2-yne thioate makes it possible to lift the inhibition of apoptosis due to the bcl2 gene in LNCaP cells.

Claims

1- Use, for the preparation of a pharmaceutical composition intended to remove the inhibition of a trait resistant to the induction of apoptosis of transformed cells, this trait being due to the bcl2 gene present in these cells, of at least an aminothiolester derivative of formula (I) below

(I) in which R1, R2 and R3, independently, represent an alkyl radical, linear or branched, saturated or unsaturated, of C1-C6

2 - Use, for the preparation of a pharmaceutical composition intended to remove the inhibition of the character resistant to chemotherapy or to antiandrogens of transformed cells, of at least one aminothiolester derivative of formula (I) below

(l) in which R1, R2 and R3, independently, represent an alkyl radical, linear or branched, saturated or unsaturated, of C1-C6

3 - Use according to claim 2, characterized in that the character resistant to chemotherapy or antiandrogens of transformed cells is due to the bcl2 gene present in these cells

4- Use according to one of claims 1 to 3, characterized in that the aminothiolester derivative has a formula (I) in which R1, R2 and R3, independently, represent radicals chosen from methyl, ethyl, propyl, butyl radicals , pentyl, hexyl, 2-methylbutyl, 2-methylρentyle, isopropyl, tert-butyl and allyl 5- Use according to any one of the preceding claims, characterized in that R1, R2 and R3, independently, represent an alkyl radical from C1 to C3

6- Use according to any one of the preceding claims, characterized in that R1, R2 and R3 represent the methyl radical

7- Use according to any one of the preceding claims, characterized in that the compound of formula (I) has the amino radical in the form of ammonium, preferably in the form of organic ammonium and advantageously in the form of formate or acetate

8- Use according to any one of the preceding claims, characterized in that the pharmaceutical composition is intended to treat the pathologies chosen from breast cancers, B cell lymphomas, leukemias, neuroblastomas, adenocarcmomas of the prostate, prolactinomas and other pituitary adenomas

9- Pharmaceutical composition, characterized in that it comprises at least one compound of formula (I), defined according to one of claims 1 to 7, and at least one compound chosen from methional and an antomeoplastic agent

10- Pharmaceutical composition according to the preceding claim, characterized in that it comprises methional and at least one compound of formula (l), defined according to one of claims 1 to 6

11- Composition according to one of claims 9 or 10, characterized in that it is in the form of a kit, the compounds of this kit being packaged separately

12- Pharmaceutical composition as defined in one of claims 9 to 11 intended to treat, preventively or curatively, diseases linked to a cellular hyperproliferation, more particularly cancers, autoimmune or allergic diseases

13- Pharmaceutical composition according to the preceding claim, characterized in that it is intended to treat, preventively or curatively, diseases chosen from breast cancers, B cell lymphomas, leukemias, neuroblastomas, adenocarcmomas of prostate, prolactinomas and other pituitary adenomas