Modulating apoptosis of a composition comprising a factor influencing the intracellular content of rationale or malonic dialdehyde acid

 

(57) Abstract:

The invention relates to the field of medicine and for the connection of rationale, malonic dialdehyde acid and any factor affecting the intracellular content of rationale or malonic dialdehyde acid as a drug to modulate the phenomenon of programmed cell death (apoptosis). The invention also relates to pharmaceutical or cosmetic modulating apoptosis of a composition containing as active agent a compound selected among Nationale, malonic dialdehyde acid and any factor affecting the intracellular content of rationale or malonic dialdehyde acid, in combination with pharmaceutically or cosmetically acceptable base. Finally, the object of the invention is a method of preventing and/or against photoinductive age or aging skin. The proposed compositions are more effective for the treatment of various dermatological lesions. 3 C. and 16 h.p. f-crystals, 8 tab., 1 Il.

The invention relates to a compound chosen among Nationale malonic dialdehyde acid and any factor affecting wew the public funds. This drug is mainly intended to modulate the phenomenon of programmed cell death (apoptosis).

The invention also relates to modulating apoptosis composition. Finally, the object of the invention is also a method of preventing and/or against photoinductive age or aging skin.

There are two types of mechanisms involved in cell death. The first classic type called necrosis. Morphologically necrosis is characterized by an excessive increase in mitochondria and cytoplasm and nuclear changes (damage) with the subsequent destruction of the cells and its autolysis, and this is accompanied by the phenomenon of inflammation. Necrosis occurs passively and randomly. Tissue necrosis is usually, for example, a consequence of physical injury to cells or chemical poison.

Another form of cell death called apoptosis (Kerr, J. F. R. and Wyllie, A. N. , Br.J. Cancer, 265, 239 (1972)), but, in contrast to necrosis, apoptosis is not the consequence of the phenomenon of inflammation. Described that apoptosis may occur in different physiological conditions. This is a highly selective form of cell suicide (suicide), which is characterized by easily detecting the associated or not with their endonuclease activity, the formation of apoptotic bodies and fragmentation of deoxyribonucleic acid (DNA) due to the activation of endonucleases to DNA fragments by size of 180-200 base pairs (these fragments can be detected using gel electrophoresis agarose).

Apoptosis can be considered as programmed cell death involved in the development, differentiation and homeostasis of tissues. Therefore, believe that the differentiation, growth and maturation of cells closely associated with apoptosis. So, a person with good health there is a balance between the combination of these phenomena.

In medicine, a number of pathological situations associated with modified, even chaotic, the mechanism of apoptosis or apoptotic mechanism, which does not contribute to the deregulation of other biological phenomena to achieve equilibrium. So, describes what free modulation of apoptosis due to its inductively or due to its suppression can afford to treat numerous diseases, especially diseases associated with cellular hyperproliferation, such as in the case of cancer, autoimmune diseases or allergies, or, on the contrary, diseases associated with cell death, such as in the case of sibling Alzheimer's disease), excessive damage caused during a myocardial infarction or cerebral ischemic lesions.

Specifically, in Oncology found that many anti-cancer drugs such as adriamycin and cyclophosphamide, are able to indoctrinate apoptosis.

In the field of cosmetics, the signs of skin aging are the result of, mainly, dysfunction of the basic biological mechanisms occurring in the skin, especially with the connection mechanism of apoptosis. Therefore, we can assume that any product that is capable of modulating the apoptotic mechanism is the product suitable to prevent and/or fight against the appearance of aging and existing signs of aging like wrinkles and fine lines.

However, the relationship between exogenous or endogenous products in the cell and the cellular response, inductively or suppressing apoptosis is unknown.

The applicant has found that apoptosis can be induced by increasing the intracellular content of the natural metabolite, Nationale (3-methylthiophenyl) or malonic dialdehyde acid. The applicant has also disclosed that the transformation in vivo Nationale in malonic dialdehyde acid can be deregulated Express apoptosis, caused by reactive oxygendemanding species in cells and predominantly in neural cells (Kane, D. J. and other Science, 262, 1274-1277 (1993)).

Therefore, the present invention relates to a compound chosen among Nationale, malonic dialdehyde acid and any factor affecting the intracellular content of rationale or dialdehyde of malonic acid, for use as a drug. This drug is mainly intended to modulate the phenomenon of programmed cell death.

The object of the present invention is also modulating apoptosis, pharmaceutical or cosmetic composition, characterized in that it contains as active agent a compound selected among Nationale, malonic dialdehyde acid and any factor affecting the intracellular content of rationale or malonic dialdehyde acid, in combination with pharmaceutically or cosmetically acceptable base.

National, malonic dialdehyde acid and any factor affecting the intracellular content of rationale, can be used in the present izobretenii, or inductively apoptosis, or to suppress apoptosis.

Thus, according to a special variant of the invention, inductively apoptosis composition characterized in that it contains as active agent a compound selected among Nationale, malonic dialdehyde acid and any factor that increases the intracellular content of rationale or malonic dialdehyde acid, in combination with pharmaceutically or cosmetically acceptable base.

And, according to another particular variant embodiment of the invention that inhibits apoptosis composition characterized in that it contains as active agent, any factor that reduces the intracellular content of rationale or malonic dialdehyde acid, in combination with pharmaceutically or cosmetically acceptable base.

In the case of metabolism rationale it is known that 4-methylthio-2-oxobutanoic acid can be metabolized in vivo by using complex dehydrogenase-oxo-acid with branched chain, present in the mitochondria of liver cells, heart and skeletal muscle, through national to get methylthiopropionate - CoA (see G. Wu and S. J. Yeaman, Biochem. J., 257, 281-284 (1989); Haussinger D. , T. Stehle, and Gerok W., J. Biol. Chem., 366, 5 is ü metabolized in vivo by transamination in methionine (see Ogier G., Chantepie J., C. Deshayes, Chantegrel Century, C. Charlot, Doutheau A. and Quash G., Biochem. Pharmacol., 45, 1631-1644 (1993)). National, if necessary, can also be restored or oxidized, respectively, to mational using aldehyde-reductase or to methylthiopropionate acids with aldehyde-dehydrogenase. National in combination with radical HO.finally, can give dialdehyde malonic acid and methyl by reaction-hydroxylation. So, in order to better determine the location of rationale, malonic dialdehyde acid and factors that may affect their intracellular contents, the drawing illustrates the metabolism of rationale that, however, does not limit the scope of protection of the invention.

In the drawing:

MTAB means 4-methylthio-2-oxobutanoic acid;

MTPT means methylthiopropionate acid;

E1 means decarboxylase complex dehydrogenase-oxo-acid with a branched chain, a cofactor which is tiaminpirofosfat (TPF);

E2 means transacylase complex dehydrogenase-oxo-acid with a branched chain, a cofactor which is lipoic acid (La);

ALDR means aldehyde-reductase;

ALDH means aldehyde-dehydrogenases.

lots according to the invention understand the connection, choose among the predecessors of the products of metabolism of rationale or malonic dialdehyde acid, inhibitors and activators of enzymes involved in the metabolism of rationale or malonic dialdehyde acid. So, considering illustrated in the drawing, the metabolism of rationale or malonic dialdehyde acid, it is easy to understand how these connections, national or malonic dialdehyde acid introduced into the cell system can modify the intracellular content of rationale or malonic dialdehyde acid, temporarily or for a long period.

Precursors or products of metabolism rationale or malonic dialdehyde acid may represent precursors or products of intracellular metabolism rationale, such as 4-methylthio-2-oxobutanoic acid, methionine, national, methylthiopropionate acid, methylthiopropionate.

Precursors or products of metabolism rationale or malonic dialdehyde acid can also represent products that are in situ have the ability to release national or malonic dialdehyde acid or release factor complex and thioethers of rationale or malonic dialdehyde acid, which in situ can select national, malonic dialdehyde acid or 4-methylthio-2-hydroxybutanoic acid, which in turn can then be metabolismin in situ in 4-methylthio-2-oxobutanoic acid. In the pharmaceutical field, these products are usually called "prepreparatory".

In the case where I want to inductivity apoptosis, national, dealdeal malonic acid, a precursor or metabolic products of rationale or malonic dialdehyde acid is preferably used in combination with inhibitors of enzymes involved in metabolic reactions, enabling the removal of rationale, other than reaction - hydroxylation, the above (as a result of which mational into malonic dialdehyde acid), or in combination with activators - hydroxylase, which is an enzyme involved in the conversion of rationale in malonic dialdehyde acid. So, as an example, can be called a combination of 4-methylthio-2-oxobutanoic acid transaminase inhibitor involved in transformation of 4-methylthio-2-oxobutanoic acid methionine, such as removal of inhibitors.

You can also use the Association IU the W free radical HO.. You can also call BHRM (N,N-bis(2-chloroethyl)-N-nitrosamino), as it is an inhibitor of glutathione reductase, which allows to increase the intracellular content of reactive oxygenated species such as radical HO.that is one of the substrates of the reaction - hydroxylation order to obtain the dialdehyde of malonic acid.

This Association is particularly interested in the case of pathologies that are characterized by overexpression of the gene bcl2. Such pathologies are particularly cancers of the breast, lymphoma, B-cell leukemia, neuroblastoma, adenocarcinoma of the prostate, prolactinoma and other pituitary adenomas. Overexpression of the gene bcl2makes the cells resistant to chemotherapy character. This Association is, therefore, partially, completely, to inhibit this resistant to chemotherapy character.

This Association is preferably possible to add at least one transaminase inhibitor involved in transformation of 4-methylthio-2-oxobutanoic acid methionine, such as described below and in particular the compound of the formula (9).

Of inhibitors or activators of periksa dehydrogenase-oxanilate branched chain, participating in the transformation of 4-methylthio-2-oxobutanoic acid in methylthiopropionate through national, or inhibitors or activators transaminases, participating in the transformation of 4-methylthio-2-oxobutanoic acid methionine, or inhibitors or activators aldehyde-reductase, responsible for the restoration of rationale in methanol, or aldehyde-dehydrogenase, responsible for the oxidation of rationale in methylthiopropionate acid, or more activators or inhibitors - hydroxylase, which is an enzyme involved in the conversion of rationale in malonic dialdehyde acid.

Thus, as an inhibitor of the dehydrogenase complex-oxanilate branched chain and, therefore, as a factor reducing the intracellular content of rationale or malonic dialdehyde acid, can be called 2-oxybutyrate, which is a well-known substrate of this complex (see Jones S. M. A. and A. I. Cederbaum, Arch. of Biochem and Biophys., 199, 438-447 (1980)), as ketolainen, kitasamycin and Catalin, which are other substrates of this complex.

Also, as inhibitors transaminases, participating in the transformation of 4-methylthio-2-octobot is the NAHL or malonic dialdehyde acid, you can call the products described G. Ogier, Chantepie J., C. Deshayes, Chantegrel Century, C. Charlot, Doutheau A. and Quash G. , Biochem. Pharmacol., 45, 1631-1644 (1993).

Mainly inhibitors transaminases, participating in the transformation of 4-methylthio-2-oxobutanoic acid methionine, choose among the products of formulas (1)-(9) (at the end of the description), in which X represents the radical-OH or the radical-OPO3H2.

Especially preferred is the compound of formula (9), where X stands for the radical-OH. As inhibitors transaminases, participating in the transformation of 4-methylthio-2-oxobutanoic acid methionine and, therefore, as factors that increase the intracellular content of rationale or malonic dialdehyde acid, which may be called hydroxamate amides of amino acids, such as D-asparagine-hydroxamate formula CONHOHCH2CHNH2COOH and L-glutamine-hydroxamate formula CONHOHCH2CH2CHNH2COOH.

When you want to indoctrinate apoptosis, according to the invention, preferably used as a compound chosen among Nationale, malonic dialdehyde acid products that are in situ have the ability to release national or dialdehyde of malonic acid, such as esters and complex tienie of rationale in malonic dialdehyde acid.

From representing especially of interest for inductively apoptosis compounds can in particular be called the Association of Nationale at least one inhibitor of transaminases, participating in the transformation of 4-methylthio-2-oxobutanoic acid methionine, and preferably with the compound of the formula (9).

When you want to suppress apoptosis, according to the invention preference is to use inhibitors of enzymes involved in the production of rationale or dialdehyde of malonic acid, such as 2-oxybutyrate, and/or activators of enzymes involved in the removal of Nationale other way than by the reaction - hydroxylation above.

The composition according to the invention primarily is intended for the following types of treatment:

1) For treating dermatological diseases associated with disorders of keratinization leading to differentiation and hyperproliferation, especially for the treatment of ordinary (youth), polymorphic, red acne; nodular, nodular acne; senile acne, secondary acne such as solar, drug or occupational acne.

2) For treating other types of disorders of keratinization, especially ichthyosis, it is ostani, cutaneous lichen or lichen mucous membranes (oral herpes).

3) For treating other dermatological diseases associated with disorders of keratinization with inflammatory and/or immuno-allergic component, and especially for the treatment of all forms of psoriasis, which happens to be cutaneous, mucosal or ungual, and likewise for the treatment of psoriatic arthritis, or cutaneous atopy, such as eczema or respiratory atopy or more gingival hypertrophy.

4) For treating all dermal or epidermal of hyperproliferate which may be malignant or benign, viral or not, such as ordinary simple warts, flat warts and resembling a wart epidermodysplasia, oral or intraductal papilloma, and hyperproliferate, which can be caused by ultraviolet rays, especially in the case of basal cell carcinoma and pinnacleone of epithelium.

5) For treating other dermatological disorders such as bubble dermatoses and collagen diseases.

6) To treat certain eye disorders, especially corporate.

7) To restore or against starane is osov, or to deal with any pathologies associated with age or actin aging.

8) To prevent or feleciana stigmata of epidermal and/or termicheskoi atropia caused by local or systemic corticosteroids, or any other form of cutaneous atrophy.

9) For the prevention or treatment of impaired wound healing or to prevent or repair of scars on the skin when it is stretched.

10) For treating disorders of adipose function, such as Hyperborea acne or ordinary seborrhea.

11) For the treatment or prevention kantseroznoy or premalignant conditions.

12) For the treatment of painful inflammatory conditions, such as aritra.

13) For the treatment of any morbid state of viral origin, at the level of the skin or General, such as hepatitis.

14) For the prevention or treatment of alopecia.

15) For the treatment of dermatological or General diseases with an immunological component.

16) For the treatment of diseases of the cardiovascular system such as arteriosclerosis and thrombocytopenia.

17) For the treatment of neurodegenerative diseases, such as illness of al is respectfully may include other active agents, such as retinoids, agents against free radicals, derivatives of vitamin D, corticosteroids or estrogens, antioxidants, hydroxy or keto-acids and their derivatives, or blockers of potassium channels.

The object of the present invention is also the use of a composition according to the invention in or for cosmetic or pharmaceutical composition intended for the modulation of apoptosis and mainly intended for the above-described protective and/or therapeutic treatments.

Retinoids can be of natural or synthetic origin. Of retinoids can be called 9-CIS-retinoic acid, all-TRANS configuration retinoic acid.

Of vitamin D or derivatives thereof, especially, can be called a derivative of vitamin D2or D3and, in particular, 1,25-dihydroxy-vitamin D3.

Agent against free radicals especially can be called - tocopherol, dissatisified, ubiquinol or some agents formation of chelate compounds of metals.

From hydroxy or keto acids or derivatives thereof, especially, can be called ketolainen, kitasamycin, Catalin, 2-oxybutyrate, 4-methylthio-2-the th acid or salicylic acid derivatives or their salts, amides or esters.

Of blockers of potassium channels especially can be called Minoxidil (2,4-diamino - piperidinedione-3-oxide) and its derivatives.

The composition according to the invention it is possible to introduce enteral, parenteral, local or ocular route. Preferably the composition is packaged in a form suitable for the application of the system by (injection or infusion).

For administration by enteral, composition, mainly the pharmaceutical composition may be in the form of tablets, gelatin capsules, pills, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid or polymeric vesicles, which allow to happen in controlled drug release. For administration by parenteral composition may be in the form of solutions or suspensions for infusion or for injection.

The drug according to the invention is usually administered in a daily dose of about 0.001 to 100 mg/kg of body weight in 1-3 reception.

With the introduction of the topic by the composition according to the invention primarily is intended for treatment of skin and mucous membranes and may be in the form of ointments, creams, shoditsa in the form of microspheres or nanospheres or lipid or polymeric vesicles or polymer "flaps" and hydrogels, allowing to happen to the controlled release of drugs. For the introduction of local by this composition may be either in anhydrous form or in aqueous form.

For the introduction of the eyepiece by suitable mainly lotions for the eyes.

The pharmaceutical composition intended for the introduction of local or ocular (eye) through contains national, malonic dialdehyde acid or any factor affecting the intracellular content of rationale or malonic dialdehyde acid, in a concentration of preferably 0.001 to 5% based on the total weight of the composition.

The composition according to the invention also finds application in the field of cosmetics, in particular for body care and hair and especially for treatment of acne-prone skin, for growing hair against hair loss, for protection against the harmful effects of the sun or for the treatment of physiologically dry skin, to prevent and/or combat photoinduction age or aging.

Finally, an object of the present invention is a method of preventing and/or against photoinductive or age ageing of the skin, characterized in that the Asti cosmetics, the composition according to the invention preferably may contain retinoids, vitamin D or derivatives thereof, with corticosteroids, agents against free radicals, hydroxy or keto-acids and their derivatives, or blockers of ion channels. These various products used in the compositions of the present invention, are as described above.

Cosmetic composition according to the invention can be especially in the form of a cream, a milk, a lotion, a gel, microspheres or nanospheres or lipid or polymeric vesicles, a soap or shampoo.

The concentration of rationale, malonic dialdehyde acid or any factor affecting the intracellular content of rationale or malonic dialdehyde acid in cosmetic compositions is preferably of 0.0001-3 wt.% based on the weight of the composition.

Cosmetic or pharmaceutical composition according to the invention, can also contain inert or pharmacodynamically or cosmetically active additives or combinations of these additives, and especially: wetting; depigmenting agents, such as hydroquinone, azelaic acid, caffeic acid or kojic acid; me is its derivatives or urea; antisubmarine agents or anti-acne agents such as S-carboxymethylcysteine, S-benzylcyanide, their salts and their derivatives, or benzoyl peroxide; antibiotics such as erythromycin and its esters, neomycin, wedge-damycin and its esters, tetracyclines; antifungal agents such as ketoconazole or polymethylene-4,5-isothiazolin-3-ones; agents that promotes hair regrowth, such as Minoxidil (2-,4-diamino-6-piperidino-pyrimidine-3-oxide) and its derivatives, diazoxide (7-chloro-3-methyl-1,2,4-benzothiadiazine-1,1-dioxide) and phenytoin (5,4-diphenyl-imidazolidin-2,4-dione); anti-inflammatory agents, non-steroidal type, carotenoids and especially - carotene; antipsoriatics agents, such as anthralin and its derivatives and, finally, eicosa-5,8,11,14-Terranova acid and eicosa-5,8,11-three-new acid, their esters and amides.

The composition according to the invention may also contain improves the taste of agents; preservatives such as esters of parahydroxybenzoic acid, stabilizers, regulating humidity agents, regulating pH agents, osmotic pressure modifiers, emulsifiers, UV-a and UV-B filters, antioxidants, such as-tocopherol, butylhydroxyanisole or equivalent.

Example 1

The impact of metabolites on the growth of HeLa cells

HeLa cells (0.5 to 106per Petri dish) were cultured in 3 ml minimal support eagle medium containing 10 wt.% cialisbuynow fetal calf serum. After 4 hours in each of the three cups add mational (from 10 mmol to 1 mmol), methanol (from 10 mmol to 20 mmol), methylthiopropionate acid (MTPT, from 1 mmol to 20 mmol), L-methionine (from 1 mmol to 20 mmol) or 4-methylthio-2-oxobutanoic acid (MTOB, from 1 mmol to 20 mmol). After aging for three days at 37oC cells are washed twice in phosphate buffered saline (SFR) with pH 7.5 and extracted from the same type of buffer.

Cell growth is assessed by determining the protein content (see the method described by Lowry O. H., Rosebrouch N. J., Farr A. L. and Randall R. J., J. Biol. Chem. , 193, 265-275 (1951)) or DNA content in the lysates using the solution bisbenzimidazole (HOECHST 33258) (see Jarvis, W. D., Kolesnick, R. N., F. Fornari, A., Traylor, R. S., Gewirtz, D. A., and Grant, S., Proc. Natl. Acad. Sci. U. S. A., 91, 73-77 (1994)), or by determining the activity of lactic dehydrogenase, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) (see Mosmann T., J. of Immunology Methods, 65, 55-63 (1983)).

The results are presented in table. 1. Each prevideo.

IR50concentration of product required to achieve inhibition of cell growth by 50%.

The evaluation results on the measurement of DNA content or determination of the activity of lactic dehydrogenase comparable with those listed in table. 1.

In table. 1 shows the difference IR50for Nationale compared with IR50his products or precursors.

Example 2

HeLa cells (0.5 to 106per Petri dish) were cultured in 3 ml minimal support eagle medium without methionine (Met), containing 10 wt.% cialisbuynow fetal calf serum. After 4 hours add 2-oxybutyrate (from 10 mmol to 40 mmol) in the presence of 2 mmol of 4-methylthio-2-oxobutanoic acid (MTOB). After aging for three days at a temperature of 37oC cells are washed twice in phosphate buffered saline (SFR) with pH 7.5 and extracted from the same type of buffer. Cell growth is determined in the same manner as in the previous example.

2-Oxybutyrate is a well-known substrate of the dehydrogenase complex-oxanilate branched chain (BCOADC) (18 Km Microm). Used individually at a concentration of 2 mmol MTAB causes suppression of R is ol 2-oxybutyrate inhibition is reduced by 2-3 times. Therefore, establish that the BCOADC activity against turning MTAB in national shifted to another substrate, such as 2-oxybutyrate, which reduces inhibition of growth.

Example 3

The impact of rationale on DNA fragmentation in cells BAF3

Used cells correspond lymphocytic cell line mouse BAF3, which requires interleukin-3 (IL-3) for growth and which undergoes apoptosis (more than 80% of the cells) in the absence of IL-3 for 16 hours (see Collins, M. K. L., Marvel J., Malde P. And Lopez-Rivas, A., J. Exp. Med., 176, 1043-1051 (1992)).

3 106The BAF3 cells were cultured in 6 ml of culture medium containing IL-3, in Petri dishes in the presence of various concentrations (from 200 to 800 µmol) rationale or propanal, which is a substrate of aldehyde-dehydrogenase. After contacting within 8 hours the cells are washed three times in SFR with pH 7.5. Cells are lysed in 2 ml of 0.1% Triton X-100, 20 mmol add (ethylenediaminetetraacetic acid), 5 mmol TRIS pH 8 and then centrifuged at acceleration 30,000 g at 4oC for 30 minutes. Supernatant separated by decantation and subjected to the following tests:

As a comparison, the same cultivation is carried out in the environment or with IL-3 is particularly described in the publication Jarvis W. D., Kolesnick, R. N., F. Fornari, A., Traylor, R. S. , Gawirtz D. A., and Grant, S., Proc. Natl. Acad. Sci. U. S. A., 91, 73-77 (1994). Supernatant treated with ribonuclease A (20 μg/ml) for 1 hour at 37oC and with proteinase K (100 µg/ml). DNA is purified by extraction with phenol and precipitation with ethanol. In the final extract, the DNA fragments with low molecular masses analyzed by electrophoresis on 1% agarose gel. After electrophoresis (60 volts, 3 hours) gels stained with ethidiumbromid.

The results clearly show that apoptosis Indochinese rationalem; the resulting gel has the appearance of a ladder of multiple DNA fragments with sizes ranging from 180 to 200 base pairs, typical for the induction of apoptosis. If propanal gel does not have this characteristic.

The analysis is carried out using spectrofluorometry to determine the number of the obtained DNA fragments (with values less than 3 thousand nucleotides). To 2 ml of supernatant was added 1 ml of bisbenzimidazole (XEXCT 33258) (1 μg/ml) in 3 mmol of sodium chloride, 1 mmol add, 10 mmol TRIS pH 8. The resulting solutions analyzed by fluorometry ( excitation = 365 nm, emission = 460 nm). The amount of DNA expect relatively high purity DNA (0.1 to 1 μg in 2 ml of buffer for lysis), education results, presented in table. 2.

Thus, increasing the number of DNA fragments detected in cells treated with IL-3 and rationalem, similar to that found in the case when cells were placed in medium without IL-3.

Example 4

The impact of N, N-bis(2-chloroethyl)-N-nitrosoanatabine (BGNM) and transaminase inhibitor involved in transformation of 4-methylthio-2-oxobutanoic acid methionine, on the production of reactive oxygenated species (H2O2and HO.)

1,5105the BAF3 cells (cell line, such as described above), inoculated in 3 ml of culture medium containing IL-3 (modified by way of Dulbecco Wednesday Needle containing 6% fetal calf serum and 5% medium conditioned by cells Wehi-3B, which is used as a source of IL-3, described by Collins and others , J. Exp. Med, 176, 1043-1051 (1992)), is treated with 10 µm 2',7'-dichlorofluorescein (usually called DCFH-DA and the resulting Molecular Probes Inc.). After incubation for 1 hour at 37oC cells are washed with SFR. 10 minutes before analysis by fluorescence dichlorofluorescein (DHF) using flow cytometry add 3 μg/ml of propyliodide. Incubatio µmol (which corresponds to the compound of formula (9), when X represents the radical-OH). This method allows to obtain the percentage of fluorescence produced by the reaction of reactive oxygenated species (H2O2and HO.with DCFH-DA. Flow cytometry implement using the flow cytometer FA-Cscan (Becton Dickinson, San Jose, CA, USA). The results are presented in table. 3.

Thus, these results show that compound A, which allows to increase the content of rationale, suppresses the increase in the number of HO.caused by increasing concentrations PHNM. Thus, it is likely the fact that in this case the reaction occurs Nationale with the existing radicals HO.leading to the formation of malonic dialdehyde acid, which then triggers apoptosis.

Example 5

Impact BHRM and transaminase inhibitor involved in transformation of 4-methylthio-2-oxobutanoic acid methionine, apoptosis

Breaks DNA strands labeled with biotinylated dUTP (uridinediphosphate) (defined using haveinfluenced) in situ in permeabilizing and fixed cells using the test terminal, deoxynucleotidyl-transferase DNA (TdT), such as described Gorczyca and other Cancer R is turalei environment, containing IL-3 (modified by way of Dulbecco Wednesday Needle containing 6% fetal calf serum and 5% conditioned cells Wehi-3B environment, used as a source of IL-3). Cells incubated in the presence of 50 μmol of compound A, 40 μg/ml PHNM, a mixture of these two products (50 µmol connection A+40 μg/ml BGNM) or in the absence of these two compounds (control). After incubation for 24 hours, the cells washed with SFR and fixed in formaldehyde, then in ethanol, and left for 3 days at -20oC. the cells are Then re-suspended in 50 μl of a solution containing 0.1 μmol of cacodylate sodium, pH 7.5, 1 mmol of cobalt chloride, 0.1 mmol of dithiothreitol, 0.05 mg/ml bovine serum albumin, 10 units of TdT (from calf thymus; Boeringer), 0.5 nmol of Biotin-16 dUTP (from calf thymus; Boeringer), 0.5 nmol adenosine triphosphate (ATP), 0.5 nmol of citydistrict (TTF) and 0.5 nmol GTP-independent (GTP), and incubated for 1 hour at 37oC. After washing with ZFR cells again suspended in 100 μl of a fourfold volume of SSC buffer (0.15 mol of sodium chloride, 0.015 mol of sodium citrate) containing 2.5 µg/ml avidin-fluorescein-isothiocyanate (Sigma), 0.1% (weight/volume) of ribonuclease A, 0.1% Trinote. These cells are washed, then suspended in 1 ml SPR containing 3 μg/ml of propyliodide. Flow cytometry implement using a FACScan flow cytometer (Becton, Dickinson, San Jose, CA, USA).

The results are presented in table. 4.

In these doses of individual products cause slight apoptosis, on the contrary, their mixture induct in a high degree of apoptosis (synergism). This result confirms shown in table. 3 results and related conclusions.

Example 6

Establish the most effective therapeutic compositions for the treatment of mice that transplanted melanoma cells

On the day of 0.105murine B16F1 melanoma cells injected B6D2F1 mice (firm IFFA CREDO, France). On day 1, and it continuously for 15 days, once a day, these mice injected through the injection of various therapeutic compositions listed in table. 3. The control corresponds to the mice, which is not administered therapeutic composition.

In table. 5 presents the results, which correspond to average values obtained in the case of three-treated mice.

For structures of several compounds used doses of each of the compounds identical to those doses when connections and is only average for mice, not having lived more than 60 days.

BHRM corresponds to N,N-bis(2-chloroethyl)-N-nitrosoanatabine.

Compound a corresponds to the compound of formula (9), where X stands for the radical-OH.

Thus, notice that the last two composition is particularly effective, because they significantly increase the survival time of these mice. From these two last (supplemented by rationalem) allows third of the mice to live longer than 60 days.

Example 7

Proceed as in the previous example, changing the composition and the number of treated mice. In table. 6 presents these data and the results obtained.

Survival time is considered, starting from day 0, the above, and it is in line with the average for mice, not having lived more than 60 days.

For the last composition, doses correspond to those compounds used individually. The percentage of survivors with the long-life corresponds to the percentage of mice lived more than 60 days. Thus, notice that the last two composition and especially the last, are very effective.

Example 8

The impact of rationale on the induction of apoptosis in BAF3 cells-b0 and BAF3-bcl2

Used cells correspond to the lymphocytic kletochnoi bc12; cells BAF3-b0 correspond to the BAF3 cells, retrospectively using gene bcl2. As clarified above, the cells BAF3-b0 undergo apoptosis (more than 80% of the cells) in the absence of IL-3 for 16 hours. In contrast, cells BAF3-bcl2, which, consequently, transfection using gene bcl2, show no sign of apoptosis in the absence of IL-3. Therefore, they represent a good model for establishing a phase of apoptosis is blocked in these cells, and its possible correlation with the inhibition of the synthesis of malonic dialdehyde acid.

Cells BAF3-b0 or BAF3-bcl2 labeled according to the method described by S. Wright and others, J. of Cell. Bio-chem., 48, 344-355 (1992), incubare 2,5 105cells/ml with 0.5 microcurie [3H]-thymidine for 40 hours at 37oC. After two washes with culture medium 2,5 106cells cultured in the presence of rationale.

After incubation for 8 hours these cells is extracted by centrifugation at acceleration 400 g for 5 minutes and washed 3 times with SFR. Extracted in the form of sediment after centrifugation, the cells are lysed in 2 ml of a solution containing 0.1% Triton X-100, 20 mmol add, 5 mmol TRIS pH 8, and centrifuged at acceleration 30 000 g at 4oC for 30 minutes. Supernatant separated and wasp is 1 ml), supernatant (0.3 ml) and dissolved sediment after centrifugation (0.1 ml) analyze in a scintillation counter. The percentage of DNA fragments is calculated as follows:

the number of decays per minute (culture medium +

the number of decays per minute for supernatant divided by

the number of decays per minute (culture medium +

the number of decays per minute for supernatant +

the number of decays per minute for the dissolved precipitate.

The results:

adding increasing amounts of rationale(0; 50; 100; 200; 300; 400 umol) in the incubation medium increases the percentage of DNA fragments up to a maximum to 400 µmol of rationale, 26% compared with 7% for cells BAF3-b0 not handled by rationalem (control). For cells BAF3-bcl2 treated with equivalent amounts of rationale, DNA fragments reach a maximum 7% for 400 µmol of rationale, compared with 3% for the control.

These results clearly show that the addition of rationale does not resolve the inhibition of apoptosis caused bc12 gene in cells BAF3-bcl2.

During the induction of apoptosis due to the lack (shortage) of IL-3 determines that mational into malonic dialdehyde acid due reacts is UP>.in cells BAF3-b0 and BAF3 - bcl2.

Example 9

The definition of reactive oxygenated species (H2O2and OH.) by flow cytometry

Used cells correspond lymphocytic cell line BAF3, such as described above. Cells BAF3-bcl2 correspond to the BAF3 cells, transfections using the bc12 gene; cells BAF3-b0 correspond to the BAF3 cells, not transfections using the bc12 gene.

1.5 to 106Cells BAF3-b0 or-bc12, inoculated in 3 ml of culture medium containing IL-3, treated with 10 µmol of dichlorofluorescein (DCFH-DA). After incubation for 1 hour at 37oC cells are washed with SFR; 10 minutes before analysis by fluorescence dichlorofluorescein (DHF) by flow cytometry add 3 μg/ml of propyliodide as in example 4. Flow cytometry implement using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA).

The results are presented in table. 7

Therefore, even if the cells BAF3-bcl2, there tends to be more reactive oxygenated species, than in the case of cells BAF3-b0, then this difference is insignificant.

Therefore, it is possible that cells BAF3-bcl2 both regional and labeled14C products, derivatives of [14C] MOB in cells BAF3-b0 and BAF3-bcl2

Used cells, therefore, are identical to those in the preceding example.

[14C] -MTAB produced by oxidative deamination [14C]-methionine, such as described Ogier G. and others, Biochem. Pharmacol., 45, 1631-1644 (1993). [14C]-MTAB (5 OF 106disintegrations per minute, which corresponds to 38.2 nmol) is added to the cell suspension BAF3-b0 or BAF3-bcl2 (2,4 106cells/ml) in modified according to the method of Dulbecco environment Needle containing 6% fetal calf serum and 5% IL-3. After 39 hours, cells are washed twice in SFR and again suspended in 9105cells / ml TO 108cell type [U14C]-MTAB (1,9106disintegrations per minute, which corresponds to 14.5 nmol) and after 8 hours the cell extract by centrifugation at acceleration 400 g for 5 minutes. Precipitation cells after centrifugation solubilizing 1.5 ml of 0.5 n sodium hydroxide solution for 30 minutes at 60oC. Existing cells full radioactivity determined on the aliquot of 10 µl. Then, to the obtained alkaline hydrolysate add perchloric acid at a final concentration of 0.5 h, then 200 Microm 2,4-dinitrophenylhydrazine (issued by farmout. The precipitate is dissolved in 1 ml of 0.5 n sodium hydroxide solution, and then determine the radioactivity and protein content. To the supernatant add the sodium hydroxide to reach pH 10. Labeled metabolites extracted with ethyl acetate and the organic phase is dried, acidified and extracted with dichloromethane. Labeled metabolites in the organic phase is separated and identified by high performance thin-layer chromatography, comparing with the derivatives of 2,4-dinitrophenylhydrazine standard connections, then quantificare by chromatography with radiometric detection and scavenging.

The results are presented in table. 8.

Thus, it appears that the total radioactivity attributed to the same amount of DNA that is similar in the two types of cells after incubation for 47 hours. The same counts for the activity of methionine cellular proteins. In terms of free methionine, an increase of 40% of the radioactivity detected in the cells BAF3-bcl2 as compared to that in cells BAF3-b0. Present in rationale radioactivity 80% more cells BAF3-b0, than in cells BAF3-bcl2. This allows you to emphasize that in cells BAF3-bcl2 reduction education metio the formation of malonic acid, it corresponds to 800 CPM per mg DNA in cells BAF3-b0, whereas no radioactivity for the formation of malonic acid was not detected in extracts of cells BAF3-bcl2. Cells BAF3-bcl2, therefore, also reduce the formation of malonic dialdehyde acid from Nationala. Example 8 clearly demonstrates that this decline is not caused due to the reduction of reactive oxygenated species in cells BAF3-bcl2 compared with cells BAF3-b0. Therefore, we can conclude that the gene bcl2 in addition, what causes the decrease in the transformation MOB in national, also negatively affects the response-hydroxylation, which resulted in national turns into a dialdehyde of malonic acid. Therefore, any product that modulates the intracellular content of rationale or its metabolic product, malonic dialdehyde acid, can be considered as a modulator of apoptosis.

1. The use of compounds selected among Nationale, malonic dialdehyde acid and any factor affecting the intracellular content of rationale or malonic dialdehyde acid as a drug that modulates apoptosis.

2. Farmatsevticeski the agent an effective amount of at least one connection, choose among Nationale, malonic dialdehyde acid and any factor affecting the intracellular content of rationale or malonic dialdehyde acid, in combination with pharmaceutically or cosmetically acceptable base.

3. The composition according to p. 2, characterized in that national, malonic dialdehyde acid and any factor affecting the intracellular content of rationale or malonic dialdehyde acid is used in the form of a mixture.

4. The composition according to p. 2 or 3, characterized in that the factors influencing the intracellular content of rationale or malonic dialdehyde acid is chosen among the predecessors of the products of metabolism of rationale, inhibitors and activators of enzymes involved in the metabolism of rationale.

5. The composition according to p. 4, characterized in that the precursors or products of metabolism rationale or malonic dialdehyde acid is a precursor or products of intracellular metabolism rationale or dialdehyde of malonic acid, such as 4-methylthio-2-oxobutanoic acid, methionine, national, methylthiopropionate acid and methylthiopropionate SOA.

6. Composition the acid are products, which in situ is capable of releasing national or malonic dialdehyde acid or release factor influencing the intracellular content of rationale or dialdehyde of malonic acid, such as esters and complex thioethers of rationale or malonic dialdehyde acid.

7. The composition according to p. 4, characterized in that the inhibitors or activators of enzymes involved in the metabolism of rationale, choose among the inhibitors or activators of complex dehydrogenase-oxanilate branched chain, involved in the transformation of 4-methylthio-2-oxobutanoic acid in methylthiopropionate SOA through national; inhibitors or activators transaminases, participating in the transformation of 4-methylthio-2-oxobutanoic acid methionine; inhibitors or activators aldehyde-reductase, responsible for the restoration of rationale in methanol, or aldehyde-dehydrogenase, responsible for the oxidation of rationale in methylthiopropionate acid and inhibitors or activators-hydroxylase.

8. Composition, inductively apoptosis, according to any one of paragraphs.2 to 7, characterized in that it contains as active agent at least one compound selected among MEA or malonic dialdehyde acid, in combination with a pharmaceutically or cosmetically acceptable base.

9. The composition according to p. 8, characterized in that the factors that increase the intracellular content of rationale or malonic dialdehyde acid are inhibitors of transaminases, participating in the transformation of 4-methylthio-2-oxobutanoic acid methionine, choose among the products of the following formulas (1) to (9):

< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
in which X IS-HE-or-ORO3H2.

10. The composition according to p. 9, wherein the transaminase inhibitor involved in transformation of 4-methylthio-2-oxobutanoic acid methionine, is a compound of the formula (9), in which X is the radical HE is.

11. The composition according to p. 8, characterized in that the factors that increase the intracellular content of rationale or malonic dialdehyde acid are inhibitors of transaminases, participating in the transformation of 4-methylthio-2-oxobutanoic acid methionine, choose among hydroxamates amides of amino acids.

12. The composition according to p. 8, characterized in that it contains an Association connection, choose among Nationale, malonic dialdehyde acid, precursors and products of metabolism the reactions, enabling selection of rationale, or with activators-hydroxylase.

13. The composition according to p. 12, characterized in that the Association meets the 4-methylthio-2-oxobutanoic acid transaminase inhibitor involved in transformation of 4-methylthio-2-oxobutanoic acid methionine, such as the products listed in any of paragraphs.9 - 11.

14. The composition according to p. 8, characterized in that the Association complies with nationally or factor that increases the intracellular content of rationale, at least one compound that increases the amount of free radical BUT+and this connection is preferably a N,N-bis(2-chloroethyl)-N-nitrosamino.

15. The composition according to p. 14, characterized in that it contains at least one transaminase inhibitor involved in transformation of 4-methylthio-2-oxobutanoic acid methionine, such as the products according to any one of paragraphs.9 - 11.

16. Composition according to any one of paragraphs.2 to 7, characterized in that it contains as active agent, any factor that reduces the intracellular content of rationale or malonic dialdehyde acid, in combination with pharmaceutically or cosmetically acceptable base.

18. The composition according to p. 16, characterized in that it contains inhibitors of enzymes involved in the production of rationale, and/or activators of enzymes involved in the removal of rationale.

19. The way to prevent and/or fight against photoinductive or age ageing of the skin, characterized in that it is applied to the skin a cosmetic composition, industrous apoptosis, according to any one of paragraphs.2 - 15.

 

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