Medical applications selective estrogen receptor modulators in combination with steroid sex hormone precursors

FIELD: medicine.

SUBSTANCE: method involves administering selective modulator of steroid sex hormones being in particular compounds of general formula(I) and some quantity of steroid sex hormones precursor selected from a group composed from dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-en-3β,17β-diol and compounds in vivo transformable into one of cited precursors. Bisphosphonates combined with selective estrogen receptor modulators and/or steroid sex hormones precursor are additionally introduced for medically treating and/or inhibiting osteoporosis progress.

EFFECT: enhanced effectiveness of treatment; excluded adverse side effects.

41 cl, 13 dwg, 4 tbl

 

The scope of the invention

The present invention relates to a method of treating or reducing the likelihood of acquiring osteoporosis, hypercholesterolemia, hyperlipidemia or atherosclerosis using a new combination therapy in susceptible warm-blooded animals, including humans. In particular, the combination includes the introduction of a selective modulator of estrogen receptors (SERM) and increase the patient's level predecessor of sex steroid hormones, and the specified precursor selected from the group consisting of dehydroepiandrosterone (DHEA), dehydroepiandrosterone (DHEA-S) and androst-5-ene-3β ,17β -diol (5-diol). The invention also relates to kits and pharmaceutical compositions for use in practice, a combination of the above.

Prior art

People, along with some other primates, is unique in that it has the adrenals that secrete large quantities of precursor steroids dehydroepiandrosterone (DHEA-S) and dehydroepiandrosterone (DHEA), which are converted to Androstenedione (4-dione) and then into active androgens and/or estrogens in peripheral tissues (Labrie et al., In: Important Advances in Oncology. Edited by V.T. de Vita, S. Hellman, S.A. Rosenberg, J.B. Lippincott, Philadelphia, 193-217, 1985; Labrie, Mol. Cell. Endocrinol., 78: C113-C118, 1991; Labrie et al., In Signal Transduction in Testicular Cells. Ernst Schering Research Fundation Workshop, Edited by V.Hansson, F.O. Levy, K. Tasken. Springer-Verlag, Berlin-New York (Suppl. 2), pp. 185-218, 1996; Labrie et al., Steroids, 62: 148-158, 1997). In recent work (Labrie et al., J.Clin. Endocrinol. Metab., 82: 2403-2409, 1997), the applicants have described a significant drop in circulating levels of dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEA-S), androst-5-ene-3β ,17β -diol (5-diol), 5-diol-S (5-disulfate), esters of 5-diol and fatty acids and Androstenedione in men and women aged 20 to 80 years.

Despite significant reduction of endogenous androgens in women with age, the use of androgens in postmenopausal period in women is limited due to concerns of increased risk of cardiovascular disease based on the results of earlier studies showing adverse lipid profile with androgens. However, recent studies did not show significant effects of combined therapy with estrogens and androgens on serum cholesterol, triglyceride, HDL-C (HDL), LDL cholesterol (LDL) and the ratio of HDL/LDL compared with the same estrogen (Sherwin et al., Am. J. Obstet. Gynecol., 156: 414-419, 1987). In accordance with these observations, the applicants have shown that DHEA, a compound which has a predominantly androgenic effect, clearly does not have a detrimental impact on the profile of serum lipids (Diamond et al., J. Endocrinol., 150: S43-S50, 1996). Similarly did not observe changes in the oxygen the radio cholesterol, its sub-fractions or triglycerides during the processing of a single estradiol after 6 months of treatment with implants estradiol + testosterone (Burger et al., Br. Med. J. Clin. Res. Ed., 294: 936-937, 1987). It should be noted that the study in humans showed a negative correlation between serum DHEA-S and low-density lipoprotein (Parker et al., Science, 208: 512-514, 1980). Later found a correlation between low testosterone levels and DHEA in serum and increased internal fat, parameter increased risk for cardiovascular system (Tchernof et al., Metabolism, 44: 513-519, 1995).

5-diol is a compound, biosynthetically of DHEA with the participation of restorative 17β -hydroxysteroid dehydrogenase (17β -HSD) and is a weak estrogen. He has the affinity of 85-fold lower than the 17β -estradiol (E2for estrogen receptor in rat in the cytosol of the anterior part of the pituitary gland (Simard and Labrie, J. Steroid Biochem., 26: 539-546, 1987), further confirming the data obtained with the same parameter in the myometrium of man and cancerous breast tissue (Kreitmann and Bayard, J. Steroid Biochem., 11: 1589-1595, 1979; Adams et al., Cancer Res., 41: 4720-4926, 1981;

Poulin and Labrie, Cancer Res., 46: 4933-4937, 1986). However, at concentrations that are within the levels in the blood plasma, established in adult women, 5-diol increases cell proliferation and the levels of progesterone receptor in tumor cells ZR-75-1 breast jelly the s man (Poulin and Labrie, Cancer Res., 46: 4933-4937, 1986) and increases dependent on estrogen synthesis of glikoproteid size 52 kDa in MCF-7 cells (Adams et al., Cancer Res., 41: 4720-4926, 1981).

As mentioned above, it is known that levels of DHEA, DHEA-S and 5-diol in the serum decreases with age and, accordingly, there is a significant, dependent on the age of the reduction in the formation of androgens and estrogens in peripheral target tissues. Such changes in the secretion of DHEA-S and DHEA lead to a significant reduction of the biochemical and cellular functions, stimulated gonadal steroid hormones. In the recently DHEA and DHEA-S has been used to treat a variety of diseases associated with decrease and/or imbalance of the levels of sex steroid hormones.

Osteoporosis, a disease that affects men and Jenssen, and is associated with reduced levels of androgens and estrogens. It has been shown that estrogen reduces the rate of degradation of the bone, while androgens help to build bone mass. However, for estrogen replacement therapy, commonly used for osteoporosis requires adding progestins to prevent proliferation of the endometrium and reduces the risk of endometrial cancer induced by estrogen. In addition, since it is believed that both estrogens and progestins increase the risk of breast cancer (Bardon et al., J. Clin.Endocrinol. Metab., 60: 692-697, 1985; Colditz et al., N. Engl. J. Med., 332: 1589-1593, 1995) the use of replacement therapy with estrogen-progestin Pets from a limited number of women and usually within a very short period of time.

Based on several studies it has been suggested that osteoporosis is a clinical manifestation of lack of androgens in men (Baran et al., Calcif. Tissue Res. 26: 103-106, 1978; Odell and Swerdloff, West J. Med. 124: 446-475, 1976; Smith and Walker, Calif. Tissue Res. 22 (Suppl.): 225-228, 1976). It was found when observing with mantraonline.com that treatment with androgens increases bone mineral density of vertebrae in women in the postmenopausal period (Need et al., Arch. Intern. Med., 149: 57-60, 1989). Treatment of postmenopausal women with nandrolone increased content of mineral substances in cortical bone (Need et al., Clin. Orthop. 225: 273-278, 1987). However, side effects in the use of androgens have been reported in 50% of patients. Such data are of interest because at that time, as all existing therapies are limited to reducing bone loss, when anabolic steroid nandrolone was found to increase bone mass. It has been suggested similar stimulation of bone formation under the action of androgens in men with hypogonadism (Baran et al., Calcif. Tissue Res. 26: 103, 1978). Labrie et al. reported stimulation of the formation to the values in postmenopausal women during DHEA treatment within 12 months (J. Clin. Endocrinol. 82: 3498-3505, 1997).

DHEA (450 mg/kg body weight, 3 times per week) significantly reduced the occurrence of breast cancer in mice SN who have a genetic predisposition to develop breast cancer (Schwartz, Cancer Res. 39: 1129-1132, 1979). In addition, it was found that the risk of developing bladder cancer increases among men who have low levels of DHEA in serum (Gordon et al., Cancer Res. 51: 1366-1369, 1991).

The patent application U.S. 5550107 relates to a method of treatment of breast cancer and endometrial cancers in susceptible warm-blooded animals, which may include inhibition of ovarian hormonal secretion by surgical methods (oophorectomy) or chemical means (use of LHRH agonist, e.g., [D-Thr6, des-Gly-NH

10
2
]LR of ethylamide, or antagonist) as part of combination therapy. Discusses antiestrogens, androgens, progestins, inhibitors of education of sex steroid hormones (especially production of sex steroid hormones catalyzed 17β -hydroxysteroid dehydrogenase or aromatase)inhibitors of secretion of prolactin and growth hormone secretion and the secretion of ACTH (ACTH). An integral part of it was published under the international publication number WO 90/10462.

Cu is IU, cardiovascular disease is associated with reduced serum levels of DHEA and DHEA-S, and as DHEA and DHEA-S have been proposed for the prevention and treatment of these diseases (Barrett-Connor et al., N. Engl. J. Med. 315:1519-1524, 1986).

In adult rats, Sprague-Dawley, Schwartz (in Kent, Geriatrics 37: 157-160, 1982) observed that body weight was reduced from 600 to 550 g under the action of DHEA without affecting feed intake. Schwartz (Cancer 39: 1129-1132, 1979) observed that mice SN, which was introduced DHEA (450 mg/kg, 3 times per day)significantly decreased weight gain, they grew faster than the control animals had a lower number of body fat and were more active. The weight loss was achieved without loss of appetite or restrictions in the diet. In addition, DHEA can prevent weight gain in animals intended for manifestations of obesity in adult age (in Kent, Geriatrics 37: 157-160, 1982).

Introduction DHEA depleted rats Zucher resulted in lower increases in weight, despite the increased feed intake. The treated animals were smaller fat accumulation, on the basis of what in General it can be assumed that DHEA increases the metabolism of feed, leading to a lower increase in weight and fat accumulation (Svec et al., Proc. 2ndInt. Conf. Cortisol and Anti-Cortisols, Las Vegas, Nevada, USA, p.56 abst., 1997).

It was found that obesity is treated mice mutants Andvy(Yen et al., Lipids 12: 409-413, 1977) and in rats Zucher (Ceary and Zisk, Fed. Proc. 42: 536, 1983). Processed DHEA mouse SN looked younger in comparison with the control animals (Schwartz, Cancer Res. 39: 1129-1132, 1979).

DHEA reduced the frequency of occurrence of atherosclerosis in rabbits fed cholesterol (Gordon et al., J.Clin. Invest. 82: 712-720, 1988; Arad et al., Arteriosclerosis 9: 159-166, 1989). In addition, it was reported that high concentrations of DHEA-S serum protects against death from cardiovascular disease in humans (Barrett-Connor et al., N.Engl. J.Med. 315: 1519-1524, 1986). It was found that the levels of circulating DHEA and DHEA-S is inversely correlated with mortality due to cardiovascular disease (Barrett-Connor et al., N. Engl. J. Med. 315: 1519-1524, 1986) and reduced in parallel with the suppression of the immune system (Thoman and Weigle, Adv. Immunol. 46: 221-222, 1989). Research on humans has shown there is an inverse correlation between DHEA-S in fetal serum and low-density lipoprotein (LDL) (Parker et al., Science 208: 512, 1980).

The use of DHEA, as well as positive effects of treatment with androgens and estrogens are discussed in the publication of international patent WO 94/16709.

Based on the correlations observed in the previous prior art, it was impossible to suggest methods of treatment or prevention that is effective or do not have undesirable side effects, such as combination therapy disclosed here.

A brief description of the image is possible

Therefore, the aim of the present invention is the provision of effective methods of treatment of osteoporosis, hypercholesterolemia, hyperlipidemia, atherosclerosis, breast cancer, endometrial cancer, ovarian cancer and uterine cancer, at the same time minimizing undesirable side effects.

Another purpose is to provide ways to reduce the risk of acquisition of the above diseases.

Another purpose is the provision of kits and pharmaceutical compositions suitable for use in the above methods.

In one embodiment of the invention relates to a method of treating or reducing the risk of acquiring osteoporosis, comprising increasing levels of a sex steroid precursor hormones selected from the group consisting of dehydroepiandrosterone (DHEA), dehydroepiandrosterone (DHEA-S) and androst-5-ene-3β ,17β -diol(5-diol), in a patient in need of a specific treatment or a specified reduction, and further including the introduction of the indicated patient a therapeutically effective amount of a selective modulator of estrogen receptors (SERM)as part of combination therapy.

In another embodiment the invention provides a method of treating or reducing the risk of acquisition of hypercholesterolemia, including increased levels predecessor of sex steroid hormones in the alarm group, consisting of dehydroepiandrosterone, dehydroepiandrosterone and androst-5-ene-3β ,17β -diol, in a patient in need of a specific treatment or a specified reduction, and further including the introduction of the indicated patient a therapeutically effective amount of a selective modulator of estrogen receptors as part of combination therapy.

In another embodiment the invention provides a method of treating or reducing the risk of acquiring hyperlipidemia, comprising increasing levels of a sex steroid precursor hormones selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone and androst-5-ene-3β ,17β -diol, in a patient in need of a specific treatment or a specified reduction, and further including the introduction of the indicated patient a therapeutically effective amount of a selective modulator of estrogen receptors as part of combination therapy.

In another embodiment the invention provides a method of treating or reducing the risk of acquisition of atherosclerosis, including increased levels of the precursor of sex hormones selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone and androst-5-ene-3β ,17β -diol, in a patient in need of a specific treatment or a specified reduction, and further comprising entered the e indicated patient a therapeutically effective amount of a selective modulator of estrogen receptors, as part of a combination therapy.

In another embodiment the invention provides a method of treating or reducing the risk of acquiring breast cancer, comprising increasing levels of a sex steroid precursor hormones selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone and androst-5-ene-3β ,17β -diol, in a patient in need of a specific treatment or a specified reduction, and further including the introduction of the indicated patient a therapeutically effective amount of a selective modulator of estrogen receptors as part of combination therapy.

In another embodiment the invention provides a method of treating or reducing the risk of acquiring cancer of the endometrium, including increased levels predecessor of sex steroid hormones selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone and androst-5-ene-3β ,17β -diol, in a patient in need of a specific treatment or a specified reduction, and further including the introduction of the indicated patient a therapeutically effective amount of a selective modulator of estrogen receptors as part of combination therapy.

In another embodiment the invention provides a method of treating or reducing the risk of acquiring cancer of the uterus, including increasing levels of pre is restonica sex steroid hormones selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone and androst-5-ene-3β ,17β -diol, in a patient in need of a specific treatment or a specified reduction, and further including the introduction of the indicated patient a therapeutically effective amount of a selective modulator of estrogen receptors as part of combination therapy.

In another embodiment the invention provides a method of treating or reducing the risk of acquiring ovarian cancer, comprising increasing levels of a sex steroid precursor hormones selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone and androst-5-ene-3β ,17β -diol, in a patient in need of a specific treatment or a specified reduction, and further including the introduction of the indicated patient a therapeutically effective amount of a selective modulator of estrogen receptors as part of combination therapy.

In another embodiment the invention provides a kit comprising a first container containing a therapeutically effective amount of at least one precursor of sex steroid hormones selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone and androst-5-ene-3β ,17β -diol, and any prodrugs, which in vivo turns the I into any of the above precursors; and further comprising a second container containing a therapeutically effective amount of at least one selective modulator of estrogen receptors.

In another embodiment the invention provides a pharmaceutical composition comprising: a) a pharmaceutically acceptable excipient, diluent or carrier; b) a therapeutically effective amount of at least one precursor of sex steroid hormones selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone and androst-5-ene-3β ,17β -diol, and the prodrug, which in vivo is transformed into any of the above precursors of sex steroid hormones; and (C) a therapeutically effective amount of at least one selective modulator of estrogen receptors.

In another embodiment the invention provides a method of reducing the risk of acquiring breast cancer, comprising the administration to a patient in need of such reduction, prophylactically effective amount of a selective modulator of estrogen receptors.

In one embodiment reduce the likelihood of acquiring cancer of the breast, it is desirable to combine the introduction of a SERM with the introduction of the predecessor of sex steroid hormones. However, the invention also includes the introduction of one SERM which is shown, for example, f is the Gora 1 and 2, to ensure a significant prophylactic effect, even in the absence of appointed predecessors. Preferred SERM for this purpose are the same as discussed here for other uses. Preferred dosages and routes of administration are also the same.

As used herein, the selective modulator of estrogen receptors (SERM) is a compound which, either directly or through its active metabolite acts as an antagonist of the estrogen receptor (“antiestrogen”) in the breast tissue, and even provides estrogenic or estrogenically effect on bone and cholesterol in the serum (i.e. reducing serum cholesterol). Non-steroidal compounds that act as antagonists of the estrogen receptor in vitro or in the breast tissue of a human or rat (especially if the connection behaves as an antiestrogen in the cancerous cells of the breast man), probably functioning as a SERM. On the contrary, steroidal antiestrogens do not tend to act as a SERM because they do not tend to show any positive effect on cholesterol levels in serum. Applicants tested the nonsteroidal antiestrogens and found that they function as a SERM, is key EM-800, EAT-01538, raloxifene, tamoxifen and raloxifen. Applicants tested the steroidal antiestrogen ICI 182780 and found that it does not function as a SERM. SERM according to the invention can be entered at the same dose, known in this area, when these compounds are used as an antiestrogen.

The applicants also noted the correlation between the positive SERM effects on cholesterol levels in serum and positive estrogen and estrogen-like effects on bone and serum lipids. In the study of applicants SERM, has shown himself as the acting positively on all parameters, including bone mass, levels of cholesterol and triglycerides. Not intending to be bound by theory, it is believed that the SERM, many of which preferably have two aromatic rings connected by one or two carbon atoms, interact as expected with the estrogen receptor through the above portion of the molecule, which is best recognized by the receptor. Preferred SERM have side chains that can selectively induce antagonistic properties in breast tissue without showing significant antagonistic properties in other tissues. Thus, SERM can function in the desired direction as antiestrogens in breast, at the same time surprisingly functional is of irua in the desired direction as estrogen (or providing estrogenically activity) in the bones and blood (where they have a positive effect on the concentration of lipids or cholesterol). Favorable effect on cholesterol and lipids transferred to a beneficial effect against atherosclerosis, the development of which, as you know, negatively acting unusual levels of cholesterol and lipids.

All diseases treatable according to the invention discussed here, a positive response to androgens. To a greater extent than the use of androgens in themselves, applicants use the predecessors of sex steroid hormones such as DHEA, DHEA-S, 5-diol or prodrug into any such predecessors of sex steroid hormones. In vivo DHEA-S converted into DHEA, which in turn, turns into a 5-diol. It is believed that any tissue respond positively to one, likely to respond positively to others. Proletarienne form active metabolites are well known in this area. See, for example, H.Bundgaard “Design and Application of Prodrugs” (In.: A Textbook of Drug Design and Development. Edited by H.Bundgaard and P.Krogsgaard-Larsen; Harwook Academic Publishers GmfH, Chur: Switzerland, 1991), the content of which is incorporated herein by reference. In particular, see pages 154-155, which describes various functional groups of active metabolites and the corresponding group of prodrugs that are converted in vivo into each functional group. In cases, when the predecessors of sex steroid hormones in patients p is increased in accordance with the invention, this can usually be achieved with the introduction of such predecessor or the introduction prodrugs of such predecessor. When applying predecessors instead of androgens decreases unwanted androgenic activity in tissues other than misheneva. Fabric transform precursors such as DHEA, androgens only through natural and more manageable process. A large percentage of androgens produced locally in peripheral tissues and in different tissues in different degrees.

Cancers amenable to treatment in accordance with the invention, adversely respond to estrogenic activity. On the other hand, osteoporosis, hypercholesterolemia, hyperlipidemia, and atherosclerosis positively respond to estrogenic or estrogenically activity. Using a SERM in accordance with the invention, the desired effects are provided in the target tissues without adverse effects in other tissues. For example, a SERM can have a positive estrogenic effects in bone (or on lipids and cholesterol), at the same time, they have no adverse estrogenic effect on the breast.

Therefore, as a precursor, and SERM, provide a positive effect on target tissues, at the same time minimizing adverse effects in some other parodentium is. Moreover, there is significant synergy when using the two together in accordance with the invention. For example, estrogens and androgens provide a positive effect against osteoporosis by different mechanisms (estrogen-reducing bone resorption, androgen-increasing bone formation). The combination of the present invention provides a bone positive estrogenic or estrogenically action through the activity of the SERM, and also provides positive androgen action through local conversion of the precursor to androgen in bone. It is assumed that the predecessor also provides estrogen. The same is true in relation to the regulation of lipid or cholesterol (suitable for treatment or prevention of atherosclerosis). Similar synergism is provided against breast cancer, endometrial or ovarian cancer, where SERM provides the desired anti-estrogenic effect, and the predecessor provides the desired androgenic effect (if any adverse conversion of the precursor to estrogen, being softened by the antiestrogen). Unwanted effects are also attenuated by synergistic combination used according to the invention.

For all of the diseases discussed here, any other action on breast tissue that may otherwise arise from estroge is s, formed from its predecessor (when used as a predecessor to ensure androgenic effects according to the invention), attenuated antiestrogenic effect of SERM in the breast tissue.

In some embodiments of the invention are added progestins to provide additional androgenic effect. Progestins can be used in low doses, is known in this area, without negative effects on other receptors than the androgen receptor (for example, receptors glucocorticoids).

They are also relatively free from unwanted androgenic side effects such as growth of facial hair in women).

Preferred SERM discussed here include 1) all diseases specified as sensitive to the invention); 2) as to therapeutic and prophylactic applications, and 3) the preferred pharmaceutical compositions and kits.

In one embodiment the precursor is DHEA.

In another embodiment the precursor is DHEA-S. In another embodiment the precursor is 5-diol.

A patient in need of treatment or risk reduction beginning of this disease is the principal who has either been diagnosed with such a disease, or a subject who is sensitive to acquiring such diseases.

Except where stated the Noah, the preferred dose of the active compounds (concentrations and routes of administration) according to the invention is the same for therapeutic and preventive purposes. The dosage of each active component, discussed here, is the same regardless of the disease being treated (or disease, the probability of which decreases).

Except when noted otherwise or when it is obvious from the context, dose here refer to the weight of the active compounds, without undergoing the influence of pharmaceutical excipients, diluents, carriers or other ingredients, although it is desirable to include such additional ingredients as shown in the examples. Any dosage form (capsule, tablet, injection or the like)commonly used in the pharmaceutical industry, for this to apply, and the terms “excipient”, “diluent” or “media” include inactive ingredients, which usually included together with the active ingredients in such formulations in the industry. For example, can be included regular capsules, pills, casings, solid or liquid diluents or excipients, flavorings, preservatives and the like.

All of the active ingredients used in any of the treatments discussed here, you can switch the diamonds in the form of pharmaceutical compositions, which also include one or more other active ingredients. Alternative each of them can be entered separately, but sufficiently so that the patient ultimately increased levels in the blood, or otherwise received the positive impact of each of the active ingredients (or strategies) at the same time. In some preferred embodiments of the invention, for example, one or more of the active ingredients should be included in the same pharmaceutical composition. In other embodiments of the invention provides a kit that includes at least two separate container where the contents of the at least one container differs in whole or in part from content of at least one other container in the active ingredients contained therein.

Combination therapy discussed here, also includes the use of one active ingredient (combination) in the manufacture of a medicinal product for the treatment (or risk reduction) of the disease where the treatment or prophylaxis further includes another active ingredient combination according to the invention. For example, in one embodiment the invention provides the use of SERM for obtaining a medicinal product for use in conjunction with the predecessor chaff the steroid hormones, selected from the group consisting of DHEA, DHEA-S, 5-diol, and prodrugs into any of the above precursors of sex steroid hormones in vivo, when treating any of the diseases for which, as expected, real effective combination therapy (i.e. breast cancer, endometrial cancer, uterine cancer, ovarian cancer, osteoporosis, hypercholesterolemia, hyperlipidemia, and atherosclerosis). In another embodiment the invention provides the use of a precursor of sex steroid hormones selected from the group consisting of DHEA, DHEA-S, 5-diol, and prodrugs into any of the above precursors of sex steroid hormones in vivo, for obtaining a medicinal product for use in conjunction with a SERM in the treatment of any of these diseases.

In one embodiment of the invention DHEA is not used as a predecessor. In another embodiment of EM-800 is not used as a SERM. In another embodiment does not use the combination of DHEA with EM-800.

In the preferred embodiment of DHEA used in conjunction with EM-1538.

Brief description of drawings

The figure 1 shows the treatment effect of DHEA (10 mg percutaneous, once daily) or EM-800 (75 µg orally, once daily) alone or in combination for 9 months on the frequency of DMBA induced breast carcinoma in rats in t is the treatment 279-day observation period. Data are expressed as percentage of the total number of animals in each group.

The figure 2 shows the treatment effect of DHEA (10 mg percutaneous, once daily) or EM-800 (75 µg orally, once daily) alone or in combination for 9 months on the average number of tumors in animals-carriers of tumour (A) and the average size of tumors in a rat-ofwholesale () for 279-day observation period. Data are expressed as averages ± the average error of the mean.

The figure 3 shows the treatment effect of DHEA (10 mg percutaneous, once daily) or EM-800 (75 µg orally, once daily) alone or in combination for 9 months on the levels of triglycerides (A) and cholesterol (C) in the serum of rats. Data are expressed as averages ± the average error of the mean. * * : p&λτ; 0,01 experienced versus the corresponding control.

The figure 4 shows: (A) effect of increasing doses of DHEA (0.3 mg, 1.0 mg or 3.0 mg), percutaneous insertion, twice a day, on the average size of tumors ZR-75-1 naked mice with remote ovaries (OVX)and treated with estrone. Control OVX mice treated for one medium was used as additional control. The initial size of the tumor was taken as 100%. DHEA was administered transdermally (CC) in 0.02 ml of 50% ethanol, 50% propylene glycol in which the auger back. C) effect of treatment with increasing doses of DHEA or EM-800 alone or in combination for 9.5 months on the weight of the tumors ZR-75-1 in OVX naked mice treated with estrone. * * : p&λτ; 0,01 treated versus control OVX mice with the addition of estrone.

The figure 5 shows the effect of increasing oral doses of the antiestrogen EM-800 (15 μg, 50 μg or 100 μg) (A) or percutaneous introduction of increasing doses of DHEA (of 0.3, 1.0 or 3.0 mg) in combination with EM-800 (15 µg) one or EM-800 (C) for 9.5 months on the average size of tumors ZR-75-1 naked mice with remote ovaries (OVX)and treated with estrone. The initial size of the tumor was taken as 100%. Control OVX mice treated for one medium was used as additional control. Estrone was administered subcutaneously at a dose of 0.5 mg once a day, while DHEA was dissolved in a mixture of 50% ethanol, 50% propylenglycol and was applied to the back skin twice a day in a volume of 0.02 ml Comparison was also performed with OVX animals receiving a single carrier.

The figure 6 shows the effect of 12-month treatment only dehydroepiandrosterone (DHEA) or in combination with flutamide or EM-800 on the volume of cancellous bone in rats with removed ovaries. Intact animals were added as an additional control. Data are presented as averages ± middle Osh the BKA average. * * p&λτ; of 0.01 versus OVX control.

The figure 7 shows the effect of 12-month treatment only dehydroepiandrosterone (DHEA) or in combination with flutamide or EM-800 on the number of trabeculae in rats with removed ovaries. Intact animals were added as an additional control. Data are expressed as averages ± the average error of the mean. * * p&λτ; of 0.01 versus OVX control.

The figure 8 shows metaphysi in the proximal part of the tibia from the intact control animals (A), control rats with removed ovaries (In) and in rats with removed ovaries treated with one of DHEA (S) or in combination with flutamide (D) or EM-800 (E). It is noted reduced cancellous bone (T) control animals with remote ovaries (In) and a significant increase in cancellous bone (T), induced after injection of DHEA (S). Add flutamide to DHEA partially blocked the effect of DHEA on the amount of cancellous bone (D), while the combination of DHEA and EM-800 has provided full protection against bone loss associated with oophorectomy. Modelirovanie Masson trichrome-GoIdner, increase × 80. T-spongy substance; GP, growth plate.

The figure 9 shows the effect of increasing doses (0.01, of 0.03, 0.1, 0.3 and 1 mg/kg) EM-800, EM-1538 and raloxifen is a (EAT-1105), introduced orally daily for 4 days on cholesterol levels in rats with removed ovaries.

The figure 10 shows the influence of the 34-week introduction only dehydroepiandrosterone (DHEA) or in combination with EM-1538 (EM-652· Hcl) on bone mineral density (BMD) in the lumbar vertebrae in rats with removed ovaries. Intact animals were added as an additional control. Data are expressed as mean values±the average error of the mean. * * p&λτ; of 0.01 versus OVX control.

The figure 11 shows the combined effect of the SERM (EM-652) and DHEA on the parameters of menopause. Negative effects are not expected.

The figure 12 shows the concentration of DHEA in the blood plasma (ng/ml) (Y-axis) as a function of time (X axis) after a single oral administration preferred precursors of sex steroid hormones according to the invention (150 μmol/rat) in rats-males. In the rectangle presents AUC DHEA for 24 h induced by these compounds.

EAT-760 dehydroepiandrosterone

EM-900 androst-5-ene-3β ,17β -diol

EM-1304 androst-5-ene-3β ,17β -diol 3-acetate

EM-1305-CS androst-5-ene-3β ,17β -diol diacetate

EAT-1397 androst-5-ene-3β ,17β -diol 3-acetate 17-benzoate

EAT-1400 androst-5-ene-3β ,17β -diol dibenzoate

EM-1410 androst-5-ene-3β ,17β -diol of dipropyl the NAT

EM-1474-D androst-5-ene-3β ,17β -pyrrolidone dyamically

The figure 13 shows the concentration in the blood plasma of androst-5-ene-3β ,17β -diol (ng/ml) (Y-axis)as a function of time (X-axis), after a single oral administration of a precursor of sex steroid hormones according to the invention (150 μmol/rat) in rats-males. In the rectangle presents AUC androst-5-ene-3β ,17β -diol for 24 h induced by these compounds.

EAT-760 dehydroepiandrosterone

EM-900 androst-5-ene-3β ,17β -diol

EM-1304 androst-5-ene-3β ,17β -diol 3-acetate

EM-1305-CS androst-5-ene-3β ,17β -diol diacetate

EAT-1397 androst-5-ene-3β ,17β -diol 3-acetate 17-benzoate

EAT-1400 androst-5-ene-3β ,17β -diol dibenzoate

EM-1410 androst-5-ene-3β ,17β -diol dipropionate

EM-1474-D androst-5-ene-3β ,17β -pyrrolidone dyamically.

A detailed description of the invention

It is well known that estrogens stimulate the proliferation of epithelial cells of the mammary gland, and offers itself that cell proliferation increases the risk of cancer of the accumulation of random genetic errors that can lead to neoplasia (Preston Martin et al., Cancer. Res. 50: 7415-21, 1990). Based on this concept, antiestrogens were introduced for the prevention of breast cancer with the aim of reducing the rate of cell division stimulated es what rolename.

Loss of ovarian cyclicity installed at female rats Sprague-Dawley after 10 months of age, accompanied by elevated levels of estrogen and prolactin and low concentrations of androgens and progesterone in serum (Lu et al., 61stAnnual Meeting of the Endocrine Society 106 (abst. # 134), 1979; Tang et al., Biol. Reprod. 31: 399-413, 1984; Russo et al., Monographs on Pathology of Laboratory Animals: Integument and Mammary Glands 252-266, 1989; Sortino and Wise, Endocrinology 124: 90-96, 1989; Cardy, Vet. Pathol. 28: 139-145, 1991). These hormonal changes that occur spontaneously in aging female rats, associated with multifocal proliferation and increased secretory activity of acinar/alveolar tissue, as well as the expansion of the ducts of the mammary gland and the formation of cysts (Boorman et al., 433, 1990; Cardy, Vet. Pathol. 28: 139-145, 1991).

It should be noted that hyperplastic and neoplastic changes of the mammary gland in rats often accompanied by elevated levels of estrogen and prolactin (Meites, J. Neural. Transm. 48: 25-42, 1980). Treatment of EM-800, SERM according to the present invention induces atrophy of the mammary gland, which is characterized by a reduction in the size and number of lobular structures, and no signs of secretory activity, which indicates a strong antiestrogenic activity of EM-800 in the breast (Luo et al. Endocrinology 138: 4435-4444, 1997).

Processing DHEA, a precursor of sex steroid hormones of the present invention, increases vortochny DHEA and 5-diol, at that time, as the levels of 4-dione, testosterone, dihydrotestosterone and estradiol in serum increased only in moderate or often remain unchanged, confirming thus the intracellular biotransformation of this precursor of steroid hormones in peripheral tissues (Labrie et al., Mol. Cell. Endocrinol. 78: C113-C118, 1991). However, there is a stimulating effect of oral introduced DHEA on serum androgens, such as testosterone and dihydrotestosterone, a larger range than its effect on serum estrogens, on the basis of which it can be assumed that these animals DHEA preferably converted into androgens. This observation is consistent with the data on women, in which the formation of androgens DHEA was more important way than the transformation of DHEA into estrogen (Morales et al., J. Clin. Endocrinol. Metab. 78: 1360-1367, 1994; Labrie et al., Ann. N.Y. Acad. Sci. 774: 16-28, 1995; Labrie et al., Steroids 62: 148-158, 1997).

With the popularity of the above strong antiestrogenic activity, leading to atrophy of the mammary gland, and the predominant androgenic effects of DHEA on the mammary gland, histomorphological changes that are installed on animals treated with the combination of a SERM and predecessor of sex steroid hormones that best explains the only androgenic effects of DHEA in the mammary gland of rats.

More importantly nab Eugenie, that androgens are a direct antiproliferative activity on the growth of ZR-75-1 in cancer cells human mammary gland in vitro and that this inhibitory effect of androgens is additive to that of the antiestrogen (Poulin and Labrie, Cancer Res. 46: 4933-4937, 1986; Poulin et al., Breast Cancer Res. Treat. 12: 213-225, 1988). Similar inhibiting effects were observed in vivo in xenografts ZR-75-1 naked mice (Dauvois et al., Cancer Res. 51: 3131-3135, 1991). It was also shown that androgens inhibit the growth of DMBA induced breast carcinoma in rats, and this inhibitory effect is removed with the simultaneous introduction of the net antiandrogen flutamide (Dauvois et al., Breast Cancer Res. Treat. 14: 299-306, 1989). Taken together, these data point to the involvement of androgen receptors in the inhibitory effect of DHEA on breast cancer.

As antiestrogens and predecessors of sex steroid hormones exhibit inhibitory activity against breast cancer through various mechanisms, the present invention shows that the combination of a SERM (EM-800) and the predecessor of sex steroid hormones (DHEA) shows a stronger inhibiting effects than each connection itself, on the development of DMBA-induced breast carcinoma, which is well shown in figures 1 and 2. Actually was not found DMBA-induced tumors in the end of the experiment the animals that p is were given as DHEA, and EM-800.

The present invention describes that the combination of the predecessor of sex steroid hormones (DHEA) and SERM (EM-800) kept the stimulatory effect of DHEA on bone formation and increased inhibitory effect only SERM (EM-800) on the metabolism and bone resorption, which was shown to be an additional reduction in the allocation of hydroxyproline and calcium with urine, when we were United both connections.

Applicants have shown that DHEA has a positive effect on bone in rats-females (Luo et al., Endocrinology 138: 4435-4444, 1997) and postmenopausal women (Labrie et al., J.Clin. Endocrinol. Metab. 82: 3498-3505, 1997). Thus, in intact female rats treatment DHEA increases bone mineral density (BMD) of the whole skeleton, lumbar spine and femur (Luo et al., Endocrinology 138: 4435-4444, 1997).

On the other hand, the processing of EM-800 had no significant effect on BMD in the intact animals, although the observed strong stimulating effects in rats with removed ovaries (Martel et al., unpublished data). Because EM-800 shows a similar stimulating effect on BMD of the whole skeleton, lumbar spine and femur in rats, the lack of reliable stimulating effect of EM-800 in the intact animals may be the result of the fact that sex steroid hormones are present in the intact female rats, have a maximum effect on BMD (Luo et al., Endocrinology 138: 4435-444, 1997). Similarly, no significant effect of EM-800 in rats with removed ovaries, has already received DHEA, probably, is due to the stimulating effect of responsiveness to androgens (and possibly estrogen), synthesized in bone cells from exogenous DHEA.

It is known that estrogens reduce the level of cholesterol in the serum, but increase or have no effect on the levels of triglycerides in the serum (Love et al., Ann. Intern. Med. 115: 860-864, 1991; Walsh et al., New Engl. J. Med. 325: 1196-1204, 1991; Barrett-Connor, Am. J.Med. 95 (Suppl. 5A): 40S-43S, 1993; Russell et al., Atherosclerosis 100: 113-122, 1993; Black et al., J.Clin. Invest. 93: 63-69, 1994; Dipippo et al., Endocrinology 136: 1020-1033, 1995; Ke et al., Endocrinology 136: 2435-2441, 1995). The figure 3 shows that EM-800 has as hypocholesterolemic and hypotriglyceridemic effects in rats, thus demonstrating its unique effect on the profile of serum lipids, which clearly differs from other SERM such as tamoxifen (Bruning et al., Br. J.Cancer 58: 497-499, 1988; Love et al., J.Natl. Cancer Inst. 82: 1327-1332, 1990; Dipippo et al., Endocrinology 136: 1020-1033, 1995; Ke et al., Endocrinology 136: 2435-2441, 1995), droloxifene (Ke et al., Endocrinology 136: 2435-2441, 1995) and raloxifene (Black et al., J.Clin. Invest. 93: 63-69, 1994). The combination of DHEA and EM-800 stores hypocholesterolemic and hypotriglyceridemic effects of EM-800 on the basis of which it can be assumed that such combination can show positive effects on serum lipids

It should be noted that the profile of serum lipids significantly different in rats and humans. However, because in the hypocholesterolemic action of estrogens and antiestrogens participates mechanism which is carried out through the estrogen receptors (Lundeen et al., Endocrinology 138: 1552-1558, 1997), the rat remains to be a useful model for studying holesterin lowering action of estrogens and antiestrogens” people.

Briefly, the above data clearly show the effect of the combination SERM (EM-800) and the predecessor of sex steroid hormones (DHEA) on the development of breast carcinoma induced by DMBA, as well as the protective effects of this combination, on bone mass and serum lipids. Based on these data, we can assume that there are additional positive effects of this combination for the treatment and prevention of osteoporosis, while improving lipid profile.

Applicants also investigated the potential interaction of the inhibitory effect of a new antiestrogen (EM-800) with such predecessor of sex steroid hormones (DHEA) on the growth of human xenotransplantation ZR-75-1 breast cancer in Nude mice with the combined introduction of the two drugs. In figures 4 and 5 show that DHEA itself is used in doses causes 50-80% inhibition of tumor growth, while at almost full tarmogen the e tumor growth, achieved with low-dose antiestrogen, DHEA had no effect.

Limitations when measuring bone mineral density (BMD) is well known. As an example, determination of BMD showed no differences in rats treated with steroid an antiestrogen ICI 182780 (Wakeling, Breast Cancer Res. Treat. 25: 1-9, 1993), while inhibiting changes were established by histomorphometry (Gallagher et al., Endocrinology 133: 2787-2791, 1993). Reported similar differences with tamoxifen (Jordan et al., Breast Cancer Res. Treat. 10: 31-35, 1987; Sibonga et al., Breast Cancer Treatm. 41: 71-79, 1996).

Should indicate that reduced bone mineral density is not the only anomaly is associated with reduced bone strength (Guidelines for preclinical and clinical evaluation of agents used in the prevention or treatment of postmenopausal osteoporosis. Division of Metabolism and Endocrine Drug Products, FDA, May, 1994). Therefore, it is important to analyze changes in the biochemical parameters of bone metabolism induced by various compounds and treatments to gain more knowledge about their action.

It is especially important to indicate that the combination of DHEA and EM-800 showed an unexpected positive effects on important biochemical parameters of bone metabolism. In fact, DHEA itself does not affect the ratio of hydroxyproline/creatinine in urine, a marker of bone resorption. Moreover, it was impossible to detect the effect of DHEA on a daily selection with mosaically or phosphorus (Luo et al., Endocrinology 138: 4435-4444, 1997). EM-800, on the other hand, reduced the ratio of hydroxyproline/creatinine in urine by 48%, while similar to DHEA, it has not been established effect in EM-800 on the secretion of calcium and phosphorus in the urine. In addition, EM-800 had no effect on the activity of serum Alp, a marker of bone formation, while DHEA increased the option value by approximately 75% (Luo et al., Endocrinology 138: 4435-4444, 1997).

One of the unexpected effects of a combination of DHEA and EM-800 refers to the ratio of hydroxyproline/creatinine in urine, a marker of bone resorption, which decreased by 69%when combined together DHEA and EM-800, this figure was significantly different (p&λτ; 0,01) from 48% inhibition achieved one EM-8 00, at that time, as by itself, DHEA did not show any action. Thus, the addition of DHEA to EAT-800 increases by 50% inhibitory effect of EM-800 on bone resorption. More important another unexpected effect of adding DHEA to EAT-800 was reduced by 84% of calcium in the urine (23,17± 1.55V to 3.71± 0.75 µmol/24 h/100 g (p&λτ; 0,01)) and a decrease of 55% of the phosphorus in the urine (132,72± between 6.08 to 59,06± 4.76 mmol/24 h/100 g (p&λτ; 0,01), respectively (Luo et al., Endocrinology 138: 4435-4444, 1997).

Table 1

GroupUrineSavorotocnami
 Calcium (mmol/24 h/100 g)Phosphorus (µmol/24 h/100 g)SE/cu (hydroxyproline/creatinine (µmol/mmol)OSF (total alkaline phosphatase) (IU/L)
Control23,17± 1,55132,72± between 6.0813,04± 2,19114,25± 14,04
Dehydroepiandrosterone DHEA (10 mg)25,87± 3,54151,41± of 14.5714,02± 1,59198,38± 30,76*
EM-800 (75 µg)17,44± 4,5102,03± 25,13for 6.81± 0,84* *114,11± of 11.26
NEA + EM-8003,71± 0,75* *59,06± 4,76* *4,06± 0,28* *204,38± 14,20* *

Also of interest to note that the strong inhibitory effect of EM-800 on serum cholesterol is not removed with the simultaneous introduction of DHEA (Luo et al., Endocrinology 138: 4435-4444, 1997).

Although raloxifene and similar compounds prevent bone loss and reduction of serum cholesterol (like estrogen), it should be noted that, when raloxifene compared with premarino the effect on BMD, the effect of raloxifene on BMD was less strong than that of premarin (Minutes of the Endocrinology and Metaolism Drugs Advisory Committee, FDA Thursday, Meeting # 68, November 20th1997).

These data obtained in rats, clearly show that DHEA can provide positive effects, which are not available when using a single selective modulator of estrogen receptors (SERM) such as EM-800, raloxifene, etc. while SERM has action, limited inhibition of bone resorption, it is believed that the addition of DHEA, 5-diol, DHEA-S stimulates bone formation effect was not found with SERM or estrogen) and further reduces bone resorption to a greater extent than the effect achieved with EM-800.

It is important that the combined use of EM-800 and DHEA for 12 months in rats with removed ovaries had a positive effect on bone morphometry. The amount of cancellous bone is particularly important for bone strength and prevent fractures. So, in the above experience, the volume of cancellous bone increased from 4.1± 0,7% in rats with removed ovaries to 11.9± 0,6% (p&λτ; 0.01) for introduction only DHEA, while the addition of EM-800 to DHEA has further increased the volume of cancellous bone to 14.7± 1,4%, values close to the specified in the intact control animals (6).

From 0,57± 0,08 mm in rats with removed ovaries DHEA treatment resulted in an increase of 137% in the number of trabeculae to the tee compared to control rats with removed ovaries. Thus, when stimulating action of DHEA, this indicator reached 1.27± 0,1 mm, while simultaneous treatment of EM-800 and DHEA resulted in an additional increase in the number of trabeculae bone by 28% (p&λτ; 0.01) compared with achievable only DHEA (Fig.7). Similarly, the addition of EM-800 to the introduction of DHEA resulted in an additional 15% reduction (p&λτ; 0,05) separation of bone trabeculae compared with the achievable only DHEA, thus leading to values not different from that established in the intact control animals.

In addition to numerous data shown in figures 6 and 7, figure 8 shows the increase in cancellous bone in metaphase proximal part of the tibia, DHEA induced in animals with remote ovaries (C), compared to control rats with removed ovaries (In), and partial inhibition of the stimulatory action of DHEA after adding flutamide processing DHEA (D). On the other hand, the introduction of DHEA in combination with EM-800 resulted in complete prevention induced by oophorectomy osteopenia (E), when the volume of cancellous bone, comparable to those established in the intact control rats (A).

It is believed that the bone loss observed at menopause in women is associated with an increased rate of resorption to the stay, which is not fully compensated by a secondary increase in bone formation. In fact, the parameter of bone formation and increase bone resorption in osteoporosis, and as resorption and bone formation inhibited when estrogen replacement therapy. It is assumed, therefore, that the inhibitory effect of estrogen replacement on bone formation is the result of the associated mechanism between bone resorption and bone formation so that the primary estrogenization reduction of bone resorption leads to the decrease in bone formation (Parfitt, Calcified Tissue International 36 Suppl. 1: S37-S45, 1984).

The strength of cancellous bone and subsequent resistance to fracture depend not only on the total number of cancellous bone, but also on the microstructure of trabeculae, which is determined by the number, size and distribution of trabeculae. Loss of function of the ovaries in postmenopausal women is accompanied by a significant decrease in the total volume of cancellous bone (Melsen et al., Acta Pathologica &αμπ; Microbiologica Scandinavia 86: 70-81, 1978; Vakamatsou et al., Calcified Tissue International, 37: 594-597, 1985), mainly related to the decrease in the number and, to a lesser extent, the width of trabeculae (Weinstein and Hutson, Bone 8: 137-142, 1987).

In the present study androgenic stimulatory effect of DHEA was observed in almost all the studied GIS is morphometrically parameters bones. So, DHEA resulted in a significant increase in cancellous bone, as well as the number of trabeculae, at that time, as he lowered megabecquerel area.

In order to contribute to the aspect of the combination therapy according to the invention for any discussed here indicate, the invention provides pharmaceutical compositions that include a SERM or bisphosphonate connection and predecessor of sex steroid hormones (DHEA, DHEA-S, 5-diol) in a single composition for simultaneous administration. The composition may be suitable for administration by any conventional method, including, but not limited to, by oral administration, subcutaneous injection, intramuscular injection or percutaneous introduction. In other embodiments of the provided kit, where the kit includes one or more SERM or a bisphosphonate and predecessors of sex steroid hormones in a separate or the same container. The kit can include appropriate materials for oral administration, e.g. tablets, capsules, syrups and the like, and for transdermal administration, for example, ointments, lotions, gels, creams, pieces of tape with the continuous release and the like.

Applicants believe that the introduction of a SERM and predecessors of sex steroid hormones applicable for the treatment and/or prevention of osteoporosis, breast cancer W is Lesa hypercholesterolemia, hyperlipidemia or atherosclerosis. The active ingredients according to the invention (SERM or predecessor, or a bisphosphonate, or other) can be included in the composition and introduced in various ways.

The active ingredient for delivery through the skin and mucous preferably is at a level of from 0.5% to 20% by weight relative to the total weight of the pharmaceutical composition, more preferably between 2 and 10%. You need to DHEA or 5-diol was at a concentration of at least 7% for percutaneous introduction. Alternative active ingredient can be included in the patch for transdermal delivery with patterns known in this field, for example, patterns such as in European patent No. 0279982.

When included in the composition of the ointment, lotion, cream or gel, or the like, the active compound is mixed with suitable carrier, which is compatible with the skin and mucous membranes of man, and which increases the penetration of compounds through the skin or mucous membranes. Suitable carriers are known in this field and include, but are not limited to, klocale HF and glaxal base. Some are commercially available, for example, glaxal base provides Glaxal Canada Limited Company. In Koller and Buri, S.T.P. Pharma 3(2), 115-124, 1987 you can find other suitable media. The carrier preferably is one in which rest is con siderably widening (soluble) active ingredient(you) when the ambient temperature when the concentration of the active ingredient, in which it is used. The carrier should have sufficient viscosity to maintain the precursor in a limited area of skin or mucosa to which is applied the composition, without runoff or evaporation over a period of time sufficient for the main penetration of the precursor through a limited area of skin or mucosa and into the bloodstream where it will cause the desired clinical effect. The media is usually a mixture of several components, for example, pharmaceutically acceptable solvents and thickeners. The mixture of organic and inorganic solvents can help for hydrophilic and lipophilic solubility, for example, water and alcohol, such as ethanol.

Preferred precursors of sex steroid hormones are dehydroepiandrosterone (DHEA) (provides Diosynth Inc., Chicago, Illinois, USA), its prodrug (Steraloids, Wilton, New Hampshire, USA), 5-androsten-3β ,17β -diol and its prodrugs EM-1304 and EAT-01474-D (Steraloids, Wilton, New Hampshire USA).

EAT-01474-D

Preferably, the precursor of sex steroid hormones were part of the alcoholic gel containing 2.0 to 10% of capryl-capric triglyceride (Neobee M-5); 10-20% of hexyleneglycol; 2,0-10% diethylene glycol nanometrology ether (Transutol); 2.0 to 10% cyclo is eikona (Dow Corning 345); 1,0-2% benzyl alcohol and 1.0 to 5.0% hydroxypropylcellulose (Klucel HF).

The media may include various additives commonly used in ointments and lotions, and are well known in the field of cosmetology and medicine. For example, there can be fragrances, antioxidants, fragrances, gelling agents, thickeners such as carboxymethyl cellulose, surfactants, stabilizers, emollients, dyes, and other similar agents. When used for the treatment of systemic diseases, the place for application on the skin can be changed in order to avoid excessive local concentration of the active ingredient and possible over-stimulation of the skin and sebaceous glands androgenic metabolites predecessor of sex steroid hormones.

In the pharmaceutical compositions for oral administration of DHEA or other precursor is preferably in a concentration of between 5 and 98% by weight relative to the total weight of the composition, more preferably between 50 and 98%, especially between 80 and 98%. Separate precursor such as DHEA, there can be only one active ingredient, or alternatively you can use a variety of precursors and/or their analogues (for example, a combination of DHEA, DHEA-S, 5-diol, or a combination of two or more compounds, turn in vivo in DHEA, DHEA-S or 5-diol, or a combination of DHEA or 5-diol and one or more of their counterparts, which are converted to DHEA or 5-diol in vivo, etc. Levels of DHEA in the blood is the ultimate criterion of an adequate dosage, taking into account individual differences in absorption and metabolism.

Preferably, the attending physician, especially at the beginning of treatment, followed total individual response of the patient and the level of DHEA in the blood serum (in comparison with the preferred serum concentrations discussed above) and watched the overall response of the patient to treatment, if necessary, adjusting the dose where the metabolism or response to treatment in this patient are atypical.

Treatment in accordance with the invention is suitable for indefinite duration. It is assumed that treatment with DHEA and/or 5-diola will simply maintain the levels of DHEA within the limit, similar to what usually occurs in women before menopause (concentration in serum between 4 and 10 micrograms per liter), or generally the young adult men (concentration in serum between 4 and 10 micrograms per litre).

Connection SERM or a bisphosphonate and/or the precursor of sex steroid hormones can also enter through the mouth and it can be prepared in a mixture with conventional pharmaceutical excipients, for example, spray dried lactose, microcrystalline cellulose stearate and m is fester, in tablets or capsules for oral administration.

The active substance can be included in the core of tablets or pills, mixed with powdered solid carriers, such as sodium citrate, calcium carbonate or dicalcium phosphate, and binders such as polyvinylpyrrolidone, gelatin or cellulose derivatives, possibly, adding also lubricants such as magnesium stearate, sodium lauryl sulfate, carbowax or polyethylene glycol. Of course, in the case of forms for oral administration can be added to improve the taste of the substance.

As an additional dosage forms, you can use capsules, for example, hard gelatin, and closed soft gelatin capsules, comprising a softener or plasticizer, such as glycerol. The sealed capsules contain the active substance preferably in the form of a granulate, for example, in a mixture with fillers such as lactose, sucrose, mannitol, starches such as potato starch or amylopectin, cellulose derivatives or highly dispersed silicic acid. In soft gelatin capsules the active substance is preferably dissolved or suspended in suitable liquids such as vegetable oil or liquid polyethylene glycols.

Lotion, ointment, gel or cream should be thoroughly rubbed into the skin the AK, that was not clear excess, and the skin should not be washed in this area will not occur until the main absorption through the skin, preferably for at least 4 hours, more preferably at least 6 hours

Transdermal patches can be used to deliver the precursor in accordance with known methods. It is usually applied for a longer period of time, for example, 1-4 days, but usually the active ingredient in contact with a lower surface area, allowing for a slow and continuous delivery of the active ingredient.

A number of systems for delivery of drugs through the skin, which have been developed and are suitable for delivery of the active ingredient of the present invention. The rate of release is usually governed by the diffusion matrix, or the passage of the active ingredient through regulating the membrane.

Mechanical aspects of transdermal devices are well known in this area and explains, for example, in U.S. patents 5162037, 5154922, 5135480, 4666441, 4624665, 3742951, 3797444, 4568343, 5064654, 5071644, 5071657, the contents of which are here incorporated by reference. Additional prerequisites are presented in the European patent 0279982 and Patent application in the UK 2185187.

The device may be of the General type known in this field, including the device the VA for transdermal delivery with adhesive matrix or reservoir type. The device may include containing the drug matrix comprising fibers that absorb the active ingredient and/or the media. In the device reservoir type reservoir may be separated by a polymer membrane that is impermeable to the carrier and the active ingredient.

In the transdermal device itself supports the active ingredient in contact with the desired local surface of the skin. In such a device, the viscosity of the carrier for the active ingredient has a smaller value than for the cream or gel. The solvent system for transdermal devices may include, for example, oleic acid, lactate normal alcohol and dipropyleneglycol or other solvents known in this field. The active ingredient can be dissolved or suspended in the carrier.

To attach to the skin transdermal patch can be inserted into a surgical adhesive tape having a hole in the middle. The tape is preferably covered with a removable strip for protection prior to use. Conventional materials suitable for decreasing strip include polyethylene and polyethylene coated paper and is preferably coated with silicone for easy removal. For applying the device removable strip is easily removed, and the adhesive tape on the Yu tape attached to the patient's skin. In U.S. patent 5135480, the content of which is incorporated by reference, Bannon et al. describe an alternative device having nepriklausomas means of fixation of the device on the skin.

You can also use the system for delivery through the skin or mucous membranes according to the invention as a new and improved delivery system for the prevention and/or treatment of osteoporosis or other diseases that respond favorably to treatment with androgens and/or estrogens.

Selective modulator of estrogen receptor according to the invention has a molecular formula with the following characteristics: (a) two aromatic rings separated by 1-2 intermediate carbon atoms, both aromatic rings are unsubstituted or substituted by a hydroxyl group or a group, turn in vivo in hydroxyl and (b) a side chain having an aromatic ring and the function of the tertiary amine or its salt.

One preferred SERM according to the invention is EM-800, reported in PCT/CA 96/00097 (WO 96/26201). Molecular structure of EM-800 is:

Other preferred SERM according to the invention is EM-01538:

EM-1538 (also called EM-652· Hcl) is a strong hydrochloride antiestrogen EM-652, compared with EM-800, EM-1538 is more, the issue is Oh or easy to synthesize salt. It is also easy to isolate, purify, it crystallizes and exhibits good stability in the solid state. It is assumed that the introduction of as EM-800, and EM-1538 leads to the formation of the same active substance in vivo.

Other preferred SERM according to the invention include tamoxifen ((Z)-2-[4-(1,2-diphenyl-1-butenyl)]-N,N-dimethyl-ethanamine) (Zeneca, UK), toremifene (Orion provides-it facilitates Pharmaceuicia, Finland or Schering-Plough), droloxifene and CP-336156 (CIS-1R-[4′ -pyrrolidinecarbonyl]-2S-phenyl-6-hydroxy-1,2,3,4-tetrahydronaphthalen D-(-)-tartrate) (Pfizer Inc., USA), raloxifene (Eli Lilly and Co., USA), LY 335563 and LY 353381 (Eli Lilly and Co., USA), idoxifene (SmithKline Beecham, USA), levormeloxifene (3,4-TRANS-2,2-dimethyl-3-phenyl-4-[4-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl]-7-methoxypropan) (Novo Nordisk, A/S, Denmark), which is disclosed in Shalmi, etc. in WO 97/25034, WO 97/25035, WO 97/25037, WO 97/25038; and Korsgaard and others in WO 97/25036), GW 5638 (described by Willson et al., Endocrinology, 138(9), 3901-3911, 1997) and indole derivatives (disclosed Miller and others in the European patent 0802183 A1) and TSE 424, developed by Wyeth Ayers (USA) and disclosed in Japan patent 10036347 (American home products corporation) and non-steroidal estrogenic derivatives disclosed in WO 97/32837.

You can use any SERM, used, as is required for the manifestation of efficiency, as recommended by the manufacturer. The appropriate dose is known in this field. Can be used with the accordance with the invention any other non-steroidal antiestrogen, commercially available; any connection with the activity, such SERM (example: you can use raloxifene).

SERM, in accordance with the invention, is preferably introduced at a dose of between 0.01-10 mg/kg of body weight per day (preferably 0.05 to 1.0 mg/kg) at 5 mg / day, especially 10 mg per day in two equally divided doses are preferred for a person with an average body weight when administered orally, or at a dose in the range of 0.003-3.0 mg/kg of body weight per day (preferably of 0.015-0.3 mg/kg) at 1.5 mg per day, especially 3.0 mg per day in two equal divided doses are preferred for a person with an average body weight at parenteral administration (i.e. intramuscular, subcutaneous or percutaneous introduction). Preferably SERM shall, together with pharmaceutically acceptable diluent or carrier, as described below.

Preferred bisphosphonates according to the invention include alendronate [disodium salt hydrate (4-amino-1-hydroxybutylidene)bisphosphonates acid], Merck provides Shape and Dohme under the trade name Fosamax; etidronate [(1-hydroxyethylidene)bisphosphonic acid 2,2'-aminobutanol], provides Procter and Gamble under the trade names Didrocal and Didronel; clodronate [disodium salt (dihlormetilen)bisphosphonates acid], provides Rhone-Poulenc Rorer under the trade name Bonefos or CH the Boehringer Mannheim under the trade name Astak and pamidronate (the disodium salt of 3-amino-1-hydroxypropylamino)bisphosphonates acid), provides Geigy under the trade name Aredia. Risedronate (monosodium salt of 1-hydroxy-2-(3-pyridyl)ethylidene-bisphosphonates acid) is currently in clinical studies. Can be used in accordance with the invention, other bisphosphonates, commercially available, all at the manufacturer's recommended doses. Also predecessors of sex steroid hormones can be used in doses recommended by the prior art, preferably in doses that restore circulating levels to those in healthy male 20-30 years of age, or if adult women in pre-menopausal women.

In respect of all recommended doses here doctor should monitor individual response of the patient and accordingly to adjust the dose.

EXAMPLES

Example 1

MATERIALS AND METHODS

Animals

Rats female Sprague-Dawley [Crl:CD(SD)Br] were obtained at the age 44-46 days from Charles River Canada Inc. (St. Constant, Quebec) and divided by 2 in a cage under controlled conditions of light (12 h light/day light at 07:15 h) and temperature (22± 2°). Animals were fed Purina for rodents and tap water without restrictions. Animal studies conducted with equipment approved by the Canadian Council on animal care (NACC), in accordance with the directions of the NACC for the care and use of experimental animals.

Indukti the breast tumors using DMBA

Carcinoma of the breast induced after a single intragastric administration of 20 mg DMBA (Sigma Chemical Co., St. Louis, MO) in 1 ml of corn oil to females aged 50-52 days. After 2 months of twice weekly measurements of tumors. Recorded with calipers the two largest perpendicular diameter to establish the size of the tumor, as described (Asselin et al., Endocrinology 101: 666-671, 1977). Recorded the location, size and number of tumors.

Processing

Animals were randomly divided into groups, each containing 20 rats, except for the 40 animals in the control group. Animals were treated for 282 days as follows: (1) control of the media for both DHEA and EM-800; (2) EM-800 ((+)-7-pivaloyloxy-3-(4′ -pivaloyloxymethyl)-4-methyl-2-(4″ -(2′ ″ -piperidinyloxy) phenyl)-2H-benzopyran) (75 mcg orally once a day) in 0.5 ml of the suspension from 4% ethanol, 4% polyethylene glycol-600, 1% gelatin, 0.9% NaCl); (3) DHEA (10 mg percutaneous, once daily) in 0.5 ml of 50% ethanol, 50% propylene glycol and (4) as EM-800, and DHEA. Treatment was started 3 days before oral administration DMBA. EM-800 synthesized in the Department of medical chemistry laboratory applicants, while DHEA bought in Steraloids Inc., Wilton, NH.

Many of the control animals and some processed EM-800 or DHEA were euthanized by cervical vertebrae shift under anesthesia with isoflurane che is ez 6 months after administration of DMBA due to the large size of the tumors. The values of size and number of tumors in these rats at slaughter together with those defined at a later time intervals in surviving animals, used for analysis at a later date, frequency of tumors, the average number of tumors per rat-ofwholesale and the average size of tumors in animal-ofwholesale. The remaining animals (9 rats from control and 13-19 rats from each other group) continued to receive treatment for another 3 months, in order to observe long-term prophylactic efficacy of DHEA and EM-800 alone or in combination. Rats were killed after 279 days after administration of DMBA. Immediately removed the uterus, vagina and ovaries, freed from connective and adipose tissue and weighed.

Sampling and sample processing

At the end of the experience collected 24-hour urine samples from the first 9 rats from each group after their transplantation in metabolic chambers (Allentown Caging Equipment Co., Allentown, NJ). Took two urine samples and analyzed on different days for each animal, in order to minimize the influence of diurnal variations. Therefore, each of the value shown represents the average of two determinations, carried out in two different days. In tubes for collection of urine was added 0.5 ml of toluene to prevent evaporation of urine and bacteria growth and record the volume of urine. Blood from the body collected the ri for the death and allowed to clot at 4° With overnight before centrifugation at 3000 rpm for 30 minutes

Urine analysis and biochemical indices of blood serum

Fresh samples were used for determination of creatinine, calcium and phosphorus in the urine, and the activity of total serum alkaline phosphatase (ASF), cholesterol and triglycerides. These biochemical parameters were determined automatically on the chemical system Monarch 2000 (Instrumentation Laboratory Co. Lexington, MA) in benign laboratory practice. Hydroxyproline urine was determined as described (denphant et al., Clinica Chimica Acta 142: 145-148, 1984).

The definition of cash bones

Rats were anestesiologi VBR injection cetomimidae and diazepam in doses of 50 and 4 mg/kg body weight, respectively. The whole skeleton and the right femur was scanned using deenergizes x-ray absorbtiometry (DEXA; QDR 2000-7 S, Hologic, Waltham, MA)equipped with software Regional High Resolution. The field sizes were equal 28,110× 17,805 cm and 5.0× 1,902 cm, and the resolution was 0,1511× 0,0761 cm and 0,0254× 0,0127 cm, while the scanning speed was equal 0,3608 and 0,0956 mm/sec for the entire skeleton and femur, respectively. The mineral content in the bones (IUDs) and bone mineral density (BMD) of the whole skeleton, lumbar spine and femur were determined in the images obtained when the IC is planning.

Statistical analysis

Statistical significance was determined by test multiple rows Duncan-Kramer (Biometrics 12: 307-310, 1956). Analysis of the incidence of mammary tumors was performed according to the exact test of Fisher (Conover, Practical nonparametric statistics, 2ndEdition 153-170, 1980). Data are presented as mean values±the average error of the mean.

RESULTS

Effect on the development of breast carcinoma induced by DMBA

As shown in figure 1 in 95% of the control mice developed palpable tumors in the mammary gland through 279 days after administration of DMBA. Processing DHEA or EM-800 partially prevented the development of DMBA-induced breast carcinoma, and frequency, thus, decreased to 57% (p&λτ; 0.01) and 38% (p&λτ; 0,01), respectively. Interestingly, the combination of the two compounds resulted in significantly higher inhibitory effect than those obtained with each compound separately (p&λτ; 0,01 against only DHEA or only EAT 800). In fact, only two tumors that developed in the group of animals treated with both compounds, disappeared before the end of the experience.

Processing DHEA or EM-800 has reduced the average number of tumors per animal-ofwholesale from 4.7± 0.5 tumors in control animals to 3.4± 0,7 (insignificant) and 1.4± 0,3 (p&λτ; 0,01) tumors/animal (s) is, respectively, while in animals treated with both drugs, found no tumors in the end of the experiment (p&λτ; 0,01 against three other groups (figa). One of the two tumors, which later disappeared, there were 79 for 201 days after administration of DMBA, while other tumor even royalty with 176 on day 257. On FIGU shows that only DHEA or only EAT 800 reduced the average size of tumors per animal-ofwholesale from 12.8± 1.3 cm2at the end of the experience to 10.2± 2.1 cm2(insignificant) and 7.7± 1.8 cm2(not significant), respectively, while the combined treatment led to a 0-value (p&λτ; 0,01 against the other three groups). Two tumors that developed in the group of animals treated as DHEA and EM-800, did not rise more than 1 cm2. It should be noted that the real value of the mean square of tumors, as well as the average number of tumors per animal-ofwholesale in the control group should be higher than the values presented in figure 2, as many rats were forcibly euthanized before the end of the experiment due to excessive size of tumors. The values defined at the time of the killing, were thus included as such in the calculations made in later periods of time in order to minimize bias in the control group, which remained significantly higher than what about the comparison with other groups.

Effect on bone

Long-term percutaneous introduction of DHEA to rats-females has increased by 6.9% (p&λτ; 0.01), and to 10.6% (p&γτ; 0,05) and 8.2% (p&λτ; 0,01) bone mineral density (BMD) of the whole skeleton, lumbar spine and femur, respectively (table 2). On the other hand, no statistically significant changes in the animals which were injected EM-800. In addition, when simultaneously administered both compounds, the values obtained were comparable with one DHEA.

The DHEA treatment increased the activity of total alkaline phosphatase in serum (EWF) to 74% (p&λτ; 0,05), but had no effect on daily allotment of calcium and phosphorus in the urine and on the ratio of hydroxyproline to creatinine (table 3). On the other hand, the processing of EM-800 has reduced the ratio of hydroxyproline to creatinine in urine by 48% (p&λτ; 0,01), but had no significant impact on the daily allotment of calcium and phosphorus in the urine and activity EWF in the serum. The combination of DHEA and EM-800 resulted in increased activity of serum OSF (p&λτ; 0,01) similar to the effect achieved only DHEA, and reduced the ratio of hydroxyproline to creatinine 69%, a value significantly lower (p&λτ; 0,01)than that achieved only EAT 800. In addition, the combination of the two drugs significantly reduced the daily allocation of feces is tion and phosphorus, accordingly, by 84% (p&λτ; 0.01) and 56% (p&λτ; 0,01), while significant changes were not observed with each drug separately (table 3).

Effect on the levels of lipids in blood serum

Long-term administration of EM-800 led to lower levels of triglycerides and cholesterol in serum by 72% (p&λτ; 0.01) and 45% (p&λτ; 0,01), respectively, while long-term administration of DHEA reduced the levels of triglycerides in the serum of 60% (p&λτ; 0.01), and the concentration of cholesterol remained unchanged. Moreover, in animals treated with both EM-800 and DHEA, it was found that the concentration of triglycerides and cholesterol decreased by 42% (p&λτ; 0.01) and 52% (p&λτ; 0,01) (figure 3).

Example 2

Summary

In the breast androgens are formed from precursor steroids dehydroepiandrosterone (DHEA). Clinical data indicate that androgens exert an inhibitory effect on breast cancer. Estrogen, on the other hand, stimulate the development and growth of breast cancer. The applicants have studied the effect of DHEA or in combination with newly described a pure antiestrogen, EM-800, on the growth of tumor xenografts, developed from a line of cancer cells breast cancer human ZR-75-1 polyh mice with remote ovaries.

Mice were daily injected subcutaneously with 0.5 mg of estrone (an estrogen hormone) immediately after oophorectomy. EM-800 is administered orally, once daily (15, 50 and 100 μg).

Twice a day (total dose of 0.3, 1.0 or 3.0 mg) on the skin of the back was applied either DHEA or in combination with 15 mg daily oral dose of EM-800. Changes in the size of tumors in response to treatment was evaluated periodically in relation to measurements carried out on the first day. At the end of the experiments, tumors were removed and weighed.

In mice with remote ovaries treated only estrone, observed an increase in the size of tumors 9.4 times within 9.5 months compared to mice not treated with estrone. Introduction mice with remote ovaries EM-800 doses of 15, 50 or 100 µg adding estrone resulted in reduction of tumor size, respectively, 88%, 93% and 94%. On the other hand, DHEA doses of 0.3, 1.0 or 3.0 mg reduced final weight of the tumors, respectively, 67%, 82% and 85%. A comparable reduction in the size of tumors was obtained with EM-800 in its daily dose of 15 mcg oral with or without DHEA in various percutaneous doses.

DHEA and EM-800 independently inhibited the growth estrone-stimulated murine xenotransplantation tumors ZR-75-1 naked mice. The introduction of DHEA in certain doses did not change the inhibitory action EM-800.

MATERIALS AND METHODS

Cells ZR-75-1

Cancer cells of the breast is the ne ZR-75-1 were obtained from the American model of cultural collection (Rockville, MD) and were usually cultured as monolayers in RPMI medium 1640 with the addition of 2 mm L-glutamine, 1 mm sodium pyruvate, 100 IU of penicillin/ml, 100 μg streptomycin/ml and 10% fetal bovine serum in a humid atmosphere of 95% air/5% CO2at 37° as described (Poulin and Labrie, Cancer Res. 46: 4933-4937, 1986; Poulin et al., Breast Cancer Res. Treat. 12: 213-225, 1988). The cells were massirovala every week after treatment with a mixture of 0.05% trypsin: 0.02% EDTA (weight/volume). Cell cultures used for the experiments described in this report was obtained from 93 passage cell line ZR-75-1.

Animals

Female mice of alimony homozygotes Harlan Sprague-Dawley (nu/nu) (aged 28-42 days) received from HSD (Indianapolis, Indiana, USA). Mice were placed in cells with vinyl covers, provided with an air filter, and contained in the conditions of restricted pathogens. Cages, bedding and feed autoclaved before use. Water autoclaved, acidified to pH 2.8 and provided her animals without restrictions.

Perebivka cells

Mice were performed bilateral oophorectomy (OVX) a week before inoculation of tumor cells under anesthesia, conducted by intraperitoneal injection of 0.25 ml/animal of Avertin (amyl alcohol:0.8 g/100 ml of 0.9% NaCl and tribromoethanol:2 g/100 ml of 0.9% NaCl). In the logarithmic growth phase were collected in 1.5× 106cells ZR-75-1 after treatment of monolayer composition of 0.05% trypsin/0.02% EDTA weight/volume), suspended in 0.1 ml of culture medium containing 25% of Matrigel, and inoculable subcutaneously in both flanks of the animals, using a long 1-inch needle size 20, as described previously (Dauvois et al., Cancer Res. 51: 3131-3135, 1991). In order to facilitate the growth of tumors, each animal daily subcutaneous injections were administered 10 μg of estradiol (E2) on a medium consisting of 0.9% NaCl, 5% ethanol, 1% gelatin for 5 weeks. After the appearance of palpable tumors ZR-75-1 with calipers measured the diameter of the tumors and was selected for this experience mice having a diameter of tumors between 0.2 and 0.7, see

Treatment with hormones

All animals except the control group with remote ovaries OVX, received daily subcutaneous injections of 0.5 mg of estrone (E1) in 0.2 ml of 0.9% NaCl, 5% ethanol, 1% gelatin. In these groups DHEA was administered subcutaneously twice a day in doses of 0.3, 1.0 or 3.0 mg/animal, causing the volume of 0.02 ml on the skin of the back with the outside of the site of tumor growth. DHEA was dissolved in a mixture of 50% ethanol-50% propylene glycol. EM-800 ((+)-7-pivaloyloxy-3-(4'-pivaloyloxymethyl)-4-methyl-2-(4″ -(2′ ″ -piperidinyloxy) phenyl)-2H-benzopyran) was synthesized as described previously (Gauthier et al., J.Med. Chem. 40: 2117-2122, 1997), in the Department of medical chemistry, laboratory of molecular endocrinology, CHUL research center. EM-800 was dissolved in a mixture of 4% (vol/vol) ethanol, 4%(vol/vol) polyethylene glycol (PEG) 600, 1% (weight/volume) gelatin, and 0.9% (wt/vol) NaCl. The animals of these groups were treated daily with oral only EAT 800 doses of 15 μg, 50 μg or 100 μg or in combination with DHEA, while the group of animals with remote ovaries received only the vehicle (0.2 ml 4% ethanol, 4% PEG 600, 1% gelatin, 0.9% NaCl). Tumors were measured once a week using Vernier calipers. Registered two perpendicular diameters in cm (L and W) and calculated the area of tumor (cm2) according to the formula: L/2× W/2× π (Dauvois et al., Cancer Res. 51: 3131-3135, 1991). The area defined in the first day of treatment was taken as 100%, and changes in tumor size was expressed as percentage of the original area. In General, in the case of subcutaneous tumors is impossible to accurately estimate the three-dimensional tumor volume, therefore, determined only by the area of the tumor. Through 291 day (or 9.5 months) treatment of animals umertvlâl.

Categories of responses were evaluated as described (Dauvois et al., Breast Cancer Res. Treat. 14: 299-306, 1989; Dauvois et al., Eur. J. Cancer Clin. Oncol. 25: 891-897, 1989; Labrie et al., Breast Cancer Res. Treat. 33: 237-244, 1995). Summary: partial regression corresponds to the tumors, in which the regression was equal to or exceeded 50% of their original size, stable response refers to tumors that regressed less than 50% of their original size or progressed less than 50% of their original size, in thatduring as full regression refers to those tumors, not detected at the end of treatment. Progression refers to tumors that have progressed more than 50% compared to its original size. At the end of the experiment all animals were omertvlenie by decapitation. Immediately removed the tumor, uterus and vagina, freed from connective and adipose tissue and weighed.

Statistical analysis

The statistical significance of the impact of treatments on the size of the tumors was assessed using analysis of variance (ANOVA), evaluating the effect of DHEA, EM-800 and time, and repeated measurements on the same animals at the beginning and end of treatment (subjects intra-group factor). Repeated measurements at time 0 and after 9.5 months of treatment are randomized groups of animals. Thus, time was analyzed as intra-group effect, while two types of treatments were evaluated as intergroup effects. All interactions between the main effects were included in the model. The significance of factors and their interactions were analyzed using the entities within the group as errors. Data were transferred into logarithmic values. The hypotheses underlying the ANOVA, taken normal to the nature of the difference and homogeneity of changes.

Subsequent pairwise comparisons were performed using the test for Fisher least significant difference. About the main effects and impact of a treatment on the body weight and the weight of the organs was analyzed by standard bilateral ANOVA data. All anovas were performed using the SAS program (SAS Institute, NC, USA). The significance of the differences found using a 2-tail test with a total level 5%.

Categorized data were analyzed test Kruskall-Wallis for changes ordered by categories of response (complete response, partial response, stable response and progression of tumors). After a full assessment of the effects of treatments, sbrady results presented in table 4, were analyzed by summing the critical p-value for multiple comparisons. Exact p-values were calculated using StatXact (Cytel, Cambridge, MA, USA).

Data are expressed as averages ± standard error of the mean (SEM) from 12-15 mice in each group.

RESULTS

As shown in figure 4A, tumors of human ZR-75-1 increased 9.4 times for 291 days (9.5 months) in naked mice with remote ovaries, which were daily injected 0.5 µg subcutaneously estrone, while the control mice with remote ovaries, received only the carrier, the size of the tumors decreased by 36.9% from its original size during the experience.

Treatment with increasing doses of percutaneous DHEA caused a progressive inhibition of tumor growth of ZR-75-1, stimulated E1. Within 9.5 months of treatment, inhibition amounted to 50.4%76,8% 80,0% respectively with doses of DHEA 0.3 mg,1.0 mg and 3.0 mg daily for animal (figa). Consistent with the decrease in total tumor load, DHEA treatment resulted in a marked reduction in the average weight of tumors, remaining at the end of the experience. In fact, the average weight of the tumors decreased with 1,12± 0.26 g in control of the Nude mice with remote ovaries adding E1to 0.37± 0.12 g (p=0.005), 0,20± 0.06 g (p=0.001) and 0.17± 0.06 g (p=0,0009) in groups of animals treated daily with 0.3, 1.0, and 3.0 mg of DHEA, respectively (pigv).

The daily doses of 15 mcg, 50 mcg and 100 mcg antiestrogen EM-800 has reduced the size of tumors stimulated by estrogen, respectively, 87.5% (p&λτ; 0.0001)and 93,5% (p&λτ; 0,0001) and 94% (p=0,0003) (figa) compared with the size of tumors in control animals for 9.5 months.

Reducing the size of tumors, achieved with three doses of EM-800, did not differ significantly from each other. As shown in figv, the weight of the tumors at the end of the 9.5-month experience decreased with 1,12± 0.26 g in control mice with remote ovaries adding E1to 0.08± 0,03 g 0,03± 0.01 g and 0.04± 0.03 g in animals treated with EM-800 in daily doses of 15 μg, 50 μg and 100 μg, respectively (p&λτ; of 0.0001 for all doses of EM-800 vs. OVX with the addition of E1).

As mentioned above, the antiestrogen EM-800 in a daily oral dose of 15 mcg resulted in 87.5% of the inhibition of estrone-stimulated tumor growth at 9.5 months. Adding DHEA in t is ex used doses had no significant effect on the already marked decrease in the size of tumors, reached by the antiestrogen EM-800, in a daily dose of 15 mcg (pigv). Thus, the weight of the tumors significantly decreased with 1,12± 0.26 g in control mice with the addition of estrone to 0.08± 0.03 g (p&λτ; 0,0001), 0,11± 0.04 g (p=0.0002), 0,13± 0.07 g (p=0,0004) and 0.08± 0.05 g (p&λτ; 0,0001) in animals treated daily a dose of 15 μg of one antiestrogen or in combination with 0.3, 1.0 and 3.0 mg doses of DHEA, respectively (not observed any significant differences between the 4 groups) (pigv).

Also it would be interesting to assess which categories of responses obtained at the above treatments. Thus, the processing of increasing doses of DHEA reduced, although not to the level of statistical significance (p=0,088), the number of progressive tumors with 87.5% of the control OVX animals with the addition of estrone to the values of 50.0%, 53.3% and 66,7% in animals treated with DHEA in daily doses of 0.3, 1.0 and 3.0 mg (table 4). On the other hand, a complete response was increased from 0% in mice with the addition of estrone to 28.6%, 26.7% and 20.0% of the animals treated with percutaneous DHEA daily doses of 0.3, 1.0, and 3.0 mg of Stable response, on the other hand, was determined at the level of 12.5%, 21.4% and 13.3% of the control mice when adding E1and in three groups of animals treated with the above doses of DHEA, respectively. The control mice with remote ovaries share of full, partial and stable otvet the x reactions were determined at the level of 68.8%, 6,2% and 18.8%, respectively, while progression was found in only 6.2% of tumors (table 4).

Full response or disappearance of the tumor was achieved in 29.4%, 33.3%and 26.7% and 35.3% of the tumors in the animals that received only the antiestrogen EM-800 (p=0,0006) (15 μg) or in combination with DHEA at doses of 0.3 mg, 1.0 mg or 3.0 mg, respectively (table 4). On the other hand, the progression was found in 35.3%, 44,4%, 53.3% and 17.6% of tumors, respectively, in the same groups of animals. There is no statistically significant difference between groups treated only EAT 800 or in combination with DHEA.

Not observed a significant effect of DHEA treatment or EM-800 on body weight relative to the weight of the tumors. Treatment of OVX mice with estrone increased weight of the uterus from 28± 5 mg in OVX control mice to 132± 8 mg (p&λτ; 0,01), whereas increasing doses of DHEA caused a progressive but relatively insignificant inhibition of the stimulatory effect of estrone, which reached 26% (p=0,0008) at the highest used dose of DHEA. On the same figure can be seen that the weight of estrone-stimulated uterus decreased from 132± 8 mg in control mice with the addition of estrone to 49± 3 mg, 36± 2 mg and 32± 1 mg (p&λτ; of 0.0001 for all doses vs control) with daily oral administration of EM-800 in doses of 15 μg, 50 μg or 100 μg (total p value&λτ 0,0001), respectively. When fifteen micrograms (15 µg) EM-800 in combination with 0.3 mg, 1.0 mg or 3.0 mg daily doses of DHEA, the weight of the uterus was equal to, respectively, 46± 3 mg, 59± 5 mg and 69± 3 mg.

On the other hand, treatment with estrone increased the weight of the vagina with a 14± 2 mg in OVX animals until 31± 2 mg (p&λτ; 0,01), while adding DHEA had no significant effect. The weight of the vagina then decreased to 23± 1 mg, 15± 1 mg and 11± 1 mg after treatment, EM-800 in daily doses of 15 mcg, 50 mcg or 100 mcg, respectively (overall p value and pair p&λτ; of 0.0001 for all doses vs control). In combination with DHEA EM-800 in doses of 0.3 mg, 1.0 mg or 3.0 mg weight vagina was 22± 1 mg, 25± 2 mg and 23± 1 mg, respectively (statistically insignificant for all groups compared to 15 µg EM-800). It should be noted that at the highest used dose, and it is 100 micrograms per day, EM-800 has reduced the weight of the uterus in OVX animals with the addition of estrone to values not different from that of OVX control, while the weight of the vagina decreased to values below that of OVX control mice (p&λτ; 0,05). DHEA, possibly due to the androgenic effect, which partially removes the effect of EM-800 on the weight of the uterus and vagina.

E1- estrone; DHEA - dehydroepiandrosterone; OVX - remote ovaries

Example 3

Action is the preferred connection p is the invention on cholesterol levels in rats with removed ovaries

Animals and treatment

Used female rats Sprague-Dawley (Crl:CD(SD)Br) (Charles River Laboratory, St-Constant, Canada) 50-60 days of age with a weight of about 190 g at the time of oophorectomy. Animals acclimatized to the ambient conditions (temperature: 22± 3° C, humidity: 50± 20%, cycles of 12 h light - 12 h dark, the light at 07:15 h) within 1 week before surgery. Animals placed three in the cage and gave plenty of tap water and pelleted certified forage for rodents (Lab Diet 5002, Ralston Purina, St Louis, MO). The experiment was carried out with equipment approved by the Canadian Council on animal care (NACC), in accordance with the directions of the NACC for the care and use of experimental animals.

136 female rats were subjected oophorectomy under anesthesia with isoflurane on day 0 of experience and were randomly divided into 17 groups of animals for research, itemized below:

Group 1: with remote ovaries control

Group 2: with remote ovaries + EM-800 (0.01 mg/kg orally per day)

Group 3: with remote ovaries + EM-800 (0.03 mg/kg orally per day)

Group 4: remote ovaries + EM-800 (0.1 mg/kg orally per day)

Group 5: remote ovaries + EM-800 (0.3 mg/kg orally per day)

Group 6: remote ovaries + EM-800 (1 mg/kg orally per day)

Group 7: remote ovaries + EAT-01538 (0.01 mg/kg orally per day)

the group 8: remote ovaries + EAT-01538 (0.03 mg/kg oral, day)

Group 9: remote ovaries + EAT-01538 (0.1 mg/kg orally per day)

Group 10: remote ovaries + EAT-01538 (0.3 mg/kg orally per day)

Group 11: remote ovaries + EAT-01538 (1 mg/kg orally per day)

Group 12: remote ovaries + raloxifene (EAT-1105) (0.01 mg/kg orally per day)

Group 13: remote ovaries + raloxifene (EAT-1105) (0.03 mg/kg orally per day)

Group 14: remote ovaries + raloxifene (EAT-1105) (0.1 mg/kg orally per day)

Group 15: remote ovaries + raloxifene (EAT-1105) (0.3 mg/kg orally per day)

Group 16: remote ovaries + raloxifene (EAT-1105) (1 mg/kg orally per day)

Group 17: intact control.

Introduction preparations began on the 10th day of experience and held probe for oral administration once a day to 13-day experience. Suspension for injection were prepared in 0.4% methylcellulose and concentration brought up to the average body weight for the group registered on the 10th day of the experience, in order to introduce 0.5 ml of suspension for the rat. Approximately 24 h after the last injection, sustained hungry during the night the animals were killed by exsanguination by venipuncture of the abdominal aorta under anesthesia with isoflurane, and blood samples were processed to obtain serum. Removed the uterus, freed from the remaining fat and weighed.

Analysis ur the init of cholesterol and triglycerides in serum

The total concentration of cholesterol and triglycerides were determined using the Boehringer Mannheim Diagnostic Laboratory Systems.

Example 4

Androsten-3β ,17β -diol (5-diol) has inherent estrogenic activity. In addition, as a precursor to sex steroid hormones, it can turn into active androgens and/or estrogens in peripheral intracrine tissues. In order to assess the relative importance of androgenic and estrogenic components of action of 5-diol in relation to bone mass in rats at the age of 21 weeks were subjected oophorectomy and were treated with percutaneous once daily 2.5 or 12.5 mg only 5-diol or in combination with antiandrogens with flutamide (FLU, 10 mg, CC, once a day) and/or the antiestrogen EM-800 (100 µg, CC, once daily) for 12 months. Bone mineral density (BMD) was determined after 11 months of treatment. Oophorectomy (OVX) resulted in reduction of 12.8% BMD of the femur (p&λτ; 0,01), while the introduction of 5-diol in the highest dose was recovered by 34.3% for BMD of the femur that had been reduced during the 11 months after OVX (p&λτ; 0,01). Simultaneous introduction with the FLU completely removed stimulating effect of 5-diol on BMD of the femur, while the addition of EM-800 led to further stimulation by 28.4% compared with the action of one 5-diol. At the same time, the th introduction of 5-diol, FLU and EM-800 showed only the effect of EM-800 (27%), because the effect of 5-diol was completely blocked FLU. Comparable results were obtained with BMD of the lumbar vertebrae, although BMD of the lumbar vertebrae in OVX rats receiving 12.5 mg only 5-diol, 12.5 mg 5 diol+EM-800 or 12.5 mg of 5-diol+FLU+EM-800, recovered to values not significantly different from those of intact animals. Histomorphometrical analysis showed that the stimulatory effects of 5-diol on the amount of cancellous bone, trabeculae and inhibitory effect on the separation of secondary trabeculae of the spongy substance in metaphase proximal part of the tibia are removed FLU, but additionally strengthened EM-800. Noticeable stimulation of the activity of serum alkaline phosphatase obtained after treatment of 5-diola amounted to 57% (p&λτ; 0,01 against 12.5 mg only 5-diol) and removed with simultaneous introduction with the FLU. Treatment of 5-diola not had a statistically significant inhibitory effect on the ratio of hydroxyproline to creatinine in the urine. The highest dose of 5-diol caused a significant decrease of 23% (p&λτ; 0,01) serum cholesterol, while the addition of EM-800 reduced the concentration of serum cholesterol by 62% (p&λτ; 0,01). These data clearly show the stimulatory effect of 5-diol on bone formation, and on them is the basis we can assume, that despite the fact that 5-diol is a weak estrogen, its stimulatory effect on bone formation mainly through androgenic effect. In addition, an additive stimulatory effects of EM-800 and 5-diol on bone mass show mostserious action of the antiestrogen EM-800 in rats. Cholesterol-lowering activity of 5-diol and EM-800 can be interesting when used for prevention of cardiovascular diseases.

Example 6

An example of the synthesis of preferred compounds according to the invention

Synthesis of (S)-(+)-7-hydroxy-3-(4′ -hydroxyphenyl)-4-methyl-2-(4″ -(2′ ″ -piperidinyloxy)phenyl)-2H-1-Benaojan hydrochloride EAT-01538 (EM-652· Hcl)

Stage A: BF3·Et2O, toluene; 100° C; 1 h

Stage C: 3,4-dihydropyran, p-toluensulfonate acid monohydrate, ethyl acetate; 25° C in an atmosphere of nitrogen for 16 h and then crystallization in isopropanol.

Stage D, E and F:

1) piperidine, toluene, the unit Dean-stark boiling under reflux in a nitrogen atmosphere; 2) 1,8-diazabicyclo[5,4,0]undec-7-ene, DMF, boiling under reflux for 3 h; 3) CH3gl, THF, -20 to 0° and then at room temperature for 24 hours

Stage G, H: (1S)-(+)-10-camphorsulfonic acid, acetone, water, the Wal, room temperature, 48 hours

Stage LV: 95% ethanol, 70° C, then at room temperature for 3 days.

Stage HHR: recycling the mother liquor and washing the product with the stage NN, (S)-10-camphorsulfonic acid, boiling under reflux; 36 h, then room temperature for 16 hours

Stage I:

1) DMF aq., Na2CO3the ethyl acetate;

2) ethanol, diluted Hcl;

3) is water.

Synthesis of 2-tetrahydropyranyloxy-4-hydroxy-2′ -(4′ ″ -Tetra-hydroperoxides)acetophenone (4). A suspension of 2,4-dihydroxy-2′ -(4″ -hydroxyphenyl)acetophenone 3 (97.6 g, 0.4 mol) (Chemsyn Science Laboratories, Lenexa, Kansas) 3.4-dihydropyran (218 ml, 3,39 mol) and ethyl acetate (520 ml) was treated with monohydrate p-toluensulfonate acid (0.03 g, 0,158 mmol) at about 25° C. the Reaction mixture was stirred in nitrogen atmosphere without external heating for about 16 hours Then the mixture was washed with sodium bicarbonate solution (1 g) and sodium chloride (5 g) in water (100 ml). The phases were separated and the organic phase was washed with brine (20 ml). Each leaching fraction was again extracted with 50 ml ethyl acetate. All organic phases were combined and filtered through sodium sulfate.

The solvent (about 600 ml) was removed by distillation at atmospheric pressure and was added isopropanol (250 ml). Additional dissolve the ü (approximately 300 ml) kept at atmospheric pressure and was added isopropanol (250 ml). Additional solvent (approximately 275 ml) kept at atmospheric pressure and was added isopropanol (250 ml). The solution was cooled to about 25° with stirring, and after about 12 hours, the crystalline solid was filtered substance was washed with isopropanol and dried (116,5 g, 70%).

Synthesis of 4-hydroxy-4-methyl-2-(4′ -[2″ -piperidino]ethoxy)phenyl-3-(4′ ″ -tetrahydropyranyloxy)phenyl-7-tetrahydropyranyloxy (10). A solution of 2-tetrahydropyranyloxy-4-hydroxy-2′ -(4″ -tetrahydropyranyloxy)acetophenone 4 (1 kg, 2,42 mol), 4-[2-(1-piperidino)ethoxy]benzaldehyde 5 (594 g, 2.55 mol) (Chemsyn Science Laboratories, Lenaxa, Kansas) and piperidine (82,4 g, 0.97 mol) (Aldrich Chemical Company Inc., Milwaukee, Wis.) in toluene (8 l) was boiled under reflux in nitrogen atmosphere with the unit Dean-stark have not collected one equivalent of water (44 ml).

Toluene (6.5 liters) was removed from the solution by distillation at atmospheric pressure. Added dimethylformamide (6.5 liters) and 1,8-diazabicyclo[5,4,0]undec-7-ene (110,5 g, 0,726 mol).

The solution was stirred for approximately 8 h at room temperature for the isomerization of chalcone 8 in chromanone 9 and then added a mixture of ice water (8 l) and toluene (4 l). The phases were separated and the toluene layer washed with water (5 l). Combined washing water fraction was extracted with toluene (3× 4 l). The combined toluene is xtracta finally washed with brine (3× 4 l)was concentrated at atmospheric pressure to 5.5 l and then cooled to -10° C.

When continuous external cooling and stirring in an atmosphere of nitrogen was added a 3 M solution chloromethylene in THF (2.5 l, 7.5 mol) (Aldrich Chemical Company Inc., Milwaukee, Wis.), maintaining the temperature below 0° C. After all was added the Grignard reagent, continued external cooling and the mixture was allowed to warm to room temperature. The mixture was stirred at this temperature for 24 hours

The mixture was again cooled to about -20° and continuous external cooling and stirring was slowly added to a saturated solution of ammonium chloride (200 ml), keeping the temperature below 20°C. the Mixture was stirred for 2 h and then was added a saturated solution of ammonium chloride (2 l) and toluene (4 l) and stirred for 5 minutes, the Phases were separated and the aqueous layer was extracted with toluene (2× 4 l). The combined toluene extracts were washed with diluted hydrochloric acid until the solution became homogeneous, and then brine (3× 4 l). Toluene solution was finally concentrated at atmospheric pressure to 2 liters of This solution was directly used in the next stage.

The synthesis of the salt of (2R, S)-7-hydroxy-3-(4′ -hydroxyphenyl)-4-methyl-2-(4″ -[2′ ″ -piperidino]ethoxy)phenyl)-2H-1-benzopyran (1S)-10-camphorsulfonic acid is (± 12). To the toluene solution of 4-hydroxy-4-methyl-2-(4′ -[2″ -piperidino]ethoxy)phenyl-3-(4′ ″ -tetrahydropyranyloxy)phenyl-7-tetrahydropyranyloxy (10) was added acetone (6 l), water (0.3 l) and (S)-10-camphorsulfonic acid (561 g, 2,42 mole) (available from Aldrich Chemical Company Inc., Milwaukee, Wis.). The mixture was stirred in nitrogen atmosphere for 48 h, after this time the solid salt of (2R, S)-7-hydroxy-3-(4′ -hydroxyphenyl)-4-methyl-2-(4″ -[2′ ″ -piperidino]ethoxy)phenyl)-2H-1-benzopyran (1S)-10-camphorsulfonic acid (12) was filtered, washed with acetone and dried (883 g). This substance is used at the next (HH) stage without additional purification.

Synthesis of salts of (2S)-7-hydroxy-3-(4′ -hydroxyphenyl)-4-methyl-2-(4″ -[2′ ″ -piperidino]ethoxy)phenyl)-2H-1-benzopyran (1S)-10-camphorsulfonic acid (13, (+)-EM-652(1S)-CSA salt).

A suspension of a salt of (2R, S)-7-hydroxy-3-(4′ -hydroxyphenyl)-4-methyl-2-(4″ -[2′ ″ -piperidino]ethoxy)phenyl)-2H-1-benzopyran (1S)-10-camphorsulfonic acid ± 12 (759 g) in 95% ethanol was heated with stirring to about 70° until dissolved solid. The solution was allowed to cool to

room temperature with stirring, then was made the seed of a few crystals of salt (2S)-7-hydroxy-3-(4′ -hydroxyphenyl)-4-methyl-2-(4″ -[2′ ″ -piperidino]ethoxy)phenyl)-2H-1-benzopyran (1S)-10-camphor AlfaNova acid 13. The solution was stirred at room temperature generally within three days. The crystals were filtered off, washed with 95% ethanol and dried (291 g, 76%). The product yield was 94.2 percent and a purity of 98.8 per cent.

Synthesis of (S)-(+)-7-hydroxy-3-(4′ -hydroxyphenyl)-4-methyl-2-(4″ -(2′ ″ -piperidinyloxy)phenyl)-2H-1-benzopyran hydrochloride EAT-01538 (EM-652· HCl). A suspension of a salt of compound 13 (EM-652-(+)-CSA salt, 500 mg, 0,726 mmol) in dimethylformamide (11 μl, 0.15 mmol) was treated with 0.5 M aqueous solution of sodium carbonate (7.0 ml, 3.6 mmol) and was stirred for 15 minutes, the Suspension was treated with ethyl acetate (7.0 ml) and was stirred for 4 hours the Organic phase is then washed with aqueous saturated solution of sodium carbonate (2× 5 ml) and brine (1× 5 ml), dried over magnesium sulfate and was concentrated. The solution obtained pink foam (EM-652) in ethanol (2 ml) was treated with 2 N hydrochloric acid (400 μl, 0.80 mmol), was stirred for 1 h, treated with distilled water (5 ml) and was stirred for 30 minutes the resulting suspension was filtered, washed with distilled water (5 ml), dried in air and in high vacuum (65° (C) obtaining a cream powder (276 mg, 77%): fine, no white powder; scanning calorimetry: the beginning of the melting peak at 219°, Δ N=83 j/g;in methanol 10 mg/ml;1H NMR (300 MHz, the D 3OD) δ (ppm) of 1.6 (broad, 2H, H-4′ ″ ), of 1.85 (broad, 4H, H-3″ ″ and 5″ ″ ), 2,03 (s, 3H, CH3), 3,0 and 3.45 (broad, 4H, H-2″ ″ and 6″ ″ ), 3,47 (t, J=4.9 Hz, 2H, H-3′ ″ ), 4.26 deaths (t, J=4.9 Hz, 2H, H-2′ ″ ), of 5.82 (s, 1H, H-2), 6,10 (d, J=2.3 Hz, 1H, H-8), 6.35mm (DD, J=8,4, 2,43 Hz, 1H, H-6), 6,70 (d, J=8.6 Hz, 2H, H-3′ and H-5′ ), 6,83 (d, J=8.7 Hz, 2H, H-3″ and N-5″ ), 7,01 (d, J=8.5 Hz, 2H, H-2' and H-6'), 7,12 (d, J=8,4 Hz, 1H, H-5), from 7.24 (d, J=8.6 Hz, 2H, H-2″ and N-6″ );13With NMR (CD3D, 75 MHz) δ ppm 14,84, 22,50, 23,99, 54,78, 57,03, 62,97, 81,22, 104,38, 109,11, 115,35, 116,01, 118,68, 125,78, 126,33, 130,26, 130,72, 131,29, 131,59, 134,26, 154,42, 157,56, 158,96, 159,33. Elemental composition: C, H, N, Cl: theoretically: 70,51, 6,53, 2,84, 7,18%, found: 70,31, 6,75, 2,65, 6,89%.

Example 7

In vivo studies no bioavailability of prodrugs androst-5-ene-3β ,17β -diol

1) Principle

Experiments on the bioavailability of prodrugs predecessors of sex steroid hormones were performed on rats male Sprague-Dawley when determining concentrations in plasma connections after a single oral administration of the compounds.

a) Animals and processing

Rats male Sprague-Dawley [Crl:CD(SD)Br] body weight 275-350 g were obtained from Charles River Canada Inc. and placed at 2 in the cell during the acclimatization period and individually during the experience. Animals were kept in 12 h light:12 h dark (lights on 08:00). Animals received a certified food for rodents (Lab. Diet # 5002, pellets) and tap water without limitation the. Rats were kept hungry (giving access only to water), starting from the evening before the introduction.

Each connection that has been tested, has introduced three animals in the form of a suspension in 0.4% methylcellulose probe for oral administration at a dose of 150 mcmg/rat. From the jugular vein of the rats took one blood sample in the amount of ≈ 0.7 ml under anesthesia with isoflurane at 1, 2, 3, 4 and 7 h after injection. Blood samples are immediately transferred into a cooled microcontainer capacity 0.75 ml, containing EDTA, and kept in an ice bath until centrifugation at 3000 rpm for 10 minutes Quickly separated plasma (less than 50 minutes after blood collection. Then an aliquot of plasma in 0.25 ml was transferred into a borosilicate tube (13× 100) and quickly frozen on dry ice. Plasma samples were stored at -80° to determine the concentration of the precursors of sex steroid hormones in plasma GC-MS.

Results

Absorption through the mouth and AUCs shown in figures 12 and 13.

Examples of pharmaceutical compositions

Below are presented as examples and not limitations, several pharmaceutical compositions which are preferred active SERM EM-800 or EM-1538 and preferred active precursor of sex steroid hormones DHEA, EM-1304 or EAT-01474-D. you Can use other compounds according to the invention or a combination of them instead of (or the complement) EM-800 or EM-1538, DHEA, EM-1304 or EAT-01474-D. the concentration of the active ingredient may be varied within wide limits, as discussed here. The number and types of other ingredients that can be included, well known in this field.

Example 8

Tablet

IngredientWeight % (by weight of the entire composition)
EM-8005,0
Dehydroepiandrosterone (DHEA)15,0
Gelatin5,0
Lactose58,5
Starch16,5

Example 9

Gelatin capsules

IngredientWeight % (by weight of the entire composition)
EM-8005,0
Dehydroepiandrosterone (DHEA)15,0
Lactose water65,0
Starch4,8
Microcrystalline cellulose9,8
Magnesium stearate0,4

Sample kits

Below are presented as examples and not limitations, multiple sets, which are the preferred active SERM EM-800 or EM-1538 and preferred active predecessor chaff the steroid hormone DHEA, EM-1304 or EAT-01474-D. it is Possible to use other compounds by the image-acquisition or combination of them instead of (or in addition to) EM-800 or EM-1538, DHEA, EM-1304 or EAT-01474-D. the concentration of the active ingredient may be varied within wide limits, as discussed here. The number and types of other ingredients that can be included, well known in this field.

Example 10

SERM is administered orally, while the predecessor of sex steroid hormones prescribed percutaneous.

Song SERM for oral administration (capsules)

IngredientWeight % (by weight of the entire composition)
EM-8005,0
Lactose water80,0
Starch4,8
Microcrystalline cellulose9,8
Magnesium stearate0,4

The composition of the precursor of sex steroid hormones for local application (gel)

IngredientWeight % (by weight of the entire composition)
DHEA10,0
Capryl-capric triglyceride (Neeobee-5)5,0
Hexyleneglycol15,0
Transcutol (monomial is tilby ether of diethylene glycol 5,0
Benzyl alcohol : 2,0
Cyclomethicone (Dow corning 345)5,0
Ethanol (absolute)56,0
Hydroxypropylcellulose (1500 CP) (Klucel)2,0

Example 11

SERM and predecessor of sex steroid hormones administered orally.

The composition of non-steroidal antiestrogen for oral administration (capsules)

IngredientWeight % (by weight of the entire composition)
EM-8005,0
Lactose water80,0
Starch4,8
Microcrystalline cellulose9,8
Magnesium stearate0,4

The composition of the precursor of sex steroid hormones for oral administration (capsules)

IngredientWeight % (by weight of the entire composition)
Dehydroepiandrosterone (DHEA)15,0
Lactose water70,0

Starch4,8
Pulp microcrystallites is th 9,8
Magnesium stearate0,4

You can use other SERM instead of EM-800 or EM-01538 in the above compositions, as well as other predecessors of sex steroid hormones DHEA instead, EAT-1304 or EM-01474-D. you Can include more than one SERM, or more than one predecessor, in this case, the total weight percentage is preferably the weight percentage for one predecessor or one SERM presented in the examples above.

The invention is described by way of preferred embodiments and examples, but is not limited thus. Specialists in this field will easily understand the broader applicability and scope of the invention which is limited only by the claims.

1. A method of treating or reducing the risk of acquiring a disease selected from the group consisting of endometrial cancer, uterine cancer, ovarian cancer, vaginal dryness and loss of muscle mass, by introducing a precursor of sex steroid hormones selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone, androst-5-ene-3β,17β-diol and 4-androsten-3,17-dione, and prodrug that is converted in vivo in any of the above precursors of sex steroid hormones, and a therapeutically effective amount of a selective modulator of the estrogen-receptor.

2. Pharmaceutical composition for treating or reducing the risk of acquiring a disease selected from the group consisting of atherosclerosis, osteoporosis, hypercholesterolemia, endometrial cancer, uterine cancer, ovarian cancer, vaginal dryness and loss of muscle mass, containing a) a pharmaceutically acceptable excipient, diluent or carrier; b) a therapeutically effective amount of at least one precursor of sex steroid hormones selected from the group consisting of androst-5-ene-3β,17β-diol, 4-androsten-3,17-dione, and prodrugs, which is converted in vivo in any of the above precursors of sex steroid hormones; and (C) a therapeutically effective amount of at least one selective modulator of estrogen receptors.

3. Set to treat or reduce the risk of acquiring a disease selected from the group consisting of atherosclerosis, osteoporosis, hypercholesterolemia, endometrial cancer, uterine cancer, ovarian cancer, vaginal dryness and loss of muscle mass, comprising a first container containing a therapeutically effective amount of at least one precursor of sex steroid hormones selected from the group consisting of androst-5-ene-3β,17β-diol, 4-androsten-3,17-dione and any prodrugs, which in vivo is converted to any of you who ukazannyh predecessors, and further comprising a second container containing a therapeutically effective amount of at least one selective modulator of estrogen receptor.

4. The method according to claim 1, where addition is administered a therapeutically effective amount of bisphosphonates.

5. The kit according to claim 3, comprising at least one additional container, which contains a therapeutically effective amount of at least one of bisphosphonates.

6. The pharmaceutical composition according to claim 2, where the composition further includes a therapeutically effective amount of at least one of bisphosphonates.

7. The method according to claim 1, further comprising introducing a therapeutically effective amount of progestin.

8. The method, pharmaceutical composition or kit according to one of the preceding paragraphs, where the predecessor is not 4-androsten-3,17-dione.

9. The method, pharmaceutical composition or kit according to one of preceding claims 1 to 8, wherein the selective modulator of estrogen receptor has a molecular formula with the following characteristics: (a) two aromatic rings separated by 1-2 intermediate carbon atoms, both aromatic rings are unsubstituted or substituted by a hydroxyl group or a group that convert in vivo to hydroxyl; (b) a side chain having an aromatic ring and a function treting the amine or its salt.

10. The method, pharmaceutical composition or kit according to claim 9, where the side chain is selected from the group consisting of

11. The method, pharmaceutical composition or kit according to claim 9, where two aromatic rings are both phenyl, and where the side chain is a fragment selected from the group consisting of Metin, methylene -,- O and-S, aromatic ring, and the function of the tertiary amine and its salts.

12. The method, pharmaceutical composition or kit according to claim 9, where the selective modulator of estrogen receptor selected from the group consisting of derived benzothiophene, derived triphenylethylene derived indole derivative benzopyran and derived centchroman.

13. The method, pharmaceutical composition or kit according to claim 9, where the selective modulator of estrogen receptors is derived benzothiophene the following formula:

where R1and R2independently selected from the group consisting of hydrogen, hydroxyl and groups that convert in vivo to hydroxyl;

R3and R4independently selected from the group consisting of C1-C4the alkyl or R3and R4and the nitrogen to which they are attached, together are any structure selected from the group consisting of pyrrolidino, dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, PI is uridine, hexamethylenimine, morpholine;

And selected from the group consisting of-CO-,-SNON and CH2-;

In selected from the group consisting of phenylene, pyridylimino and-ciclos4H2N2-.

14. The method, pharmaceutical composition or kit according to item 13, where a selective modulator of estrogen receptor selected from the group consisting of raloxifene, 2-(4-methoxyphenyl)-3-[4-[2-(1-piperidinyl)ethoxy]phenoxy]benzo[b]thiophene-6-ol(arzoxifene) and 2-(4-hydroxyphenyl)-3-[4-[2-(1-piperidinyl)ethoxy]-phenoxy]benzo[b]thiophene-6-ol.

15. The method, pharmaceutical composition or kit according to claim 9, where the selective modulator of estrogen receptors is derived triphenylethylene the following formula:

where D is-co2CH2N(R3R4or-CH=CH-COOH (R3and R4or is independently selected from the group consisting of C1-C4the alkyl, or R3, R4and the nitrogen atom to which they are attached, together are a ring structure selected from the group consisting of pyrrolidino, dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholino);

E and K independently are hydrogen or hydroxyl;

J is hydrogen or halogen.

16. The method, pharmaceutical composition or kit according to one of the preceding PP, where selective modulator of estrogen receptors is tamoxifen, HE-tamoxifen, droloxifene, toremifene, idoxifene and (E, Z)-(3-[4-(1,2-diphenyl-1-butenyl)phenyl]-2-propanolol acid.

17. The method, pharmaceutical composition or kit according to claim 9, where the selective modulator of estrogen receptors is an indole derivative of the following formula:

where D is selected from the group consisting of-OCH2CH2N(R7R8, -CH=CH-CON(R7R8, -CC-(CH2)4-N(R7R8(R7and R8or is independently selected from the group consisting of C1-C6the alkyl, or R7, R8and the nitrogen atom to which they are attached, together form a ring structure selected from the group consisting of ring pyrrolidino, dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholino);

X is selected from the group consisting of hydrogen and C1-C6of alkyl;

R1, R2, R3, R4, R5and R6independently selected from the group consisting of hydrogen, hydroxyl, C1-C6the alkyl and groups that convert in vivo to hydroxyl.

18. The method, pharmaceutical composition or kit according to claim 9, where the selective modulator of estrogen receptors is derived centchroman the following formula:

where R1and R2independently selected from the group consisting of hydrogen, hydroxyl and groups that convert in vivo to hydroxyl;

R5and R6independently are hydrogen or C1-C6by alkyl;

D is-co2CH2N(R3R4(R3and R4or is independently selected from the group consisting of C1-C4the alkyl, or R3, R4and the nitrogen atom to which they are attached, together are a ring structure selected from the group consisting of ring pyrrolidino, dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholino).

19. The method, pharmaceutical composition or kit according p where derived centchroman is (3,4-TRANS-2,2-dimethyl-3-phenyl-4-[4-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl]-7-methoxypropan).

20. The method, pharmaceutical composition or kit according to claim 9, where the selective modulator of estrogen receptors has the following formula:

where R1and R2independently are hydrogen, hydroxyl or a group which is converted in vivo to hydroxyl;

Z is a divalent closing group;

R100is a divalent group that separates L from In-ring 4-10 intermediate atoms;

L is a divalent or three is alentinoj polar group, selected from the group-SO-, -CON-, -N< and-SON<;

G1selected from the group consisting of hydrogen, C1-C5hydrocarbon or a divalent group which connects with G2and L with the formation of a 5-7-membered heterocyclic ring, and halogenated or unsaturated derivatives of the foregoing;

G2or is absent, or selected from the group consisting of hydrogen, C1-C5hydrocarbon or a divalent group which connects with G1and L with the formation of a 5-7-membered heterocyclic ring, and halogenated or unsaturated derivatives of the foregoing;

G3selected from the group consisting of hydrogen, methyl and ethyl.

21. The method, pharmaceutical composition or kit according to claim 20, where Z is selected from the group consisting of-O-, -NH-, -S -, and-CH2-.

22. The method, pharmaceutical composition or kit according to item 21, where the compound is a derivative of benzopyran following General structure:

where D is-OCH2CH2N(R3R4(R3and R4or is independently selected from the group consisting of C1-C4the alkyl, or R3, R4and the nitrogen atom to which they are attached, together are a ring structure selected from the ring pyrrolidino, dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, piperidine is, hexamethylenimine, morpholine);

R1and R2independently selected from the group consisting of hydrogen, hydroxyl and groups, turn in vivo in hydroxyl.

23. The method, pharmaceutical composition or kit according to item 22, where the derived benzopyran is an optically active compound having an absolute configuration S on carbon atom 2, or its pharmaceutically acceptable salt, and a specified compound has a molecular structure

where R1and R2independently selected from the group consisting of hydroxyl and groups, turn in vivo to hydroxyl;

R3is a group selected from the group consisting of saturated, unsaturated or substituted pyrrolidinyl, saturated, unsaturated or substituted piperidino, saturated, unsaturated or substituted piperidinyl, saturated, unsaturated or substituted, morpholino, nitrogen-containing cyclic group, nitrogen-containing polycyclic group, and NRaRb(Raand Rbindependently are hydrogen, straight or branched C1-C6the alkyl, straight or branched C2-C6alkenyl and straight or branched C2-C6the quinil).

24. The method, pharmaceutical composition or kit according to item 23, where the compound or Sol is essentially does not contain (2R)-enantiomer.

25. The method, pharmaceutical composition or kit according to item 22, where a selective modulator of estrogen receptor selected from the group consisting of

EM-800

EM-01520

EM 01533

EM-01518

26. The method, pharmaceutical composition or kit according to item 22, where the derived benzopyrene is a salt of an acid selected from the group consisting of acetic acid, adipic acid, benzosulfimide acid, benzoic acid, camphorsulfonic acid, citric acid, fumaric acid, itestosterone acid, Hydrobromic acid, hydrochloric acid, designsolutions acid, oximately acid, lactic acid, maleic acid, methanesulfonic acid, metallernas acid, 1,5-naphthalenedisulfonic acid, nitric acid, palmitic acid, pavlinovoi acid, phosphoric acid, propionic acid, succinic acid, sulfuric acid, tartaric acid, terephthalic acid, p-toluensulfonate acid and valerianic acid.

27. The method, pharmaceutical composition or kit according p, where the acid is hydrochloric acid.

28. The way the headlight is aseptically composition or kit according to one of preceding claims 1 to 8, where selective modulator of estrogen receptors is:

EM-L538

29. The method according to claim 1, where the precursor of sex steroid hormones is dehydroepiandrosterone.

30. The pharmaceutical composition or kit according to claim 2, 3 or 8, where the connection, turn in vivo precursor of sex steroid hormones have a General formula

where X is selected from the group consisting of N, ROC, RCO2CHRaand RbSO2- (R selected from the group consisting of hydrogen, straight or branched (C1-C8) alkyl, straight or branched (C2-C18) alkenyl, straight or branched (C2-C18) quinil, aryl, furil, straight or branched (C1-C18) alkoxy, straight or branched (C2-C18) alkenylamine, straight or branched (C2-C18) alkyloxy, aryloxy, forelocks and or halogenated carboxylic analogues of the above; Rais hydrogen or (C1-C6) alkyl and Rbselected from the group consisting of hydroxyl (or its salt), methyl, phenyl and p-toluyl);

Y is a carbonyl oxygen or Y is β-OX (X has the same meaning as above) and αN.

31. The pharmaceutical composition of the sludge is set according to claim 2, 3 or 8, where the connection, turn in vivo precursor of sex steroid hormones selected from the group consisting of:

EM-1304

EM-01474-D

32. Pharmaceutical composition for treating or reducing the risk of acquiring a disease selected from the group consisting of atherosclerosis, osteoporosis, hypercholesterolemia, endometrial cancer, uterine cancer, ovarian cancer, vaginal dryness and loss of muscle mass, including pharmaceutically acceptable excipient, diluent or carrier, a therapeutically effective amount

EM-1538

and a therapeutically effective amount of androst-5-ene-3β,17β-diol.

33. The method according to 17, where a selective modulator of estrogen receptors is TSE 424.

34. A method of treating or reducing the risk of acquiring a disease selected from the group consisting of atherosclerosis, osteoporosis, hypercholesterolemia, by introducing a precursor of sex steroid hormones selected from the group consisting of androst-5-ene-3β,17β-diol and 4-androsten-3,17-dione, and any prodrug that is converted in vivo in the above-mentioned predecessors, and a therapeutically effective amount of a selective receptor modulator ect is Oganov.

35. The method according to clause 34, further comprising the stage of introducing a therapeutically effective amount of bisphosphonates.

36. The method according to clause 34, further comprising the stage of introducing a therapeutically effective amount of progestin.

37. The method according to one of the preceding p-36, where the predecessor is not 4-androsten-3,17-dione.

38. The method according to one of the preceding PP 37, where a selective modulator of estrogen receptors is tamoxifen, HE-tamoxifen, droloxifene, toremifene, idoxifene and (E, Z)-(3-[4-(1,2-diphenyl-1-butenyl)phenyl]-2-propanolol acid.

39. The method according to one of the preceding PP 37, where the predecessor of sex steroid hormones is androst-5-ene-3β,17β-diol.

40. The method according to PP 37, where the connection, turn in vivo precursor of sex steroid hormones have a General formula

where X is selected from the group consisting of N, ROC, RCO2CHRaand RbSO2- (R selected from the group consisting of hydrogen, straight or branched (C2-C18) alkyl, straight or branched (C2-C18) alkenyl, straight or branched (C2-C18) quinil, aryl, furil, straight or branched (C1-C18) alkoxy, straight or branched (C2-C18) alkenylamine, the nternet or branched (C 2-C18) alkyloxy, aryloxy, forelocks and or halogenated carboxylic analogues of the above; Rais hydrogen or (C1-C6) alkyl and Rbselected from the group consisting of hydroxyl (or its salt), methyl, phenyl and p-toluyl);

Y is a carbonyl oxygen or Y is β-OX (X has the same meaning as above) and α-N, and Y is a carbonyl oxygen, when X represents N.

41. The method according to PP 37, where the connection, turn in vivo precursor of sex steroid hormones selected from the group consisting of

EM-1304

EM-01474-D



 

Same patents:

FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to compounds designated for applying in photochemotherapy or diagnosis and indicated compounds represent 5-aminolevulinic acid aryl-substituted esters, their derivatives or pharmaceutically acceptable salts. In particular, invention provides preparing compounds of the general formula (I): R

22
N-CH2COCH2CH2CO-OR1 wherein R1 represents aryl-substituted C1-alkyl group, preferably C1-alkyl group substituted with non-heteroaromatic aryl wherein indicated group aryl is substituted group, and especially preferable this radical is substituted with one or more alkyl groups (for examples, (C1-C2)-alkyl), alkoxy- (for example, methoxy-) groups, fluorine, chlorine atoms, nitro- or trifluoromethyl groups; R2 being each of that can be similar or different represents hydrogen atom or alkoxycarbonyloxy-; indicated alkyl group is broken optionally with one or more groups: -O-, -NR3-, -S- or -PR3- wherein R3 represents hydrogen atom or (C1-C6)-alkyl group, and their salts for applying in diagnosis and photochemotherapy of injures and disorders of internal and external surfaces of body, and products and sets for realization of this invention also.

EFFECT: valuable medicinal properties of compounds.

18 cl, 17 dwg, 2 tbl, 3 ex

FIELD: medicine, pharmaceutical, cosmetic and food industry.

SUBSTANCE: method involves destruction and hydrolysis of alga taken among the group including: laminaria, zooster and focus followed by separating the cytoplasmatic fraction and addition of food acid polyvalent metal salt. Then correction of acidity is carried out to obtain pH = 5-7 followed by drying and conversion of fraction to form useful for storage by sterilization or freezing, or drying, or preserving. The food acid polyvalent metal an aqueous solution is added preferably in the amount 0.75% of preparation mass. Invention provides expanding functional capacity of the preparation with respect to stimulation of processes in healing wounds and burns, regeneration of post-traumatic damages, oncoprotecting and photo-sensitizing activity with respect to cancer cells at maximal absorption of light radiation in the region 630-770 nm.

EFFECT: improved producing method, valuable medicinal properties of preparation.

11 cl, 3 ex

FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

SUBSTANCE: the hemopoietic protein comprises the amino acid sequence of the formula: R1-L1-R1, R2-L1-R1, R1-R2 or R2-R1 wherein R1 represents the modified ligand flt-3; R2 represents the modified human IL-3, the modified or unmodified colony-stimulating factor. Modification of R1 is carried out by addition of N-end with C-end directly or through linker (L2) that is able to join N-end with C-end to form new C- and N-ends. The modified human IL-3 is prepared by replacing amino acids at positions 17-123. The human G-CSF is modified by exchange of amino acids. The hemopoietic protein is prepared by culturing cells transformed with vector comprising DNA that encodes the hemopoietic protein. The hemopoietic protein stimulates producing hemopoietic cells and this protein is used as a component of pharmaceutical composition used in treatment of humans suffering with tumor, infectious or autoimmune disease. Invention provides preparing multifunctional hemopoietic proteins eliciting the enhanced activity with respect to stimulation of hemopoietic cells and eliciting the improved physical indices. Invention can be used for preparing chimeric multifunctional hemopoietic proteins.

EFFECT: improved preparing and producing method, valuable medicinal properties of protein.

22 cl, 19 dwg, 18 tbl, 117 ex

FIELD: organic chemistry, chemical technology, medicine, oncology, pharmacy.

SUBSTANCE: invention relates to new derivative of taxane of the formula (I):

that elicits strong antitumor effect. Also, invention relates to intermediates substances, a method for preparing compound of the formula (I), a method for preparing 1,14-β-hydroxy-1,14-carbonate-baccatin III-derivatives substituted with isoserine residue at position 3 and to pharmaceutical composition based on compounds of the formula (I). Invention provides preparing new derivative of taxane that elicits higher activity and reduced toxicity as compared with paclitaxel.

EFFECT: improved preparing method, enhanced and valuable medicinal properties of compound.

10 cl, 7 tbl, 6 ex

FIELD: organic chemistry, chemical technology, medicine, biochemistry, pharmacy.

SUBSTANCE: invention relates to new derivatives of sulfonamides of the formula (I) or their pharmaceutically acceptable salts wherein R1 means -OH or -NHOH; R2 means hydrogen atom; R3 means alkyl, alkoxyalkyl, arylalkyl, pyridylalkyl or morpholinylalkyl; A means piperidyl or tetrahydrofuranyl; n = 0; E means a covalent bond; (C1-C4)-alkylene, -C(=O)-, -C(=O)O- or -SO2-; X means hydrogen atom, alkyl, aryl, arylalkyl, alkoxyalkyl, morpholinyl or tetrahydropyranyl; each among G and G' means -C(R5)=C(R5') wherein R5 and R5' mean hydrogen atom; M means the group -CH-; z means the group -(CR7R7')a-L-R8 wherein a = 0 and each among R7 and R7' means hydrogen atom; L means a covalent bond; R8 means halogen atom or alkoxy-group. Compounds of the formula (I) are inhibitors of metalloproteases and can be used for treatment of arthritis, cancer tumors and other diseases.

EFFECT: valuable medicinal properties of compounds.

15 cl, 7 tbl, 56 ex

FIELD: metalloorganic chemistry, chemical technology.

SUBSTANCE: invention relates to the improved method for preparing platinum complexes having the formula (Ia) or (Ib) given in the description. Method involves: 1a) the first stage wherein [PtA4]2- interacts with L under corresponding conditions in the first solvent to yield [PtA3(L)]-; 1b) the second stage wherein [PtA3(L)]- interacts with L' under corresponding conditions in the second solvent to yield cis-[PtA2(L')(L)]; 1c) in the case when Y represents halogen atom or hydroxy-group the third stage wherein cis-[PtA2(L')(L)] interacts with H2O2, Y2 or halogen containing oxidant to yield c,t,c-[PtA2Y2(L')(L)]; in the case when Y represents ester of carboxylate, carbamate or carbonate the forth stage wherein intermediate compound and wherein Y represents hydroxy-group obtained at the stage 1c) is functionalized with corresponding acylating agent; and 1d) in the case when A doesn't represent halide or differs from the parent halide the additional stage (stages) wherein at the first stage halide A of intermediate compound obtained at stage 1a), 1b), 1c) or 1d) is converted to another halide and new removing group (groups) A, such as monodentate hydroxy-, alkoxy-, carboxylate or bidentate carboxylate, phosphonocarboxylate, diphosphonate or sulfate wherein L, L' and Y have values in the description.

EFFECT: improved preparing method, expanded assortment of platinum-containing medicinal agents.

33 cl, 3 tbl, 9 dwg, 23 ex

FIELD: chemistry of metalloorganic compounds, medicine, oncology.

SUBSTANCE: invention relates to derivatives of platinum tetrachloride and to a method for their preparing also. Invention proposes compounds of the formula PtCl4 x 2 Li wherein Li represents N-(2-nitroxyethyl)nicotinamide or N-(2-nitroxyethyl)isonicotinamide, or nicotine hydroxamic acid, or isonicotine hydroxamic acid. Also, invention proposes a method for preparing these compounds that involves interaction of pyridine carboxylic acid nitroxyethylamides or hydroxamic acids, or their hydrochlorides with potassium hexachloroplatinate followed by isolation of the end product. Invention provides synthesis of the unknown early chelate platinum compounds that are physiologically active substances and can be used in medicinal practice instead cisplatin as effective anti-metastatic medicinal agents with low toxicity.

EFFECT: improved preparing method, enhanced and valuable medicinal properties of compounds.

2 cl, 3 ex

FIELD: medicine, oncohematology.

SUBSTANCE: the present innovation deals with treating elderly patients with chronic lympholeukosis accompanied with cardiovascular failure. The method deals with applying chemopreparations and cytoprotector. Moreover, 1 wk before the onset of chemotherapeutic therapy one should prescribe preductal at the dosage of 105 mg daily. At this background one should sample blood out of elbow vein at the volume of 200 ml into a vial with glugicir to centrifuge it, isolate plasma, divide into two portions, add into the 1st vial - cyclophosphan 600-800 mg/sq. m, vincristin 1.4 mg/sq. m, into the 2nd vial - adriamycin 50 mg/sq. m to be incubated for 30 min at 37 C and intravenously injected by drops for patients. Simultaneously, the intake of prednisolone should be prescribed at the dosage of 60 mg/sq. m since the 1st d and during the next 5 d and preductal at the dosage of 105 mg daily during a week, and then 2 wk more at the dosage of 60 mg daily. All the procedures should be repeated in above-mentioned sequence 4-6 times. The method enables to decrease toxic manifestations of chemotherapy while applying adequate dosages of cytostatics, anthracycline antibiotics, among them, at no great manifestations of their toxicity due to preductal's cardioprotective action.

EFFECT: higher efficiency of therapy.

1 ex, 5 tbl

FIELD: medicine, oncology.

SUBSTANCE: the method deals with polychemotherapy and is fulfilled by the following technique: it is necessary to carry out blood sampling in these patients at the quantity of 25 ml: during the 1st d of polychemotherapy one should incubate 5 ml autoblood with 50 mg/sq. m metothrexate, during the 2nd, 3d, 4th and 5th d one should incubate per 5 ml autoblood with 15 mg/sq. m bleomycin daily in thermostat at 37 C for 30 min, carry out application anesthesia and introduce chemopreparations upon autoblood into endonasal tumor component or alternate intratumor introduction of chemopreparations upon autoblood at introducing them into maxillary antrum if it is affected with tumor process. The method provides local concentration and decreased toxicity of chemopreparations due to their intratumor introduction upon autoblood.

EFFECT: higher efficiency of therapy.

1 ex, 1 tbl

FIELD: medicine, oncology, amino acids.

SUBSTANCE: invention relates, in particular, to the development of an antitumor preparation based on natural substances. Invention relates to an amino acid preparation comprising at least one modified essential amino acid obtained by treatment of amino acid by ultraviolet radiation (UV) at wavelength 250-350 nm for 12-80 h at temperature 15-30oC or with ozone at temperature 15-25oC. The modified amino acid has no toxicity for health cells. Also, invention relates to a method for preparing such preparation. Invention provides the development of an antitumor preparation based on modified amino acids and expanded assortment of antitumor preparations being without cytotoxicity for normal cells.

EFFECT: valuable medicinal antitumor properties of preparation.

8 cl, 4 tbl, 2 dwg, 4 ex

FIELD: medicine, gastroenterology, phytotherapy, pharmacy.

SUBSTANCE: invention relates to solid medicinal formulations, namely capsules "Gastrobiol-TSD". Gelatin capsule contain the natural pharmacologically active component - sea-buckthorn oil concentrate, cyclodextrin, vitamin U and magnesium oxide taken in the definite ratio of components given in the invention description. Invention provides preparing a medicinal formulation eliciting an anti-ulcer, analgetic and protective effect on mucosa effect being with minimal risk for development of adverse effect.

EFFECT: valuable medicinal properties of preparation.

1 tbl, 1 ex

FIELD: organic chemistry, heterocyclic compounds, medicine, pharmacy.

SUBSTANCE: invention relates to derivatives of heteroarylalkylpiperazine of the general formula (I):

wherein m = 1, 2 or 3; q means NH or oxygen atom (O); R1, R2, R3, R4 and R5 are taken independently among the group including hydrogen atom, (C1-C15)-alkyl, OR20 wherein R20 represents hydrogen atom; R6, R7 and R8 represent hydrogen atom; R9, R10, R11, R12, R13, R14, R15 and R16 are taken independently among the group including hydrogen atom, (C1-C4)-alkyl; or R9 and R10 in common with carbon atom to which they are joined form carbonyl group; R17 means heteroaryl that is taken among the group including indolyl, benzoxazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, pyridyl, benzopyrazinyl substituted optionally with 1-2 substitutes taken among the group including hydrogen atom, CF3 group, (C1-C8)-alkyl, phenyl, CON(R20)2. Compounds elicit property as a partial inhibitor of oxidation of fatty acids and can be used in therapy for protection of skeletal muscles against results of muscular or systemic diseases. Also, invention describes a pharmaceutical composition based on the claimed compounds.

EFFECT: valuable medicinal properties of compounds.

39 cl, 3 tbl, 25 ex

The invention relates to benzimidazole derivative of the formula (I)

or its pharmaceutically acceptable salt, where Rrepresents a group of formula -(ALK)q-R1where (ALK) represents alkyl, alkenyl or quinil, q is 0 or 1, R1represents a group of formula-CO2R2where R2is hydroxyalkyl, alkoxyalkyl or toolboxitem, Rrepresents a group of the formula

where o is 0 or 1, n is 0, 1 or 2, X represents N or CH, Y is O, NR11or CHR11where R11represents hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, carboxyl, or acyl, or a group of the formula -(alkyl)p-CN, -(alkyl)p-aryl, -(alkyl)p-O-aryl, -(alkyl)p-O-aralkyl, -(alkyl)p"heterocycle", -(alkyl)p-CO2"heterocycle" or -(alkyl-CO2)s-(alkyl)t-COR5and , in these formulas, R, s and t independently of each other 0 or 1, "heterocycle" represents a 5 the n heteroatom, represents a nitrogen, oxygen or sulfur, and which may substituted once or more than once, by substituents selected from the group consisting of halogen, alkyl and oxo, R5represents a hydroxy, alkoxy, hydroxy-C1-8-alkoxy, C1-8-alkoxyalkane, Tiltonsville, aryl, or aralkyl, or a group of the formula-NR6R7or-O-alkyl-NR6R7and , in these formulas, R6and R7independently of one another represent hydrogen or alkyl, and R14and R15independently of one another represent hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, carboxyl or acyl; or where R' is a group of formula -(ALK)q-R1where (ALK) represents alkyl, alkenyl or quinil, q is 0 or 1, R1represents fornillo group; and Rrepresents -(alkyl)m-CO2R8where m is 0 or 1, R8represents a group of formula -(alkyl)p-NR9R10where R is 0 or 1, and R9and R10together with the nitrogen atom to which they are attached, form a piperazinilnom group, possibly substituted by acyl

The invention relates to organic chemistry and can find application in medicine

The invention relates to the field of pharmacy and medicine and relates to solid dosage forms such as tablets "Apolar" anabolic and act-protective action based on the nature of the pharmacologically active component of the drone brood

The invention relates to pharmacy and medicine and relates to the development of new medicines

The invention relates to substituted cyclic aminoven compounds of formula (I)

< / BR>
where Ar represents thienyl, substituted pyridine, phenyl unsubstituted or substituted with halogen, hydroxy, alkoxy, C1-C4the alkyl, phenyloxy, NO2or phenyl; R1is NHOR2where R2is hydrogen; W is one or more hydrogen atoms; Y is independently one or more members of the group consisting of hydroxy, SR3, alkoxy, NR6R7where R6and R7independently selected from hydrogen, alkyl, pyridylethyl, SO2R8, COR9or R6and R7can be combined with the formation of the ring containing the nitrogen to which they relate, formulas

< / BR>
where Y' is CH2OH , SO2; R3represents hydrogen, alkyl, aryl, benzothiazolyl, pyrazinyl, N-methylimidazole; R8represents C1-C4alkyl, phenyl; R9represents hydrogen, alkyl, phenyl; Z is hydrogen; n = 1, and its optical isomer, diastereoisomer, or enantiomer, or its pharmaceutically acceptable salt

The invention relates to the pharmaceutical industry and relates to improved pharmaceutical preparations containing dehydroepiandrosterone (DHEA), enriched to polymorphic forms I or II, for therapeutic purposes
The invention relates to pharmaceutical industry

The invention relates to the field of medicine and relates to pharmaceutical compositions for the treatment of complex and coronary heart disease, myocardial infarction, myocardiodystrophy, rhythm disturbances associated with the use of cardiac glycosides

FIELD: medicine, therapy.

SUBSTANCE: simultaneously for 20 d one should prescribe calcium D3 two times daily in the morning and in the evening per 1 tablet or Beresh Plus drops. The quantity of drops for daily intake is equal to patients body weight in kg and they should be introduced in three stages during meals by drinking them up with sufficient amount of water. Application of the suggested method optimizes calcium exchange in patients with osteoporosis, enables to achieve earlier and more stable results in normalization of calcium content in bones by preventing, thus, osteoporotic fractures.

EFFECT: higher efficiency of pharmacological correction.

1 ex

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