MXPA97004491A - Composition that modulates apoptosis, that comprises a factor that influences in the intracellular proportion of metional or malondialdeh - Google Patents

Abstract

The invention relates to a compound selected from methional, malondialdehyde and any factor that influences the intracellular proportion of methional or malondialdehyde, for use as a medicament. This medicine is destined in a more particular way to modulate the phenomenon of programmed cell death (apoptosis). It also relates to a pharmaceutical or cosmetic composition that modulates apoptosis, characterized in that it comprises, as an active agent, a compound selected from methional, malondialdehyde and any factor that influences the intracellular ratio of methional or malondialdehyde, in combination with a pharmaceutical or cosmetologically acceptable carrier. It has thus, by object, a procedure to prevent and / or fight against photoinduced or chronological aging of the pi

Description

COMPOSITION THAT MODULATES. APOPTOSIS, WHICH COMPRISES A FACTOR THAT INFLUENCES IN THE INTRACELLULAR PROPORTION OF METHOD OR MALONDIALDEHIDO The invention relates to a compound selected from methional, malondialdehyde and any factor that influences the intracellular proportion of metional or malondialdehyde for use as a medicine. This medicine is intended more specifically to modulate the phenomenon of programmed cell death (apoptosis). It also refers to a composition that modulates apoptosis. Likewise, its object is a method for preventing and / or combating the photo-induced or chronological aging of the skin. There are two types of mechanisms involved in the death of cells. The first classic type is called necrosis. Morphologically, the necrosis is characterized by a swelling of the mitochondria and the cytoplasm and by a nuclear alteration, followed by the destruction of the cell and its autolysis, this is accompanied by a phenomenon of inflammation. The necrosis occurs passively and incidentally. Tissue necrosis is usually due to a physical trauma of the cells or to a chemical poison, for example. REF: 24615 The other form of cell death is called apoptosis [Kerr, JFR and Wyllie, A. H, Br. J. Cancer, 265, 239 (1972)], but unlike necrosis, apoptosis is not a phenomenon of inflammation. It has been described that apoptosis can be performed under different physiological conditions. It is a highly selective form of cellular suicide, characterized by easily observable morphological and biochemical phenomena. Thus, a condensation of chromatin, associated or not with an endonuclease activity, the formation of apoptotic bodies and a fragmentation of deoxyribonucleic acid (A.D.N.) po is observed in a remarkable way. the activation of endonucleases and fragments of A.D.N. , of 180-200 base pairs (these fragments can be observed by electrophoresis on agarose gel). Apoptosis can be considered as a programmed death of the cells, involved in the development, differentiation and tissue homeostasis. It is therefore considered that the differentiation, growth and maturation of cells are linked to apoptosis. Thus, in a human being with good health, there is a balance between all these phenomena. Within the medical domain, a certain number of pathological situations present a mechanism of modified apoptosis, certainly irregular, or a mechanism of apoptosis that does not provide an alteration of another biological phenomenon to reach equilibrium. Thus, it is described that a voluntary modulation of induced or suppressed apoptosis can make it possible to treat numerous diseases, more particularly diseases linked to a cellular hyperproliferation, such as in the case of cancer, in autoimmune diseases or allergies, or by on the contrary, diseases related to a cellular disappearance, such as in the case of the immunodeficiency syndrome of the human immunodeficiency virus (HIV), neurodegenerative diseases (Alzheimer's disease), excessive damage induced then by myocardial infarction or of an ischemic brain injury. Specifically, it has been found in oncology that numerous anticancer drugs, such as adriamycin and cyclophosphamide, are capable of inducing apoptosis. In the domain of cos ethology, the signs of skin aging result essentially from a dysfunction of the main biological mechanisms of the skin, which make the mechanism of apoptosis intervene in a remarkable way. One might think that any product that modulates the mechanism of apoptosis, is a product suitable to prevent and / or fight against the onset of aging and the signs of aging, such as wrinkles and lines of expression.

Nevertheless, the link between the exogenous or endogenous products of the cell and its cellular response that induces or represses apoptosis is not known. The Applicant has discovered that apoptosis can be induced by an increase in the intracellular proportion of a natural metabolite, the methional (3-methylthio-propanal) or malondialdehyde. The Applicant has also discovered that the in vivo transformation of the metional into malondialdehyde can be deregulated by the expression of oncogenes, such as the bcl2 gene. This bcl2 gene is described in particular as being capable of inhibiting apoptosis induced by reactive oxygen species in cells, and more particularly, within neuronal cells (Kane, DJ et al., 1993, Science 262, 1274-1277). Thus, the present invention relates to a compound selected from methional, malondialdehyde and any factor that influences the intracellular proportion of methional or malondialdehyde, to be used as a medicament. This medicine is in a more particular way, designed to modulate the phenomenon of programmed cell death. The present invention also has for its object, a pharmaceutical or cosmetic composition, which modulates apoptosis, characterized in that it comprises, as an active agent, a compound selected from among the methional, malondialdehyde and any factor that influences the intracellular proportion of the metional or of malondialdehyde, in combination with a pharmaceutical or cosmetologically acceptable carrier. The metional, the malondialdehyde, and any factor that influences the intracellular proportion of metional, can be used in the present invention alone or preferably in a mixture, of course, the choice of products is made depending on the objective sought, is say, either to induce apoptosis, or to suppress apoptosis. Thus, according to a particular embodiment of the invention, the composition that inhibits apoptosis, is characterized in that it comprises, as an active agent, a compound chosen from methional, malondialdehyde and any factor that increases the intracellular proportion of methional or of malondialdehyde, in combination with a pharmaceutical or cosmetologically acceptable vehicle. And, according to another particular embodiment of the invention, the composition that represses apoptosis is characterized in that it comprises, as an active agent, any factor that decreases the intracellular proportion of methional or malondialdehyde, in combination with a pharmaceutical or cosmetological vehicle. acceptable.

In metabolism of the metional, it is known that 4-methylthio-2-oxobutanoic acid, can be metabolized in vivo by the oxo-branched-chain acid dehydrogenase complex, present in the mitochondria of liver, heart, and liver cells. skeletal muscle, by means of the metional to give 4-methylthiopropynyl CoA [cf. Wu, G. & Yeaman, S. J. (1989) Biochem. J. 257, 281-284; Haussinger, D., Stehle, T. & Gerok,. (1985) J. Biol. Chem. 366, 527-536; Jones, S. M. A. & Yeaman, S. J. (1986) Biochem. J. 237, 621-623]. It is also described that 4-methylthio-2-oxobutanoic acid can be metabolized in vivo by transamination in methionine [cf. Ogier, G., Chantepie, J., Deshayes, C, Chantegrel, B., Charlot, C, Doutheau, A & Quash, G. (1993) Biochem. Pharmacol. 45, 1631-1644]. The methional may optionally also be reduced or oxidized respectively in ethionol by an aldehyde reductase or in methylthiopropionic acid, by an aldehyde dehydrogenase. The methional in association with the OH radical, can thus give the malondialdehyde and the methane thiol by a β-hydroxylation reaction. Thus, in order to better position the metional, the malondialdehyde and the factors that can influence its intracellular proportion, Figure 1 illustrates the metabolism of the metional, without however limiting the scope of the invention.

In this figure 1: MTOB represents 4-methylthio-2-oxobutanoic acid; MTPA represents methylthiopropionic acid; It represents the decarboxylase of the oxo-branched-chain acid dehydrogenase complex, wherein the cofactor is thiamine pyrophosphate (TPP); E2 represents the transacilasa of the branched-chain oxo-acid dehydrogenase complex, where the cofactor is thioctic acid (TA); - ALDR represents aldehyde reductase; ALDH represents the aldehyde dehydrogenase. By factor influencing the intracellular proportion of methional or malondialdehyde, it is understood, according to the invention, the compounds chosen among the precursors, the methional or malondialdehyde products, the inhibitors and the activators of the enzymes involved in the metabolism of the metional or malondialdehyde. Thus, it is easily understood in view of the metabolism of methional or malondialdehyde illustrated in Figure 1, that these compounds, methional or malondialdehyde, introduced into a cellular system, will modify the intracellular proportion of methional or malondialdehyde in a provisional manner. or durable. The precursors or products of methional or malondialdehyde may be the precursors or products of intracellular metional metabolism, such as 4-methylthio-2-oxobutanoic acid, methionine, methionol, methylthiopropionic acid and methylthiopropionylCoA. The precursors or products of methional or malondialdehyde can also be products that in situ have the ability to release methional or malondialdehyde or to release a factor that influences the intracellular proportion of methional or malondialdehyde, such as esters and thioesters of methional or malondialdehyde, which will liberate in situ the methional, the malondialdehyde or the 4-methylthio-2-hydroxybutanoic acid, which will be metabolized in situ to 4-methylthio-2-oxobutanoic acid. In the pharmaceutical domain, these products are commonly called "prodrugs". In the case in which it is desired to induce apoptosis, the methional, the malondialdehyde, the precursors or the products of methional or of malondialdehyde are advantageously used in association with the inhibitors of enzymes involved in the metabolic reactions that favor the methionic elimination other than the β-hydroxylation reaction mentioned above (which transforms the methional into malondialdehyde) or in association with the activators of β-hydroxylase which is the enzyme involved in the transformation of methional into malondialdehyde. Thus, we can mention, as an example, an association of 4-methylthio-2-oxobutanoic acid with a transaminase inhibitor involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, such as the inhibitors indicated below. We can also consider the association of the metional or the factor that increases the intracellular proportion of metional with the compounds that increase the proportion of the free radical OH. We can also cite the BCNU (N, N-Bis (2-chloroethyl) -N-nitrosourea) because it is an inhibitor of glutathione reductase, which allows increasing the intracellular proportion of reactive oxygen species, such as the OH radical, which is one of the substrates of the β-hydroxylation reaction to give the malondialdehyde. * This association is particularly interesting in the case of pathologies that are characterized by an overexpression of the bcl2 gene.

Such pathologies are particularly breast cancers, B cell lymphores, leukaemias, neuroblastomas, adenocarcinomas of the prostate, prolactinomas and other pituitary adenomas. Overexpression of the bc: 12 gene gives the cells a chemoresistant characteristic. This association can thus allow to partially or totally inhibit this chemoresistant characteristic. Advantageously, we can add to this association at least one transaminase inhibitor involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, such as those described below and more particularly the compound of the formula (9) .

Among the inhibitors or activators of the enzymes involved in metabolism of the metional, we can mention in particular the inhibitors or activators of the oxo-branched-chain acid dehydrogenase complex, involved in the transformation of 4-methylthio-2-oxobutanoic acid into methylthiopropionylCoA , by means of the metional or the inhibitors or activators of the transaminase involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, or the inhibitors or activators of the aldehyde reductase responsible for the reduction of methionally in methionol, or of the aldehyde dehydrogenase responsible for the oxidation of methional in methylthiopropionic acid, or in addition the activators or inhibitors of the β-hydroxylase which is the enzyme involved in the transformation of the methional into malondialdehyde. Thus, we can mention, in the manner of the inhibitor of the oxo-branched-chain acid dehydrogenase complex and thus, as a factor that decreases the intracellular proportion of methional or malondialdehyde, the 2-oxobutyrate which is a well-known substrate of this complex [cf. . Jones, S. M. A. & Cederbaum, A. I. (1989) Arch. Of Biochem. and Biophys., 199, 438-447], as well as ketoleucine, ketoisoleucine and ketovalin, which are other substrates of this complex. We can also mention, as inhibitors of transaminase involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, and thus, as factors that increase the intracellular proportion of methional or malondialdehyde, the products described in Ogier , G., Chantepie, J., Deshayes, C, Chantegrel, B., Charlot, C, Doutheau, A & Quash, G. (1993) Biochem. Pharmacol. 45, 1631-1644. More particularly, the transaminase inhibitors involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine are chosen from the following products of formulas (1) to (9): (1) (2) CH, S COOCH (CH, .COOCjHj J) ', 2 (3) (4) COOCjH5 (5) (6) (7) (3) (9) formulas in which X represents a radical -OH or a radical - The compound of formula (9) in which X represents an -OH radical is particularly preferred. We can also mention, as a transaminase inhibitors involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine and thus, as factors that increase the intracellular proportion of methional or malondialdehyde, hydroxamates of amino acids , such as the hydroxamate of D-asparagine, of the formula CONHOHCH2CHNH2COOH, and the hydroxamate of L-glutamine, of the formula CONHOHCH.CH: CHNH; COOH.

When we want to induce apoptosis, it is preferred according to the invention, to use a compound chosen from methional, malondialdehyde, or products that in situ have the ability to liberate metional or malondialdehyde, such as methionic esters or thioesters. or of malondialdehyde, or the activators of the enzyme involved in the transformation of the metional into malondialdehyde. Among the particularly interesting mixtures to induce apoptosis, we can mention in particular the association of methional with at least one transaminase inhibitor involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, and preferably, the compound of formula (9). When we want to repress apoptosis, it is preferred according to the invention, to use the inhibitors of enzymes involved in the production of methional or malondialdehyde, such as 2-oxobutyrate, and / or the activators of the enzymes involved in the elimination of the methtional which does not is the β-hydroxylation reaction mentioned below. The composition according to the invention is more particularly intended for the following treatments: 1) For treating dermatological conditions linked to a keratinization disorder sustained on differentiation and on hyperproliferation, particularly for treating vulgar, comedogenic acnes, polymorphs, rosacea, nodulocystic acnes, conglobata, senile acnes, secondary acnes such as solar acne, medicated or professional. 2) To treat other types of keratinization problems, particularly ichthyosis, ichthyosiform states, Darier's disease, keratoderma palmoplantari, leukoplakias and leukoplasiform states, cutaneous lichenization or mucosa (buccal). 3) To treat other dermatological conditions linked to the problem of keratinization with an inflammatory and / or immunoallergic component and, particularly, all forms of psoriasis that are cutaneous, mucous or ungueal, and likewise psoriatic rheumatism, or even the cutaneous atopy, such as eczema or respiratory atopy or even gingival hypertrophy; 4) To treat all dermal or epidermal hyperproliferations that are benign or malignant, whether or not they are of viral origin, such as vulgar warts, flat warts and epidermis verpuciform epidermis, oral or florid papillomatosis and hyperproliferations that may be induced by ultraviolet rays, particularly in the case of epithelioma, basal and spinocellular.

) To treat other dermatological conditions such as vesicular dermatoses and collagen diseases. 6) To treat certain ophthalmological problems, particularly corneopathies. 7) To repair or fight against skin aging, which is photo-induced or chronological or to reduce pigmentations and actinic keratosis, or all the pathologies associated with chronological or actinic aging. 8) To prevent or cure the sores of epidermal and / or dermal atrophy induced by local or systemic corticosteroids, or any other form of cutaneous atrophy. 9) To prevent or treat problems of scarring or to prevent or repair stretch marks. 10) To fight against problems of sebaceous function such as acne hyperseborrhea or simple seborrhea. 11) In the treatment or prevention of cancerous or precancerous conditions. 12) In the treatment of inflammatory conditions such as arthritis. 13) In the treatment of any condition of viral origin at cutaneous or general level, such as arthritis. 14) In the prevention or treatment of alopecia.

) In the treatment of dermatological or general conditions that have an immunological component. 16) In the treatment of cardiovascular system conditions such as arteriosclerosis and thrombocytopenia. 17) In the treatment of neurodegenerative diseases, such as Alzheimer's disease. In the therapeutic domains mentioned above, the composition according to the invention can advantageously comprise other active agents, such as retinoids, free radicals, lo derivatives of vitamin D, cortisteroids or estrogens, antioxidants , the α-hydroxy or α-keto acids or their derivatives, or even the potassium channel blockers. Another subject of the present invention is the use of the composition according to the invention, in or for the manufacture of a cosmetic or pharmaceutical composition intended to modulate apoptosis and, more particularly, intended for preventive treatments and / or curatives described above. Retinoids can be of natural or synthetic origin. Among retinoids, we can mention 9-cis retinoic acid, retinoic acid after.

Among the vitamins D or their derivatives, we can mention, in particular, the derivatives of vitamin D2 or D3 and in particular 1, 25-dihydroxyvitamin D3. Among the free antiradicals, we can mention in particular, a-tocopherol, Super Oxide Dismutase, Ubiquinol or certain metal chelators. Among the a-hydroxy or a-keto acids or their derivatives, we can mention in particular ketoleucine, ketoisoleucine, ketovaline, 2-oxobutyrate, 4-methylthio-2-oxobutanoic acid, lactic acid, malic acid, citric acid , glycolic, mandelic, tartaric, glyceric ascorbic, or derivatives of salicylic acid or its salts, amides or esters. Among the blockers of potassium channels, we can mention in particular the Minoxidil (2,4-diamin-6-piperidin-pyrimidin-3-oxide) and its derivatives. The administration of the composition according to the invention can be effected by enteral route, parenteral, topical or ocular, preferably, the composition is conditioned under a convenient form to a systemic application (by injection or perfusion). By enteral route, the composition, more particularly the pharmaceutical composition, can be presented in the form of tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or Nanospheres or lipid or polymeric vesicles that allow controlled release. By parenteral route, the composition can be presented in the form of solutions or suspensions for perfusion or for injection. The medicament according to the invention is administered generally at a daily dose of about 0.001 mg / kg to 100 mg / kg per body weight in 1 to 3 doses. Topically, the composition according to the invention is intended more particularly for the treatment of the skin and mucous membranes and may be presented in the form of ointments, creams, milks, ointments, powders, embedded tampons, solutions, gels, rubbers, lotions or suspensions. They can also be present in the form of microspheres or nanospheres or lipid or polymeric vesicles, or polymer patches and hydrogels allowing a controlled release. This topical composition can be presented either under the anhydrous form, or under the aqueous form. By eye, they are mainly eye drops. The topical or ocular pharmaceutical composition comprised of methional, malondialdehyde or any factor that influences the intracellular proportion of methional or malondialdehyde at a concentration preferably between 0.001 and 5% relative to the total weight of the composition. The composition, according to the invention, also finds an application in the cosmetological domain, in particular in the corporal and capillary hygiene and particularly for the treatment of skins with acne tendency, for the growth of the hair, to avoid the fall of the same , in the protection against the harmful effects of the sun or in the treatment of physiologically dry skins, to prevent and / or fight against photoinduced or chronological aging. The subject of the present invention is therefore a method for preventing and / or combating photoinduced or chronological aging of the skin, characterized in that a cosmetic composition inducing apoptosis such as that described above is applied to the skin. In the cosmetological domain, the composition according to the invention may advantageously comprise retinoids, vitamins D or their derivatives, corticosteroids, free radicals, α-hydroxy or α-keto acids or their derivatives, or even ion channel blockers . These different products used in the composition of the present invention are those defined above.

The cosmetic composition according to the invention can be present in particular in the form of a cream, a milk, a lotion, a gel, microspheres or nanospheres or lipid or polymeric vesicles, a soap or a shampoo. The concentration in methional, malondialdehyde or any factor that influences the intracellular proportion of methional or malondialdehyde in the cosmetic composition, preferably comprises between 0.0001 and 3% by weight of the composition. The pharmaceutical or cosmetic composition according to the invention can otherwise comprise inert additives or also pharmacodynamically or cosmetologically active additives or combinations of these additives, and particularly the wetting agents; depigmenting agents such as hydroquinone, acelaic acid, caffeic acid or cojic acid; emollients, moisturizing agents such as glycerol, PEG 400, thymmorpholinone and its derivatives or urea, antiseborrhoeic or antiacne agents, such as S-carboxymethylcysteine, S-benzyl-cysteamine, its salts or its derivatives, or benzoyl peroxide, antibiotics such as erythromycin and its esters, neomycin, clindamycin and its esters, tetracyclines, antifungal agents such as ketoconazole or polymethylene-4,5-isothiazonilone-3; agents that promote the growth of hair, such as Minoxidil (2,4-diamin-6-piperidin-pyrimidin-3-oxide) and its derivatives, * Diazoxide (7-chloro 3-methyl 1, 2, 4- benzothiadiazine 1,1-dioxide) and Feniotoin (5,4-diphenyl-1-idazolidin-2,4-dione); non-steroidal anti-inflammatory agents; carotenoids and, particularly b-carotene, antipsoriatic agents such as anthralin and its derivatives and eicosa-5, 8, 11, 14-tetranoic and eicosa-5, 8, 11-trinoic acids, their esters and amides. The composition according to the invention may also contain flavor improving agents, preservative agents such as parahydroxybenzoic acid esters, stabilizing agents, moisture regulating agents, pH regulating agents, modifying agents, the osmotic pressure, the emulsifying agents, the UV-A and UV-B filters, the antioxidants such as a-tocopherol, butylated hydroxyanisole or butylated hydroxytoluene. The examples are now provided, by way of illustration and without any limiting character.

EXAMPLES Example 1 Effect of metabolites on the growth of HeLa cells HeLa cells (0.5 x 10s per Petri dish) are cultured in 3 ml of Eagle's minimal essential medium, which contains 10% by weight of dialysate fetal calf serum. After 4 hours, it is added to each of the three boxes, metional (lOμM-lmM), methionol (10μM-20mM), acid »methylthiopropionic (MTPA) (lmM-20mM), L-methionine (lmM-20mM) or 4-Methylthio-2-oxobutanoic acid (MTBO) (lM-20mM). After three days at 37 ° C, the cells are washed twice in a buffered saline medium at pH 7.5 and phosphated (PBS medium), and recovered in the same type of buffer. Cell growth is evaluated by measuring the protein content (see Method in Lo ry, OH Rosebrouh, NJ Farr, AL &Randall, RJ (1951) J. BIOL.CHEM. 193, 265-275), or the content of DNA in the Used ones using a bisbenzimide trichlorhydrate solution (Hoechst 33258) (see Jarvis, W.D., Kolesnick, R.N., Fornari, F.A., Traylor, R.S., Gewirtz, D.A. & Grant, S. (1994) Proc. Nati Acad. Sci.

E.U.A. 91, 73-77) or by determining the activity of lactic dehydrogenase, using the bromide of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium (MTT) (see Mosmann, T. (1983) J. Of Immunology, Methods 65, 55-63). The results are summarized in Table 1. Each value provided is the average of 3 or 4 experiments estimated for the protein content.

Table 1 IC50 corresponds to the concentration of product necessary to obtain an inhibition of cell growth of 50%. The results evaluated by measuring the DNA content or determining the activity of the lactic dehydrogenase are comparable to those obtained in this Table. The table shows the difference of IC 50 of methional in relation to the IC 50 of its products or precursors.

Example 2 The HeLa cells (0.5 x 106 per Petri dish) are cultured in 3 ml of an Eagle essential minimal medium, devoid of methionine (Met "), containing 10% by weight of dialysis fetal calf serum. , add 2-oxobutyrate (10mM-40mM) in the presence of 2mM of 4-methylthio-2-oxobutanoic acid (MTBO) After three days at 37 ° C, the cells are washed twice in a buffered saline medium. at pH 7.5 and phosphate (medium PBS), and are recovered in the same type of buffer Cell growth is measured in the same manner as in the preceding example 2-oxobutyrate is a well-known substrate of the oxo-acid dehydrogenase complex Branched chain (BCOADC) (Km 18μM) MTOB only at 2 mM, induces a growth inhibition of 72% .The results show that 2-oxobutyrate has a capacity to increase this inhibition of growth. from 10 mM of 2-oxobutyrate, reduced from 2 to 3 times. The activity of BCOADC for the transformation of MTOB into methional is displaced to a different substrate, such as 2-oxobutyrate, which decreases the inhibition of growth.

Example 3 Effect of the metional on DNA fragmentation in BAF3 cells The cells used correspond to a mouse lymphocytic cell line BAF3, which requires interleukin 3 (IL3), to grow and hasten the apoptosis (more than 80% of cells), in the absence of IL3, in 16 hours [cf . Collins, M.K.L., Marvel, J., Malde, P. & Lopez-Rivas, A. (1992) J. Exp. Med. 176, 1043-1051]. » 3- x 106 BAF3 cells, are cultured in 6ml of a culture medium containing IL3 in the Petri dishes, in the presence of different concentrations of metional (200-800μM), or of propanal, which is a substrate of the aldehyde dehydrogenase . After 8 hours of contact, the cells are washed three times in a saline medium buffered to pH 7.5 and phosphated (PBS medium). The cells are used in 2ml of 0.1% Triton X-100, 20mM EDTA (ethylenediamine tretraacetic acid), 5mM Tris pH 8 and then centrifuged at 30,000g at 4 ° C for 30 minutes. The supernatants are decanted and subjected to the following analyzes. By way of comparison, the same cultures are performed in a medium, either only with IL3 (+ IL3), or without IL3 (IL3-).

- A qualitative analysis of the DNA fragments obtained is carried out following the method described in particular in the publication by Jarvis, .D., Kolesnick, R.N., Fornari, F.A., Traylor, R.S., Gawirtz, D.A. & Grant, S. (1994) Proc. Nati Acad. Sci. USA 91, 73-77. The supernatants are treated with ribonuclease A (20μg / ml), 1 hour at 37 ° C and with proteinase K (100μg / l). The DNA is purified by extraction with phenol and precipitated with ethanol. In the final extract, the DNA fragments of low molecular weight are analyzed by electrophoresis on a 1% agarose gel. After the electrophoresis ^ (60V, 3 hours), the gels are stained with ethidium bromide. The results clearly show that apoptosis is induced with the metional, the gel obtained presents the image of a scale of multiple DNA fragments, from 180 to 200 base pairs, typical of an induction of apoptosis. In the case of propanal, the gel does not have this characteristic. - An analysis is performed by spectrofluorometry to measure the amount of DNA fragments obtained (<3 kilobases). Add 2 ml of supernatant, 1 ml of bisbenzimide trichlorhydrate solution (Hoescht 33258) (lμg / ml) in 3 mM of sodium chloride, imMd4EDTA, lOmM of Tris pH 8. Analyze the solutions obtained by fluorometry (? = 365nm,? Of emission = 460nm). The amounts of DNA are calculated in a manner relative to that of highly purified DNA (0.1 to 1 μg in 2 ml of lysate buffer), treated as indicated below and expressed in 10 ~ 9 g of DNA, recovered from 3 x 10 cells . The results summarized in Table 2 below are obtained.

Table 2 Thus, the increase in the amount of DNA fragments found in cells treated with IL3 and methional is similar to that found when the cells are placed in a medium devoid of IL3.

Example 4 Effect of the BCNU and a transaminase inhibitor, involved in the transformation of 4-methylthio-2-oxobutanoic acid, on the production of reactive oxygen species (H2Q2 and OH ") 1. 5 105 BAF3 cells (cell lineage as described above), seeded in 3ml of a culture medium containing IL3 [Dulbecco's modified Eagle's medium, containing 6% fetal calf serum and 5% d "medium conditioned by the ehi-3B cells, used as a source of IL3, medium described in Collins et al. (1992) J. Exp. Med. 176, 1043-1051], are treated with 10 μM of 2 ', 1' diacetate -dichlorofluorescein (later called DCFH-DA, and obtained by Molecular Probes Inc.). After 1 hour of incubation at 37 ° C, the cells are washed with a PBS buffer. 10 minutes before the fluorescence analysis of dichlorofluerescein (DCF), by flow cytometry, add 3μg / ml of propidium iodide. Incubation is carried out in the presence of different concentrations of BCNU and in the presence or absence of compound A 50 μM, (corresponding to the compound of the formula (9), where X represents an OH- radical). This method allows to provide a percentage of fluorescence obtained by the reaction of reactive oxygenated species (H202 and -OH) with DCFH-DA. Flow cytometry is performed with a FACScan flow cytometer (Becton Dickinson, San José, CA, USA). The results are summarized in Table 3 below.

Table 3 Thus, these results show that the compound A that allows to increase the proportion of metional, represses the increase of -OH, due to the increase in the concentration of BCNU. Thus, it is probable that there is a reaction of the metional with the radicals -OH, present to give malondialdehyde, which induces apoptosis.

Example 5 Effect of the BCNU and a transaminase inhibitor involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, on apoptosis.

The DNA strand sections are labeled with biotinylated dUTP (detected by avidinfluorescein) in situ, in cells permeabilized and fixed using the terminal DNA deoxynucleotidyl transferase (TdT) test, as described in Gorczyca et al. , (1993) Cancer, Res. 53, 1945-1951. 1.5 105 BAF3 cells (cell lineage as described above), are seeded in 3ml of a culture medium containing IL3 (Dulbecco's modified Eagle's medium, which contains 6% fetal calf serum and 5% medium conditioned by ehi-3B cells, used as a source of IL3). The cells are incubated in the presence of 50 μM of compound A, 40 μg / ml of BCNU, the mixture of these two products (50 μM of compound A + 40 μg / ml of BCNU), or in the absence of these two compounds (control) . After 24 hours of incubation, the cells are washed with a buffer of PBS and fixed in formaldehyde, then in ethanol and left at -20 ° C for 3 days. The cells were then resuspended in 50 μl of a solution containing 0.1 μM of sodium cacodylate, pH 7.5, litiM of CoC12, 0.1 mM of dithiothreitol, 0.05 mg / ml of BSA (Bovine Seroalbumin), 10 units of TdT (of thymus of Boerhringer calf), 0.5 nmol of dGTP for 1 hour at 37 ° C. After washing with PBS, the cells are resuspended in 100 μl of 4xSSC buffer (0.15 M NaCl, 0.015 M sodium citrate) containing 2.5 μg / ml of avidinfluorescein isocyanate (Sigma), 0.1% (w / weight). vol) of RNAase A, 0.1% Triton X100 and 5% (w / v) of skimmed milk, for 30 minutes at room temperature in the dark. These cells are washed, then suspended in lml of PBS containing 3 μg / ml of propidium iodide. Flow cytometry is performed with a FACScan flow cytometer (Becton Dickinson, San José, CA, USA). The results are summarized in Table 4 below.

Table 4 At these doses, the products alone weakly induce apoptosis, on the contrary, their mixture strongly induces apoptosis (synergy). This result confirms the results of Table 3 and the corresponding conclusions.

Example 6 Determination of the most effective therapeutic combination to treat mice infected with melanoma cells at » At t = 0 days, 105 B16 Fl murine melanoma cells are injected into B6D2F1 mice (from IFFA CREDO, France). At t = 1 day, and so continuously for 15 days, these mice are injected once a day with different therapeutic regimens specified in Table 3 above. The control corresponds to the mice that have not followed the therapeutic regimen. Table 5 gathers the results that correspond to the averages of the results on 3 treated mice.

Table 5 For the regimens of several compounds, the doses of each of the compounds used are identical to those of the regimens where the compounds are used alone. The survival time is counted from t = 0 day indicated above and corresponds to an average of mice that have not survived more than 60 days. The BCNU corresponds to N, N-Bis (2-chloroethyl) -N-nitrosourea.

Compound A corresponds to the compound of formula (9) wherein X represents an -OH radical. Thus, it is appreciated that the last two regimens are particularly effective since they significantly increase the survival time of these mice. Between these two regimes, the last regimen (added with metional) allows to obtain the third part of mice that survive more than 60 days.

Example 7 Proceed as in the preceding example, the regimens and the number of treated mice varies. Table 6 gathers these proportions and the results obtained.

Table 6 The survival time is counted from t = 0 day indicated above and corresponds to an average of mice that have not survived beyond 60 days. For the last regimen, the doses correspond to those of the compounds used alone. The percentage of long-term survivors corresponds to the percentage of mice that have survived more than 60 days. Thus, it is appreciated that the last regimes, and especially the last, are particularly effective.

Example 8 Effect of the metional on the induction of apoptosis in cells BAF3-b0 and BAF3-bcl2 The cells used correspond to a lymphocytic cell line BAF3, such as that described above. The BAF3-bcl2 cells correspond to the BAF3 cells transfected by the bcl2 gene, the BAF3-b0 cells correspond to the BAF3 cells not transfected by the bcl2 gene. As indicated above, BAF3-b0 cells undergo apoptosis (more than 80% of cells) in the absence of IL3 in 16 hours. In contrast, BAF3-bcl2 cells that are transfected by the bcl2 gene do not show any sign of apoptosis in the absence of IL3. In this way, they constitute a good model to determine the stage of blocked apoptosis in cells and their eventual correlation with the inhibition of malondialdehyde synthesis. The BAF3-bO or BAF3-bcl2 cells are marked by an adaptation of the method described in Wright, S et al (1992) J. of Cell Biochem. 48, 344-355, incubating 2.5-105 cells / ml with 0.5 μCi [3 H] -thymidine for 40 hours at 37 ° C. After two washes with a culture medium, 2.5 rl06 cells are placed in culture in the presence of methional. After incubation for 8 hours, these cells were recovered by centrifugation at 400g for 5 minutes, and washed 3 times in a PBS buffer. The cells recovered in the residue are used in 2 ml of 0.1% Triton X-100, 20 mM EDTA, 5 mM Tris at pH 8 and centrifuged at 30000 g at 4 ° C for 30 minutes. The supernatants are recovered and the residues are dissolved in 0.3 ml of 0.5 N NaOH. The aliquots of the culture medium (lml), the supernatant (0.3ml) and the solubilized residue (0, 1ml) are dosed. in the scintillation counter. The percentage of ÁDN fragments is calculated as follows: of DNA fragments = dpm of the culture medium + dpm of the supernatant dpm of the culture m-dio + dpm of the supernatant + dpm of the solubilized residue Results: The addition of an increasing concentration of methional (0; 50; 100; 200 300; 400 μM) in the incubation medium increases the percentage of DNA fragments to a maximum, for 400 μM of methional, of 26% compared to 7% for the BAF3-b0 cells not treated by the metional (control). For BAF3-bcl2 cells treated with the equivalent concentrations of methional, the DNA fragments reached a maximum of 7%, for 400 μM of methional, compared to 3% for the control. These results clearly show that adding methional does not increase the inhibition of apoptosis due to the bcl2 gene in BAF3-bcl2 cells. In the induction of apoptosis due to lack of IL3, it is observed that the metional is transformed into malondialdehyde, by a β-hydroxylation reaction with the help of -OH. We have put the dose of H202 and HO in the BAF3-bO and BAF3-bcl2 cells.

Example 9 Measurements of the reactive oxygen species (H2Q2 and -OH) by the flow cytometry method The cells used correspond to a lymphocytic cell lineage of BAF3 such as that described above. BAF3-bcl2 cells correspond to BAF3 cells transfected by the bcl2 gene, BAF3-bO cells correspond to BAF3 cells not transfected by the bcl2 gene. 1.5-106 cells of BAF3-bO or -bcl2, seeded in 3ml of a culture medium containing IL3, are treated with 10 μM of dichlorofluoroacetin diacetate (DCFH-DA). After 1 hour of incubation at 37 ° C, the cells are washed with a PBS buffer. 10 minutes before the fluorescence analysis of dichlorofluorocein (DCF) by flow cytometry, 3 μg / ml of propidium iodide is added, as eff example 4. Flow cytometry is performed with a flow cytometer EACScan (Becton Dickinson , San José, CA; USA). The results are gathered in table 7 below.

Table 7 Accordingly, if there is a tendency for BAF3-bcl2 cells to have more reactive oxygen species than BAF3-bO cells, this difference is not significant. It is possible that BAF3-bcl2 cells have a failure in the synthesis of methional and / or malondialdehyde.

Example 10 Analysis of 14C-labeled products derived from [14C] MTOB in BAF3-bO and BAF3-bcl2 cells The cells used are identical to those of the previous example. The MTOB [14C] is prepared by the oxidative deamination of the [14C] methionine as described in Ogier G. et al. (1993) Biochem. Pharmacol. 45, 1631-1644. The MTOB [LC] (5,106 dpm corresponding to 38.2 nmol) is added to a cell suspension of BAF3-b0 or BAF3-bcl2 (2.4-105 cells / ml) in DMEM containing 6% borino serum fetal and 5% IL3. 39 hours later, the cells are washed twice with PBS and resuspended at 9.105 cells / ml. Add 108 MTOB [U1C] cells (1.9-106 dpm corresponding to 14.5 nmol) and 8 hours later, the cells are recovered by centrifugation at 400g for 5 minutes. Cellular debris is solubilized with 1.5 ml of 0.5 N NaOH for 30 minutes at 60 ° C. The total radioactivity present in the cells is measured on the aliquots of 10 μl. After this, an alkaline hydrolyzate obtained from perchloric acid is added to a final concentration of 0.5 N, then 200 μmol of 2,4-DNPH (2,4-dinitrophenylhydrazine, obtained from Merck). The mixture is heated at 70 ° C for 30 minutes, then centrifuged at 400g for 5 minutes. The precipitate is solubilized in -1 ml of 0.5 N NaOH, the radioactivity in the protein content is determined. The supernatant is added to NaOÍT at pH 10. The labeled metabolites are extracted in ethylene acetate and the organic phase is dried, acidified and extracted in dichloromethane. The labeled metabolites in the organic phase are separated and identified by ccf.ar (high performance thin layer chromatography), and compared with the 2,4-DNPH derivatives, the compounds of the reference are then quantified by scanning radiochromatography. The results are gathered in the following table 8: Table 8 1 Radio means total radioactivity present in the cells. MDA means malondialdehyde.

Thus, it appears that the total radioactivity reported in an identical amount of DNA is similar in the two cell types after 47 hours of incubation. The same is for the methionine activity of cellular proteins. As regards free methionine, a 40% increase in radioactivity is observed in BAF3-bcl2 cells, compared to that in BAF3-b0. The radioactivity present in the metional is 80% higher in the BAF3-bO cells than in the BAF3-bcl2 cells. This demonstrates that in BAF3-bcl2 cells, the decrease in metional formation from MTOB is accompanied by a concomitant increase in methionine formation. When the formation of labeled MDA is measured, this corresponds to 800 dpm / mg of DNA in the BAF3-bO cells, no radioactivity for MDA is detected in the extracts of the BAF3-bcl2 cells. BAF3-bcl2 cells also exhibit a decrease in the formation of MDA from methional. Example 8 clearly shows that this decrease is not due to a decrease in the reactive oxygen species in the BAF3-bcl2 cells, in comparison to those of the BAF3-bO cells. We can conclude then that the bcl2 gene in addition to reducing the transformation of MTOB in metional, also negatively affects the ß-hydroxylation reaction that transforms the metional into malondialdehyde. Thus, any product that modulates the intracellular proportion of methional or its metabolism product, malondialdehyde, can be considered as a modulator of apoptosis. It is noted that with regard to this date, the best method known to the applicant to carry out the present invention is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (20)

1. A compound characterized in that it is chosen from methional, malondialdehyde and any factor that influences the intracellular proportion of methional or malondialdehyde for use as a medicine.

2. A pharmaceutical or cosmetic composition that modulates apoptosis, characterized in that it comprises, as an active agent, at least one compound selected from the methional, malondialdehyde and any factor that influences the intracellular proportion of methional or malondialdehyde, in combination with a pharmaceutical or cosmetologically acceptable carrier.

3. The composition according to claim 2, characterized in that the methional, malondialdehyde and any factor that influences the intracellular proportion of methional or malondialdehyde, are used in a mixture.

4. The composition according to one of claims 2 or 3, characterized in that the factors that influence the intracellular proportion of methional or malondialdehyde are chosen among the precursors, the metional products, the inhibitors and the enzyme activators involved in the metabolism of the metional.

5. The composition according to the preceding claim, characterized in that precursors or products of methional or malondialdehyde are the precursors or the products of the intracellular metabolism of methional or malondialdehyde, such as 4-methylthio-2-oxobutanoic acid, methionine , methionol, methylthiopropionic acid and methylthiopropylCoA.

6. The composition according to claim 4, characterized in that the precursors or products of the methional or malondialdehyde are the products that in situ have the ability to liberate the metional or the malondialdehyde, or to release a factor that influences the intracellular proportion of metional or malondialdehyde, such as esters and thioesters of methional or malondialdehyde.

7. The composition according to claim 4, characterized in that the inhibitors or the activators of the enzymes involved in the metional metabolism, are chosen among the inhibitors or activators of the oxo-acid dehydrogenase complex, which has a branched chain, involved in the transformation of 4-methylthio-2-oxobutanoic acid in methylthiopropylCoA, by means of methion, inhibitors or activators of transaminase involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, the inhibitors or activators of the aldehyde reductase responsible of the metional reduction in methional or of the aldehyde dehydrogenase, responsible for the oxidation of methional in methylthiopropionic acid, and the inhibitors or activators of the β-hydroxylase.

8. The composition that induces apoptosis according to one of claims 2 to 7, characterized in that it comprises, as an active agent, at least one compound selected above the methional, or malondialdehyde, and any factor that influences the intracellular proportion of metional or malondialdehyde, in combination with a pharmaceutical or cosmetologically acceptable carrier.

9. The composition according to claim 8, characterized in that the factors that increase the intracellular proportion of methional or of malondialdehyde are the transaminase inhibitors, involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, chosen among the products of formulas (1) to (9) below: C-H (2) (3) (5) (7) (8) CM, S, COOC (CH.) 3 (9) formulas in which X represents -OH or -OPO H

10. The composition according to claim 9, characterized in that the transaminase inhibitor involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine is the compound of formula (9), in which X represents the -OH radical.

11. The composition according to claim 8, characterized in that the factors that increase the intracellular proportion of methional or of malondialdehyde are the transaminase inhibitors involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, chosen from the hydroxamates of the aminoamidated acids.

12. The composition according to claim 8, characterized in that it comprises an association of a compound selected from methional, malondialdehyde, precursors and products of methional or malondialdehyde with the inhibitors of the enzymes involved in the metabolic reactions that favor elimination. of methional or with the activators of the ß-hydroxylase.

13. The composition in accordance with the. claim, characterized in that the association corresponds to 4-methylthio-2-oxobutanoic acid, with a transaminase inhibitor involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, such as the products mentioned in one of the claims 9 to 11.

14. The composition according to claim 8, characterized in that the association corresponds to the metional or a factor that increases the intracellular proportion of metional with at least one compound that increases the proportion of the free radical -OH, the compound is preferably the BCNU.

15. The composition according to the preceding claim, characterized in that it comprises at least one transaminase inhibitor involved in the transformation of 4-methylthio-2-oxobutanoic acid into methionine, such as the products mentioned in one of claims 9 to 11.

16. The composition according to one of claims 2 to 7, characterized in that it comprises, as an active agent, any factor that decreases the intracellular proportion of metional or malondialdehyde, in combination with a pharmaceutical or cosmetologically acceptable carrier.

17. The composition according to the preceding claim, characterized in that the factor that decreases the intracellular proportion of methional is an inhibitor of the oxo-acid dehydrogenase complex, which has a branched chain, such as 2-oxobutyrate, ketoleucine, ketoisoleucine and ketovaline

18. The composition according to claim 16, characterized in that it comprises the inhibitors of the enzymes involved in the production of methional and / or the activators of the enzymes involved in the elimination of the methional.

19. The use of a composition defined according to one of any of claims 2 to 18, in a cosmetic composition or for the manufacture of a pharmaceutical composition, characterized in that the composition is intended to modulate apoptosis.

20. A method for preventing and / or combating photoinduced or chronological aging of the skin, according to one of claims 2 to 15, characterized in that a cosmetic composition that induces apoptosis is applied to the skin.