Methods of treating and/or suppressing weight growth

FIELD: medicine.

SUBSTANCE: invention relates to a method of treating or reducing insulin resistance in susceptible warm-blooded animals, including people. Method includes introduction of a selective estrogen receptor modulator (SERM).

EFFECT: described is SERM combination with an amount of estrogen or a precursor of a sexual steroid hormone, selected from a group, consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-en-3b,17b-diol and compounds, converted in vivo into one of the said precursors or estrogen.

2 cl, 13 ex, 13 tbl, 8 dwg

 

This application claims the priority of provisional patent application U.S. No. 60/142407 filed July 6, 1999.

The technical field

This invention relates to a method of treatment and/or prevention of obesity (especially abdominal obesity) and to a method for the treatment or suppression suppression acquisition abnormal insulin resistance in susceptible warm-blooded animals, including humans. The methods include the introduction of compounds of General formula I, below, or pharmaceutical compositions based on them. In other embodiments, implementation methods include the introduction of a selective modulator of estrogen receptor (SMRA“, "SERM") in combination with a precursor of the sex steroid hormone.

Prior art

Obesity, a condition characterized by excessive body fat mass, is a well-known risk factor for the development of many diseases, such as cardiovascular disease, hypertension, diabetes, and breast cancer. In addition, personal appearance is an important part of a comfortable well-being of most people.

Conventional methods of treating obesity, such as a varied diet (including diet restriction in nutrition), a program that provides loss of excess weight, and exercise, many people find success the different degree of the final result. However, there remains a need for other methods for those who are experiencing dissatisfaction in the results when using methods (treatment) prior art or the methods used in addition to methods of prior art.

For the last time for treatment or prevention of obesity have been proposed some agonists/antagonists of estrogen: Raloxifene (Raloxifene) and related compounds in European patent application No. 0659423A1; agonists of estrogen with benzothiophene the core, in European patent application No. 0716855A2; 3,4-diphenylpropane in international application no PCT/DK96/00011; agonist/antagonist naphthylacetate in international application no PCT/IB95/00286.

It was also reported that Tamoxifen (Tamoxifen), another agonist/antagonist of estrogen, prevents the increase in body weight induced by sulpiride, female rats (Baptista et al., Pharmacol., Biochem. Behav. (1997), 57(1/2), 215-222). In addition, it was reported that tamoxifen mimics the action of estradiol on feed intake, body weight and body structure in rats (Wade et al., American Journal of discrimination 1993, 33(6), R1219-1223.)

In addition, DHEA (DHEA) has a beneficial effect in the treatment and/or prevention of obesity. In adult rats, Sprague-Dawley Schwartz (see Kent, Geriatrics 37: 157-160, 1982) observed a decrease in body weight from 600 to 550 g under the action of DHEA without affecting food intake. Schwartz (Cancer 39:1129-1132, 1979) observed that mouse strain C3H treated with DHEA (450 mg/kg, 3 times per week), gain significantly less weight, and age faster than control animals, and have less fat and more active. The decrease in body weight was achieved without loss of appetite or food restrictions. In addition, DHEA can prevent weight gain in animals,susceptibility to obesity in adulthood (see Kent, Geriatrics 37: 157-160, 1982).

The introduction of DHEA lean rats Zucher led to a decrease in body weight despite an increase in food intake. Animals subjected to treatment had a lower fat layer as a whole, thereby indicating that DHEA increases food metabolism, which leads to less weight gain and reduced accumulation of fat (Svec et al., Proc. 2ndInt. Conf. Cortisol and Anti-Cortisols, Las Vegas, Nevada, USA, p. 56 abst., 1997).

It was found that obesity is suspended in Avymutant mice (Yen et al., Lipids 12: 409-413, 1977) and rats Zucker (Cleary and Zisk, Fed. Proc. 42: 536, 1983). Mouse lines SN subjected to treatment with DHEA, looked younger than the control (Schwartz, Cancer Res. 39: 1129-1132, 1979).

Abdominal fat is associated with metabolic risk factors for coronary heart disease breast cancer (Imbault et al., Metabolism 1999, 48 (3), 355 - 62; Ledoux et al. (CMAJ 1997, 157 Suppl.1; 46-53).

The invention

Accordingly, the purpose of this invention consists in obespechennosty adipose tissue, in particular abdominal fat.

Another purpose of this invention is to achieve reduction in the risk of coronary heart disease and other diseases and conditions for which the development of obesity and the emergence of excess adipose tissue are risk factors.

In one embodiment, the present invention is to develop a new method for the treatment or suppression of weight gain in susceptible warm-blooded animals, including humans, and this this method includes the introduction of the subject, in need of such treatment or suppression, a therapeutically effective amount, with or without a pharmaceutical diluent or carrier, at least one compound of General formula I:

The formula I

where R1and R2independently selected from the group consisting of hydrogen, hydroxyl, -OM (where M is selected from the group consisting of straight or branched C1-C4of alkyl, straight or branched C3-C4alkenyl, straight or branched C3-C4the quinil) and part turnin vivoin hydroxyl,

where G represents-H or-CH3and

where R3indicates the type (substituent)selected from the group consisting of pyrrolidinyl, piperidino, morpholino and NRaRb (where Ra and Rb represent, not avisio, hydrogen, straight or branched C1-C6alkyl, straight or branched C3-C6alkenyl and straight or branched C3-C6quinil).

A method of treating or inhibiting the development of obesity, comprising administration to a subject in need of such treatment or suppression, a therapeutically effective amount of the compound or its pharmaceutically acceptable salt following General formula:

where R1and R2independently selected from the group consisting of hydrogen, hydroxyl, -OM (where M is selected from the group consisting of straight or branched C1-C4of alkyl, straight or branched C3-C4alkenyl, straight or branched C3-C4the quinil) and part turnin vivoin hydroxyl,

where G represents-H or-CH3and

where R3denotes a Deputy selected from the group consisting of pyrrolidinyl, piperidino, morpholino and NRaRb (where Ra and Rb represent, independently, hydrogen, straight or branched C1-C6alkyl, straight or branched C3-C6alkenyl and straight or branched C3-C6quinil),

where R1or R2are not pivaloyloxy group.

In another embodiment, the selective modulator of receptor estrogen is whether its pharmaceutically acceptable salt is administered to reduce abdominal fat or reduce the accumulation of abdominal fat.

In another embodiment, for the treatment of obesity or suppression of weight gain in addition to the selective modulator of estrogen receptor (SMRA, SERM) introducing the precursor of the sex steroid hormone (e.g., dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3b,17b-diol). People aged fifty or over years, as I believe, responds well to combination therapy, probably due to the fact that the levels predecessor tend with age, it is undesirable to decrease.

Thus, in this aspect of the invention provides a method for the treatment or suppression of weight gain, including introduction to the subject, if necessary, such suppression or treatment, a therapeutically effective amount, with or without a pharmaceutical diluent or carrier, at least one of SMRA (SERM) and an effective amount of at least one precursor of sex steroid hormone selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3b,17b-diol and compounds, turnin vivoin any of the above-mentioned predecessors.

In another aspect the invention provides a method of treating or reducing the risk of development of insulin resistance, comprising introducing to a subject, in need of such treatment or reduction, those who piticescu effective amount, at least one SMRA. In some embodiments of the invention as part of a combination therapy is administered effective amount of at least one precursor of sex steroid hormone selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3b,17b-diol and compounds that convert in vivo to one or the other of them.

In another aspect the invention provides a kit for the treatment of obesity, having a first container that includes at least one SMRA, and a second container that includes at least one precursor of sex steroid hormone selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3b,17b-diol and compounds that convert in vivo to one or the other.

In addition, in one or more containers may be provided in a pharmaceutical excipient, carrier or diluent, and they can include preservatives and other additives known in the field. The above-mentioned excipients can be included with any active component used in any variant of the embodiment described here, various inventions.

Used here is a selective modulator of estrogen receptor (SMRA, SERM) represents the connection, cotoroaia directly either through its active metabolite functions as an antagonist of the estrogen receptor ("antiestrogen") in the breast tissue, in addition, this compound has astrogeddon effect on body fat, bone and cholesterol levels in the serum (i.e. reducing serum cholesterol). Nesteroidnyi compounds that function as antagonists of the estrogen receptor in vitro or in the breast tissue of a person or the breast tissue of the rat (in particular, if the connection acts as an antiestrogen in human breast cancer cells), probably function as SMRA (SERM). To nonsteroidal antiestrogens, which had been tested and was found to function as SMRA-s (SERMs), are EM-800, EM-652, EM-652.HCl (EM-01538), Raloxifene, Tamoxifen, Idoxifene, Toremifene, LY 353381, LY 335563, GW 5638 and Droloxifene (described in more detail below). SMRA-s (SERMs), in accordance with any embodiment of the invention, preferably administered at the same dose, as is well known in the field of application of these compounds as antiestrogens.

Without going into theoretical reasoning, it is believed that SMRA-s (SERMs), many of which preferably have two aromatic rings connected by one or two carbon atoms, presumably interact with the estrogen receptor through Viseu Omanthai part of the molecule, which is best recognized by the receptor. In addition, such SMRA-s have side chains that can selectively be antagonistic properties in breast tissue and endometrial tissue without any significant antagonistic properties in other tissues, particularly bone. Thus, SMRA-s can operate in accordance with the desire, as antiestrogens in breast and endometrium, showing unexpectedly and in accordance with the desire estrogenically activity in relation to the fat body.

In addition, the invention includes, in accordance with the desire, suppression of additional weight gain or, in accordance with the desire, providing the weight decrease, even if the mass corresponding to the norm, is not achieved.

Used herein, the term " obesity means an excess of adipose tissue, which leads to an increase in body mass. Methods of prevention and treatment according to the invention include inhibition of weight gain and inducing loss of body weight. The invention includes the treatment of obese people, reducing their weight up to (and supporting mass) corresponding to the normal values. In addition, the invention includes the prevention of obesity for people who are susceptible to developing this disease. Patients interested in this image is hetenyi, are those who have excess body weight (compared with the established medical standards), or people with a high risk of gaining excess weight.

In addition, in accordance with the invention SMRA can be used to lower the levels of triglycerides in the blood. For example, it is assumed that EM-800 (described here) is effective for this purpose.

In another variant embodiment for the invention, the new compounds and pharmaceutical compositions based on them.

A patient in need of such treatment or reduction of risk of occurrence of a specified disease or condition is any one who is diagnosed with this disease, or who is predisposed to the development of such disease. The invention particularly useful for individuals who due to heredity, environmental factors or other recognized risk factors have a higher risk to acquire the status, which relates to this invention, than the General population.

Except where otherwise noted, the preferred dose of the active compounds of this invention are identical as for use in therapeutic purposes, and prevention. The dose of each active component, discussed here, is identical, n is dependent on the disease, subject to the treatment (or prevention).

In those cases, when dealing with two or more different active agents as part of combination therapy(for example, enzyme inhibitor and antiandrogen), use a multitude of different compounds, not a single connection with a variety of activity.

Unless otherwise noted, the term "connection" and any associated molecular structure can include any possible stereoisomers, in the form of a racemic mixture or in optically active form.

Unless otherwise stated or where the context clearly doses mentioned herein refer to the weight of active compounds, including pharmaceutical excipients, diluents, carriers or other components, although such additional components include, in accordance with the desire, as shown in the attached examples. Any dosage form (capsule, tablet, injection or the like)commonly used in the pharmaceutical industry, suitable for use here, and the terms "filler", "diluent" or "media" include inactive components, which are usually included, along with active components, such dosage forms in the industry. For example, using conventional capsules, pills, interline-active coating, solid or liquid diluents Il the fillers, flavors, preservatives or other

In some embodiments, embodiments using prodrugs of the active ingredients discussed here (i.e. compounds that becomein vivoin active ingredients). As is well known in the pharmaceutical industry, many functional groups becomeinvivoin a functional group of the active compounds discussed here. See, for example, Chapter 5 "Design and Application of Prodrugs"in A Textbook of Drug Design & Development, Bundgaard & Larsen, Ed., Harwood Academic Publishers GmbH, Chur, Switzerland, 1991). Prodrugs can often provide better bioavailability, stability during storage and/or ease of access to relevant active compounds.

All active ingredients used in any of therapies discussed here, can be included in the formulation of pharmaceutical compositions which additionally contain one or more other active ingredients. Alternatively, they can be entered, each, separately, but at the same time so that the patient ultimately had elevated levels of each active component in the blood or benefited in a different ratio from each of the active components (or strategies) at the same time. In some preferred embodiments the embodiment of the invention, for example, one or more active components must be combined in the formula used in the same pharmaceutical composition. In other embodiments, the embodiment of the invention provides a kit that includes at least two separate container where the contents of the at least two separate containers, where the contents of the at least one container differs in whole or in part from content of at least one other container in relation to the active ingredients contained in them. In the combination therapies of the invention use two or more different containers. Combination therapy described here also include the use of one active component combinations to obtain drugs for the treatment (or prophylaxis) of disease and it's treatment or prevention in addition includes other active component or strategy combination.

Abdominal fat is considered to be other than General obesity of the body, and it can occur in the absence of obesity. Also, consider that abdominal fat is a higher risk factor for the occurrence of heart disease. Abdominal fat reacts positively to this invention.

The preferred SMRA-s of the invention, for example, SMRA s above formula 1, do not have unwanted estrogenic action in the endometrium, a very important improvement in comparison with therapies involving SEE the uh, used in some methods of the prior art.

SMRA-s used in the invention are believed to favorably reduce the levels of triglycerides in the blood and also insulin resistance.

In preferred embodiments, embodiments of the invention discussed here, the effect of SMRA increases DHEA or similar predecessor of sex steroid hormone.

Without delving into the details of theoretical explanation, based on one explanation for the synergistic action obtained by combining SMRA s and predecessors, can be at least partial differences in their mechanisms of action. For example, DHEA, apparently, increases food exchange, not by suppressing the appetite. EAT-652.l, SMRA probably suppresses food intake.

Brief description of drawings

Fig. 1. Shows the effect of the 35-week treatment depending on increasing doses(0,01, 0,03, 0,1, 0,3, 1 mg/kg, orally, once daily) SMRA s EM-800, raloxifene, tamoxifen and inactive enantiomer, EM-776 (enantiomer EM-800) on the content of total fat in ovariectomized rats. Data are expressed as mean ± S (standard error of the mean). **: P<0.01 experiment versus the corresponding control.

Fig. 2. Shows the effect of the 20-day treatment on weight gain (A), increased protein (B) and increase in fat (C) in the akt rats; ovariectomised rats; ovariectomized rats subjected to treatment SMRA EM-652.HCl, or estradiol. Data are expressed in grams (g) as mean ± S. * p < 0,05 vs intact group; †p < 0,05 vs. OVX + E2group (EM-652.HCl group only).

Fig. 3. Shows the effect of the 20-day treatment on total feed intake for the intact rats, ovariectomized rats and ovariectomized rats treated with EM-652.HCl, or estradiol. Data are expressed in grams (g).

Fig. 4. Shows the effect of the 20-day treatment on the energy of the body (organism) in the form of proteins (A) and the energy of the body as fat (B) for intact rats, ovariectomized rats and ovariectomized rats treated with EM-652.HCl, or estradiol. Data are expressed in kilojoules (kJ, Kj) as mean ± S. * p < 0,05 vs intact group; †p < 0,05 vs. OVX + E2group (only EM-652.HCl group).

Fig. 5. Demonstrates the effect of the 20-day treatment on the levels of serum insulin and serum levels of glucose for intact rats; ovariectomised rats and ovariectomized rats treated with EM-652.HCl, or estradiol. Data are expressed in nmol/l as mean ± S. *p < 0,05 vs intact group.

Fig. 6. Demonstrates the effect of the 20-day treatment on the characteristics of white adipose tissue: inguinal mass (A) and retroperitoneal (C) adipose TC is neither (data are expressed in grams (g) as mean ± S); the activity of lipoprotein lipase inguinal (In) and retroperitoneal (D) white adipose tissue (data expressed in µa/g of protein as mean ± S; * p < 0,05 vs intact group; †p < 0,05 vs. OVX + E2group (only for EM-652.HCl group), to intact rats; ovariectomised rats and ovariectomized rats treated with EM-652.HCl or estradiol.

Fig. 7. Demonstrates the effect of the 20-day treatment on the characteristics of interscapular brown adipose tissue (brown fat): (A) the mass of brown adipose tissue (data are expressed in grams (g) as mean ± S, * p < 0,05 vs intact group; †p < 0,05 vs. OVX + E2group (only for EM-652.HCl group); (C) protein content (data expressed as % mass tissue as mean ± S; * p < 0,05 vs intact group; †p < 0,05 vs. OVX + E2group (only for EM-652.HCl group) and (C) the activity of lipoprotein lipase (data expressed in µa/g protein).

Fig. 8. Demonstrates the effect of the 20-day treatment on the weight of the soleus muscle (data are expressed in grams (g) as mean ± S) and lipoprotein lipase (data expressed in µa/g of protein as mean ± S; * p < 0,05 vs intact group); for the intact rats, ovariectomized rats and ovariectomized rats treated with estradiol and EM-652.HCl.

Detailed description of the invention

Preferably the introduction of pharmaceutical compositions containing f is rmaceuticals acceptable carrier, the diluent or carrier and a therapeutically effective amount of a selective modulator of estrogen receptor having the following General formula I:

The formula I

where R1and R2independently selected from the group consisting of hydrogen, hydroxyl, -OM (where M is selected from the group consisting of straight or branched C1-C4of alkyl, straight or branched C3-C4alkenyl, straight or branched C3-C4the quinil) and part turnin vivoin hydroxyl;

where G represents-H or-CH3;

where R3indicates the type (substituent)selected from the group consisting of pyrrolidinyl, piperidino, morpholino and NRaRb (where Ra and Rb represent, independently, hydrogen, straight or branched C1-C6alkyl, straight or branched C3-C6alkenyl and straight or branched C3-C6quinil),

and the predecessor of the sex steroid hormone selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3b,17b-diol and compounds, turninvivoin one or the other.

SMRA the above formula I provides the unexpected advantage that little or no estrogenic actions on the endometrium in the profile is from SMRE s, known from the prior art.

Preferably, SMRA invention optically active compound and contained more than 50% of the stereoisomer having an absolute configuration S on carbon 2.

In addition, for reasons of stability and solubility in water (biological availability), it is preferable that SMRA invention the salt of the compounds of formula I and the acid is selected from the group consisting of acetic acid, adipic acid, benzosulfimide acid, benzoic acid, camphorsulfonic acid, citric acid, fumaric acid, idiscovered acid, Hydrobromic acid, hydrochloric acid, hydrochlorothiazidebuy acid, hydroxy-naphthoic 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-toluenesulfonic acid and valerianic acid.

One preferred compound of the invention is EM-800, disclosed in PCT/CA96/00097 (WO 96/26201). Molecular structure EM-800 is:

Another more preferred compound of the invention is the I EM-652.HCl (also called EM-01538):

EM-652.HCl provides additional advantages in comparison with other SMRA-AMI, such as EM-800, because it does not contain pivaloyl groups, which can raise the risk connected with decrease of serum levels of carnitine.

Other preferred SMRA-s of the invention include Tamoxifen ((Z)-2-[4-(1,2-diphenyl-1-butenyl)]-N,N-dimethylethanamine) (available from Zeneca, UK), Toremifene (available from Orion-it facilitates Pharmaceuticl, Finland, or Schering-Plough), Droloxifene and CP-336156 (CIS-1R-[4'-pyrrolidino-ethoxyphenyl]-2S-phenyl-6-hydroxy-1,2,3,4-tetrahydronaphthalen D-(-)-tartrate salt) (Pfizer Inc., USA, described in U.S. patent 5889042) (also called Lasofoxifene), Raloxifene (Eli Lilly and Co., USA), LY 335563 and LY 353381 (Eli Lilly and Co., USA, described in international publications WO 98/45287, WO 98/45288 and WO 98/45286), 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 described Shalmi et al. in international publications WO 97/25034, WO 97/25035, WO 97/25037, WO 97/25038; Korsgaard et al. WO 97/25036), GW5638 (described Willson at al., Endocrinology, 138(9), 3901-3911, 1997) and indole derivatives (disclosed Miller et al. EP 0802183A1) and TSE 424 and ERA 923, developed by Wyeth Ayerst (USA) and disclosed in Japan patent 10036347 (American home products corporation), and nesteroidnyi estrogen derivatives described in WO 97/32837. Other SMRA-s of the invention described in: WO 99/07377; WO 98/48806; EP 0823437A2; EP 08384641; EP 0835867A1, EP 0835868A1; EP 0792641A1; EP 0873992A1 and EP 0895989A1.

You can use any SMRA when you want to achieve effectiveness (as recommended by the manufacturer for the treatment and/or prevention of osteoporosis or breast cancer). The appropriate dose is known in this field. According to the invention can be used with any other commercially available non-steroidal antiestrogen. Can be used any compound having activity similar to SMRA-am (e.g., Raloxifene).

SMRA-s used in accordance with the invention are preferably administered in doses ranging from 0.01 to 10 mg/kg of body weight per day (preferably from 0.05 to 1.0 mg/kg), and when administered orally for the average human body weight, preferably 60 mg / day, especially 20 mg / day, or in doses ranging from 0.003 to 3.0 mg/kg of body weight per day (preferably between 0.015 to 0.3 mg/kg of body weight), and 20 mg per day, especially 10 mg per day, preferably for the average human body weight at parenteral administration (i.e. intramuscular, subcutaneous or percutaneous introduction). Preferably SMRA-s administered together with a pharmaceutically acceptable diluent or carrier, as set forth below.

Preferred precursors of sex steroid hormones are dehydroepiandrosterone (DHEA) (available from Diosynth Inc., Chicago, Illinois, USA), its poleca the STV (available from Steraloids, Wilton, New Hampshire, USA), 5-androsten-3b,17b-diol and its prodrugs, androst-5-ene-3b,17b-diol 3-acetate and androst-5-ene-3b,17b-diol dyamically (available from Steraloids, Wilton, New Hampshire USA).

androst-5-ene-3b,17b-diol 3-acetate

androst-5-ene-3b,17b-diol dyamically

The predecessor of the sex steroid hormone can be represented in the form of an alcoholic gel containing from 2.0 to 10% of the triglyceride Caprylic-capric acid (Neobee M-5); from 10 to 20% of hexyleneglycol; from 2.0 to 10% diethylene glycol nanometrology ether (Transutol); from 2.0 to 10% of Cyclomethicone (Dow Corning 345); from 1.0 to 2% benzyl alcohol and from 1.0 to 5.0% hydroxypropylcellulose (Klucel HF).

In addition, the carrier (or SMRA or predecessor) may include various additives commonly used in the pharmaceutical industry. For example, there can be fragrances, antioxidants, fragrances, gelling agents, thickeners, such as carboxymethylcellulose, surfactants, stabilizers, agents for softening, dyes, and other similar tools. When the active components are administered transdermal, application site on the skin should be changed in order to avoid excessive local concentration of the active ingredient and possible overstimulation skin and sebaceous glands androgenic m is the metabolites predecessor of sex steroid hormone.

In the pharmaceutical composition for oral administration of DHEA or other precursor is preferably present in a concentration in the range from 5 to 98% of the mass. relative to the total weight of the composition, more preferably from 50 to 98 wt. -%, in particular from 80 to 98% of the mass. Can be used one predecessor, such as DHEA, as the sole active ingredient, or alternatively, you can use a variety of precursors and/or their analogues (for example, a combination of DHEA, DHEA-S (DHEA-S), 5-diol, or a combination of two or more connections, turn thein vivoin DHEA, DHEA 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-diolin vivoand so the Level of DHEA in the blood is the ultimate criterion of an adequate dose, taking into account individual variation, due to differences in absorption and metabolism.

Preferably doctor should, especially at the beginning of treatment, to monitor the overall response of the individual patient and the levels of DHEA in serum (compared to the preferred serum concentrations, above), and to control the overall patient response to treatment, adjusting the dose, as necessary, in those cases when the metabolism of the patient data or the response to treatment becomes atypical.

Treatment in accordance with the invention is applicable for an indefinite period of time. Suggest that treatment with DHEA and/or 5-diola or other predecessor, it will be easy to maintain levels of DHEA within the range, similar to what is, by nature, in women before menopause (concentration in serum between 4 and 10 micrograms per liter) or which is, by nature, young adult males (concentration in serum between 4 and 10 micrograms per litre).

Connection-SMRA and/or the precursor of the sex steroid hormone can be introduced through the mouth and can be included in the formulation of tablets or capsules for oral administration together with conventional pharmaceutical excipients, for example, spray dried lactose, microcrystalline cellulose and magnesium stearate.

The active substance may be incorporated into tablets or dragee cores by mixing with solid, powdered substances-carriers, such as sodium citrate, calcium carbonate or dicalcium phosphate, and binders, such as polyvinylpyrrolidone, gelatin or cellulose derivatives, in addition, you can add lubricants such as magnesium stearate, sodium lauryl sulfate, "Carbowax" or glycol. Of course, in the case of forms for oral administration can add substances that improve the taste.

As additional forms you can use solid the capsule, for example, hard gelatin, and closed soft gelatin capsules, including razmestiteli or plasticizer, such as glycerol. Hard 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.

Possible forms in the form of a lotion, ointment, gel or cream, and should be carefully rubbed into the skin so that there was no clearly shows no excess, and the skin in the area of application should not be cleaned up until most of the drug will not penetrate into the skin, preferably at least for 4 hours and more preferably at least 6 hours.

You can use transdermal patch for delivery of the predecessor or SMRA in accordance with known methods. Usually it is used for a much longer period of time, for example from 1 to 4 days, but usually the active ingredient in contact with a lower surface area, allowing a slow and constant all the qu of the active component.

A number of transdermal drug-delivery systems, which were developed and used, suitable for delivery of the active component of the present invention. The rate of release is typically controlled by diffusion through the matrix or passage of the active component by regulating the membrane.

Mechanical aspects of transdermal devices known to be a rat (model), and they set forth, for example, in U.S. patents 5162037, 5154922, 5135480, 4666441, 4624665, 3742951, 3797444, 4568343, 5064654, 5071644, 5071657, the descriptions of which are incorporated herein as prior art. Additional materials prior art there are in the European patent 0279982 and the application for patent in the UK 2185187.

The device may be any of those generally accepted in the field types, including transdermal delivery device based adhesive matrix transdermal device on the basis of the tank. The device may include containing the drug matrix comprising fibers that absorb the active ingredient and/or the media. In the device type of the tank, the tank can be limited to a polymer membrane that is impermeable to the carrier and an active component.

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

To attach to the skin transdermal patch can be mounted on a surgical adhesive tape having a hole perforated in the middle. The above adhesive is preferably covered by a removable strip to protect it before use. Conventional material suitable for the removal, includes polyethylene and paper coated with polyethylene and preferably coated with silicone for easy removal. In the case of use of the device the strip intended for deletion is removed and the adhesive attached to the patient's skin. In U.S. patent 5135480, disclosure of which is included as prior art, Bannon et al. describe an alternative device having a non-adhesive means for securing the device to the skin.

In addition, you can use percutaneous delivery systems or delivery system through the mucous membrane of the present invention as a new and improved delivery system is cartonnage means for the prevention and/or treatment of obesity.

EXAMPLES of the EFFECTIVENESS of different INVENTIONS

Example 1

The effect of the 35-week treatment with the compounds of the invention on total body fat and body weight ovariectomised (OVX) rats.

Materials AND METHODS

Animals and treatment

Were used rat female Sprague-Dawley (Crl:CD(SD)Br) (Charles River Laboratory, St-Constant, Canada) at the age of ten to twelve weeks, weighing approximately 225-250 g at the beginning of treatment. Animals were subjected to acclimatization to environmental conditions (temperature: 22±3°C; humidity: 50 ± 20%; series photoperiod: 12 h light - 12 h dark, lights 07:15 h) within 1 week before beginning of the experiment. Animals were placed three in a cage and given free access to tap water and granulated certified food for rodents (Lab Diet 5002, Ralston Purina, St Louis, MO). The experiment was carried out on the equipment, approved by the Canadian Council on animal care in accordance with the CCAC guide to the care and use of experimental animals.

Two hundred seventy-six rats were statistically distributed in 23 groups of 12 animals each as follows: 1) intact control; 2) OVX (ovariectomized) control; 3) to 7) OVX + EM-800 (0,01; 0,03; 0,1; 0,3 or 1 mg/kg) ; 8) to 12) OVX + Raloxifene (0,01; 0,03; 0,1; 0.3 or 1 mg/kg); 13) 17) OVX + Tamoxifen (0,01; 0,03; 0,1; 0.3 or 1 mg/kg); 18) 22) OVX + EM-776 (0,01; 0,03; 0,1; 0,3 or 1 mg/kg); 23) OVX + variety the diol (E 2the implant). On day 1 of the study the animals of the respective groups were subjected to bilateral oophorectomy (OVX) under anesthesia with isoflurane. One implant from organosilicone elastomer with estradiol (E2) inserted subcutaneously in the dorsal region of each animal group 13. The implants were below the concentration of E2and size: E2(E2: cholesterol (1:100, mass./mass.)), 0.5 cm (length), 0.125 (0,314 cm) (outer diameter) and 0,062 inch (0,156 cm (inner diameter). During the experiment implants E2replaced once a month. Treatment of EM-800, Raloxifene, Tamoxifen and EM-776 or filler (4% ethanol, 4% polyethylene glycol-600, 1% gelatin and 0.9% such as NaCl) was started on day 2 of the study. The corresponding connection or only the filler was injected once a day a catheter for oral administration in the amount of 0.5 ml/rat for 37 weeks. After approximately 24 hours after the last dosing of animals deprived of food during the night, scored by exsanguination through the abdominal aorta under anesthesia with isoflurane.

The composition of fat body

After 35 weeks of treatment in individual rats under anesthesia under the action of izoflurana, scanning was assessed full structure of the body, as well as their right thigh, using the method of two-level x-ray absorptiometry (DEXA; QDR 450A, Hologic, Waltham, MA) and software scanning area of the body with high resolution. The size of the scanning field is used for the whole body, was 30,492×17,912 cm, the resolution was 0,1512×0,0640 cm, and the scan rate was $ 2,499 mm/sec. Was determined the composition of body fat of the entire body.

Statistical analysis

Data are expressed as mean ± SEM. Statistical significance was determined in accordance with the criteria Duncan-Kramer for multiple ranges (1956).

Results

Fig. 1 illustrates the effect of increasing daily oral doses of EM-800, raloxifene, tamoxifen, and EM-776 on body fat measuredin vivothe DEXA method. You can see that the EM-800 at the lowest dose of 0.01 mg/kg decreases by 78% OVX-induced stimulation of body fat, raloxifene was less active than the EM-800, while EM-776, enantiomer EM-800, no significant action. Table 1 presents data on the effect of EM-800 or raloxifene on body weight ovariectomised rats. Compared with the intact and ovariectomized control rats. In addition, you can see that small doses of EM-800 provides body weight, similar which can be achieved at significantly higher amounts of raloxifene (for example, 0.03 mg/kg EM-800 gives you the same results as 0.3 mg/kg raloxifene). Both fundamentally change induced ova what ectopia weight gain at much higher doses.

Table 1
GroupBody weight at autopsy, g
Intact control356 ± 16**
OVX control491 ± 16
OVX + EM-800 (0.01 mg/kg)385 ± 17**
“ (0.03 mg/kg)364 ± 10**
“ (0.1 mg/kg)369 ± 17**
“ (0.3 mg/kg)359 ± 7**
“ (1 mg/kg)368 ± 6**
OVX + Raloxifene (0.01 mg/kg)439 ± 13*
“ (0.03 mg/kg)451 ± 14*
“ (0.1 mg/kg)389 ± 14**
“ (0.3 mg/kg)367 + 9**
“ (1 mg/kg)342 ± 12**

Example 2

11-month combined treatment with the compounds of the invention

1. Test substances

1.1 Test connections:

DHEA: Available from teraloids Inc. Wilton, NH. Lot: H137;

EM-800 synthesized in UCPO Department of Lab. of Mol. Endo., CHUL.

1.2 Filler

a) Gelatin: Lab-Grade, ACP Chemicals Inc., Montreal, Qc. Lot# F0195

b) Ethanol: Commercial Alcohols Inc., Brampton, Ontario. Lot # 11296

c) a polyethylene Glycol-600 (PEG-600, PEG-600): Omega

Chemical Company Inc., Lėvis, Quebec. Lot#: 00-0117-EZ

d) Standard saturated salt solution: 0.9% sodium chloride irrigation USP. Abbott Laboratories Limited, St-Laurent, Qc.

e) Propylene glycol (1,2-Propandiol, PG (PG)):

Sigma Chemical Co., St-Louis, MO.

g) Filler 1: 4% EtOH - 4% PEG-600 - 1% gelatin - 0.9% NaCl-H2For subcutaneous injection.

h) Filler 2: 50% EtOH - 50% PPG for local use.

2. Characteristics of experimental animals:

2.1 Species: Rattus norvegicus

2.2 Line: Sprague-Dawley Rat (Crl:CD®(SD) BR VAF/Plus™)

2.3 Gender: female

2.4 Source: Charles River Canada Inc. 188 Lasalle Road St. Constant, Quebec 1-800-561-4975

2.5 Age at the beginning of dosing: rats G18-29 at the age of 16 weeks

2.6 Maintenance

a) Content: the Rats were kept for 2-3 birds in the cage in Shoe boxes. Each cell was equipped with a corresponding label that indicates the Protocol number, group number and number of the animal.

b) During the research environment in the room is usually kept under control (the specified conditions: temperature 20 to 25°C; humidity: 50±20%). Photoperiod (day length) is usually lighting 12 hours: 12 hours dark.

c) Feeding ration and water: Diet for rodent is in (pellets) and tap water were presented ad libitum .

2.7 the acclimatization Period: At least two weeks.

2.8 Randomization: Upon arrival, rats generally assign groups randomly.

2.9 Method of euthanasia: General anesthesia, induced by isoflurane.

3. Methods and experiment plan

1. Intact control 11 rats/group

2. OVX control only 44 rats

3. OVX + DHEA 12.5 mg

4. OVX + DHEA 12.5 mg + EM-800 (100 µg)

3.1 Preparation of media and products for testing

a) Obtaining EM-800:

EM-800 or both of them dissolved in EtOH:PEG-600 (1:1) under stirring at laboratory heating tile Fisher, then add 1% GNS to the desired volume.

b) Receiving DHEA:

DHEA dissolved in 50% EtOH, 50% of BCPs (PPG) under stirring.

3.2 Preparation of animals and treatment:

Animals are usually treated once per day, as specified in paragraph 6.1.

DHEA prepared in 50% EtOH, 50% of BCPs (PPG), is usually injected percutaneous (CK, P.C.) corresponding to the animal once a day (I.D., I.D.) in 0.5 ml of EM-800 is usually prepared in 4% EtOH, 4% PEG-600, 1% gelatin, 0.9% of such as NaCl and injected subcutaneously animal related groups once a day (P.K.(S.C.), I.D. (I.D.)in a volume of 0.5 ml. of Rats subcutaneously injected with 0.5 ml of the filler 1, if they are not treated subcutaneously how any test compound, and applied locally 0.5 ml of the filler 2, if they are not treated with 5-diola and not treated with DHEA.

3.3 Monitoring is occurring and to measure

a) Clinical observations:

Each rat is subjected to inspection at least once a day to detect any manifestations.

b) body Weight:

Rats are weighed at the beginning and end of the test report and every three months during treatment.

4. The results:

As follows from table 2, the effect of DHEA treatment with a daily dose of 12.5 mg is reduced by 87% stimulate body weight induced by oophorectomy (OVX). Introduction EM-800 with a daily dose of 100 mcg results in a synergistic effect and is accompanied by 140% inhibition of the stimulation of body weight caused by OVX.

Table 2
ProcessingThe total body weight (g)
The intact479,5±20,4
OVX567,4±25,7
OVX+DHEA (12,5g/kg/day/rat)490,8±24,1
OVX+DHEA (12.5 mg/day/rat)+EM-800 (100 μg/day/rat)444,5±20,5

Example 3

The effect of the 20-day treatment with EM-652.HCl on the parameters of body fat and body mass ovariectomised rats

MATERIALS AND METHODS

Animals and treatment

Were used CR is si-female line Sprague-Dawley (Crl:CD(SD)Br) (Charles River Laboratory, St-Constant, Canada), at the age of eight to ten weeks, weighing approximately 200-225 g at the beginning of treatment. Animals were subjected to acclimatization to environmental conditions (temperature: 22±3°C; photoperiod cycles: 10 h light - 14 h darkness, the light from 06:00 h) for 1 week before the start of the experiment. Animals were housed individually in cages stainless steel standard design and had free access to tap water and mixed enriched carbohydrate diet, consisting of (g/100 g): corn starch 31,2; dextrose 31,2 ; casein 20,0; corn oil 6,4; dl-methionine, 0.3; the vitamin mixture 1,0; mineral mix AIN-76 4.9 and fiber (cellulose) of 5.0. The experiment is carried out on the equipment approved by the Canadian Council on animal care in accordance with the requirements of the NACC for the care and use of experimental animals.

Forty rats were statistically distributed in four groups of 10 animals each as follows: 1) intact control; 2) OVX control; 3) OVX + EM-652.HCl (0.5 mg/rat, ~2.5 mg/kg); 4) OVX + estradiol (E2the implant). On day 1 of the study the animals of the respective groups were subjected to bilateral oophorectomy (OVX) under conditions of anesthesia under the action of izoflurana. In the dorsal region of each animal group 4 subcutaneously implanted one implant, made of organosilicone the CSOs plastic, with estradiol (E2). The implants, which were chosen in preliminary experiments to ensure that physiological levels of E2had the following concentrations of steroid and size: E2: cholesterol (1:50, mass.:mass.), 0.5 cm (length diluted steroid in the flexible tube from elastica), 0.125 (0,314 cm) (outer diameter flexible tubes from elastica) and 0,062 inch (0,156 cm (inner diameter flexible tubes from elastica). The implants of estradiol kept in 0.9% such as NaCl at 37°C over night before subcutaneous introduction of animals. The processing of EM-652.HCl, or only one filler (0.4% methylcellulose in water) was started on day 2 of the study. Connection or filler was given once a day via oral catheter in the amount of 0.5 ml/rat for 20 days. Body weight and feed consumption were recorded every 2 days.

Approximately 24 hours after the last dosing of animals deprived of food during the night, was anestesiologi ketamine-xylazine and the blood was collected cardiac puncture. Collected blood, and took a white and brown adipose tissue.

Measurement of body weight, feed consumption and increase energy, increase fat and measurement of protein content

Body weight, feed consumption and increase body energy, increase fat and protein content was determined according Deshaies t al. Am. J. Physiol., 1997; 273, E355-E362 (1997).

Measurement of tissue

Lipoprotein lipase activity was determined in accordance with the methodology Deshaies et al., Am. J. Physiol., 1997; 273, E355-E362 (1997).

Results

In figures 2A and you can see that the 20-day treatment with EM-652.HCl completely transforms a 4-fold increase of the body mass and 3-fold increase of fat, respectively, observed after oophorectomy, while this effect is seen to a lesser extent on the growth of the protein (figure 2B). Estradiol had a smaller impact. Total food consumption is presented in figure 3. The effect of the treatment has an effect on weight gain. Estradiol partly prevented due to OVX increased demand in food intake, while EATING 652.Cl was in this sense more effective than estradiol, reducing the intake of food to the value lower than the value of intact animals.

The main parameters of the energy balance are summarized in table 3. OVX increases the digestible energy consumption by 44%. Estradiol reduces energy consumption in OVX animals, but the total energy consumption remains at 17% higher than the total energy consumption in intact animals, while EM-652.HCl completely prevents OVX-induced increase in energy consumption. The increase in energy is proportional to the consumption of food. And again estradiolelisaut increase energy levels, not significantly different from levels in intact rats), however, EM-652.HCl is more effective (significantly different from estradiol).

Power efficiency increases with OVX reduced in OVX animals under the action of E2and further reduced under the action of EAT-652.Cl.

As can be seen in figures 4A and b, in the treatment of EATING 652.Cl completely transformed 30% increase in energy coming from protein, after oophorectomy, and 80% increase in the body's energy coming from fat. As shown in figure 5A, the development of insulin resistance is confirmed by the presence of hyperinsulinemia in starvation (which is usually proportional to the degree of insulin resistance) and manifested fasting hyperglycemia (reflection loss of efficacy of insulin to maintain normal glucose levels in the blood). As estradiol and EM-652.HCl, both, prevents insulin resistance induced by OVX (oophorectomy).

Figures 6A and 6C show that OVX increases the mass (quantity) inguinal and retroperitoneal adipose tissue, which reflect the observed change in the content of total body fat. This increase is completely eliminated by treatment of EATING 652.l. In the case of estradiol such action is significantly less. The same picture is presented in Figure 6B and 6D, which shows the activity of lipoprotein lipase. This is t the enzyme modulates intravascular hydrolysis of triglycerides and thereby the flow of fatty acids into fat reserves.

Brown adipose tissue is the main thermogenic effector in rodents. Oophorectomy different effect on the parameters of the interscapular brown adipose tissue and white adipose tissue. The mass of brown adipose tissue is increased two-fold (figure 7A) compared to the mass observed in the case of white adipose tissue. However, the protein content (figure 7B) and the activity of lipoprotein lipase activity (figure 7C) reduced by 33% and 30%, respectively, after OVX. With respect to these three parameters, the treatment of EM-652.HCl completely cancels the effect of oophorectomy.

The weight of the soleus muscle is usually a good indicator (index) of the status of the protein mass of the body. As expected, the weight of the soleus muscle, as shown in Figure 8, increases with oophorectomy (the increase in the demand for feed intake leads to increased deposition of energy in the form of fat and protein). Treatment as estradiol and EM-652.HCl prevented the increase in muscle mass. Lipoproteinlipase in the muscle is often directly related to insulin sensitivity (the higher the sensitivity, the higher lipoprotein lipase activity in muscle). OVX decreased activity of lipoprotein lipase activity in the soleus muscle, indirectly proving the development of insulin resistance in the muscle. As treatment with estradiol and treatment of EM-652.HCl prevents the reduction of lipoprotein lipase activity in muscle.

Table 3Energy balanceThe condition of the ovariesThe intactOVXProcessingnoE2EM-652Digestible energy consumption (kJ)47086758a5511and4479bGrowth energy (kJ)5321677and903278bApparent consumption (kJ)41765081and4609a4201bThe efficiency of food (%)10,224,1a15,95,9baother than intak the data,
p < 0,05bother than ovariectomised + E2,
p < 0,05

Digestible energy consumption is the total energy absorbed during the 20-day treatment (including unassimilated substances, such as fiber).

Increase energy represents the number of kilojoules, put in the form of fat + protein during the 20-day treatment.

Apparent consumption represents the amount of energy spent on metabolic needs and locomotion (calculated from energy consumption and increase energy). It is expected to be greater when the body mass without fat anymore (as, for example, in OVX animals).

Power efficiency is the efficiency with which the absorbed energy is stored as fat and protein (in kJ, put on 100 kJ absorbed).

Example 4

An example of the synthesis of preferred compounds of the invention

Synthesis of hydrochloride of (S)-(+)-7-hydroxy-3-(4'-hydroxyphenyl)-4-methyl-2-(4"-(2"'-piperidinyloxy)phenyl)-2H-1-benzopyran EAT-01538 (EAT-652.l)

Scheme 1

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

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

Stages D, E and F:

(1) piperidine, toluene, device, Dean-stark, boiling under reflux in a nitrogen atmosphere; (2) 1,8-diazabicyclo[5,4,0]undec-7-ene, DMF (DMF), boiling under reflux for 3 hours;

(3) CH3MgCl, THF (THF), -20° to 0°C and then at room temperature for 24 hours;

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

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

Stage HHR: Recycling the mother liquor and the washing stage LV.

(S)-10-camphorsulfonic acid, boiling under reflux; 36 hours, then room temperature for 16 hours.

Stage I:

(1) DMF (DMF) aq., Na2CO3the ethyl acetate;

(2) ethanol, diluted Hcl;

(3) is water.

Synthesis of 2-tetrahydropyranyloxy-4-hydroxy-2'-(4"-tetrahydropyranyloxy)acetophenone (4)

A suspension of 2,4-dihydroxy-2'-(4"-hydroxyphenyl)acetophenone 3 (97.6 g, 0.4 mole) (available from Chemsyn Science Laboratories, Lenexa, Kansas) 3.4-dihydropyran (218 ml, 3,39 mol) and ethyl acetate (520 ml) is treated with monohydrate p-toluenesulfonic acid (0.03 g, 0,158 mmol) at approximately 25°C. the Reaction mixture was stirred in nitrogen atmosphere without external heating for about 16 hours. The mixture is then washed with sodium bicarbonate solution (1 g) and sodium chloride (5 g) in water 100 ml). Share phase and the organic phase is washed with saturated salt solution (20 ml). Each rinse again extracted with 50 ml ethyl acetate. All organic phases are combined and filtered through sodium sulfate.

The solvent (about 600 ml) is removed by distillation at atmospheric pressure and add isopropanol (250 ml). Added solvent (300 ml) is distilled off at atmospheric pressure and add isopropanol (250 ml). Added solvent (approximately 275 ml) is distilled off at atmospheric pressure and add isopropanol (250 ml). The solution is cooled at approximately 25°C with stirring, and after about 12 hours, filtered crystalline solid, 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"-Tetra-hydroperoxides)acetophenone 4 (1 kg, 2,42 mol), 4-[2-(1-piperidino)ethoxy]benzaldehyde 5 (594 g, 2.55 mol) (available from Chemsyn Science Laboratories, Lenexa, Kansas) and piperidine (82,4 g, 0.97 mole) (available from Aldrich Chemical Company Inc., Milwaukee, Wis.) in toluene (8 l) was refluxed in nitrogen atmosphere in the apparatus of the Dean-stark until then, until you collect one equivalent of water (44 ml).

From the solution by distillation at atmospheric pressure to remove the toluene (6.5 liters). Add dimethylformamide (6.5 liters) and 1,8-diazabicyclo[5,4,0]undec-7-ene (110,5 g, 0,726 mol). The solution is stirred for about 8 hours at room temperature to isomerizate Halcon 8 in chromanone 9 and then add in a mixture of water and ice (8 l) and toluene (4 l). The phases are separated and the toluene layer washed with water (5 l). The combined aqueous washings extracted with toluene (3×4 l). In conclusion, the combined toluene extracts are washed with saturated salt solution (3×4 l), concentrated under 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 of methylmagnesium in THF (THF) (2.5 l, 7.5 mole) (available from Aldrich Chemical Company Inc., Milwaukee, Wis.), maintaining the temperature below 0°C. Upon complete addition of the Grignard reagent external cooling is removed and the mixture allow to warm to room temperature. The mixture is stirred at the same temperature for about 24 hours.

The mixture is again cooled to about -20°C and in continuous outer cooling and stirring, slowly add a saturated solution (200 ml) of ammonium chloride, keeping the temperature below 20°C. the Mixture is stirred for 2 hours and then add saturated solution (2 l) of ammonium chloride and toluene (4 l) and stirred for five minutes. The phases are separated the aqueous layer was extracted with toluene (2×4 l). The combined toluene extracts are washed with dilute hydrochloric acid until then, until the solution becomes homogeneous, and then washed with saturated salt solution (3×4 l). Finally, the toluene solution was concentrated at atmospheric pressure to 2 L. the resulting solution was used directly in the next stage.

The synthesis of the salt of (1S)-10-camphorsulfonic acid and (2R,S)-7-hydroxy-3-(4'-hydroxyphenyl)-4-methyl-2-(4"-[2"'-piperidine]ethoxy)phenyl)-2H-1-benzopyran (±12)

To a solution of 4-hydroxy-4-methyl-2-(4'-[2"-piperidino]ethoxy)phenyl)-3-(4"'-tetrahydropyranyloxy)phenyl-7-tetrahydropyranyloxy (10) in toluene 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 is stirred under nitrogen atmosphere for 48 hours and at the end of this time, filtered off solid, salt of (1S)-10-camphorsulfonic acid and (2R,S)-7-hydroxy-3-(4'-hydroxyphenyl)-4-methyl-2-(4"-[2"'-piperidine]ethoxy)phenyl)-2H-1-benzopyran (12), washed with acetone and dried (883 g). The resulting substance use next (HH) stage without additional purification.

The synthesis of the salt of (1S)-10-camphorsulfonic acid and (2S)-7-hydroxy-3-(4'-hydroxyphenyl)-4-methyl-2-(4"-[2"'-piperidine]ethoxy)phenyl)-2H-1-benzopyran(13, (+)-EM-652(1S)-salt CSC(CSA))

Suspension Sol is (1S)-10-camphorsulfonic acid and (2R,S)-7-hydroxy-3-(4'-hydroxyphenyl)-4-methyl-2-(4"-[2"'-piperidine]ethoxy)phenyl)-2H-1-benzopyran 12 (759 g) in 95% ethanol is heated under stirring to about 70°C until until the solid has dissolved. Solution allow to cool to room temperature under stirring, and then make a seed - a few salt crystals (1S)-10-camphorsulfonic acid and (2S)-7-hydroxy-3-(4'-hydroxyphenyl)-4-methyl-2-(4"-[2"'-piperidine]ethoxy)phenyl)-2H-1-benzopyran 13. The solution was stirred at room temperature in the end for about three days. The crystals are filtered, washed with 95% ethanol and dried (291 g, 76%). De product is 94,2% and the purity is 98.8%.

Synthesis of hydrochloride of (S)-(+)-7-hydroxy-3-(4'-hydroxyphenyl)-4-methyl-2-(4"-(2"'-piperidinyloxy)phenyl)-2H-1-benzopyran EAT-01538 (EAT-652.Cl)

A suspension of compound 13 (EM-652-(+)-CSC salt, 500 mg, 0,726 mmol) in dimethylformamide (11 μl, 0.15 mmol) is treated with 0.5 M aqueous solution of sodium carbonate (7.0 ml, 3.6 mmol) and stirred for 15 minutes. The suspension is treated with ethyl acetate (7.0 ml) and stirred for 4 h Then the organic phase is washed with aqueous saturated sodium carbonate solution (2×5 ml) and saturated salt solution (1×5 ml), dried over magnesium sulfate and concentrated. The solution obtained pink foam (EM-652) in ethanol (2 ml) is treated with 2 N hydrochloric acid (400 μl, 0.80 mmol), stirred for 1 h, treated with distilled water (5 ml) and stirred for 30 minutes Received suspense is filtered, washed with distilled water (5 ml), dried in air and in high vacuum (65°C), receiving a cream coloured powder (276 mg, 77%): fine whitish powder; Scanning calorimetry: the beginning of the melting peak at 219°C, MON(DH, direct heating) = 83 j/g; [α]24D= 154° in methanol 10 mg/ml;1H NMR (300 MHz, CD3OD) δ (h/mn) 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,Hz, 2H, H-3"'), 4.26 deaths (t, J=4,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,Hz, 2H, H-3'and H-5'), 6,83 (d, J=8,Hz, 2H, H3 ' and H-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,Hz, 2H, H-2 and H-6");13With NMR (CD3OD, 75 MHz) d h/million 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 5 demonstrates the prevention of the increase of fat-induced LHRH-A, with EAT-652.l, the compounds of this invention, one or in combination with DHEA, a precursor hormone, intact female rats. You can see that 6-month treatment LHRH-A increases significantly, 58%, percentage of body fat of intact female rats, whereas if it is accompanied by the introduction of EAT-652.l or DHEA this increase was only 35% and 19% accordingly is O. In the case of a combination of both drugs no increase in fat was not observed after treatment of LHRH-A.

Example 6 presents the results of the prevention of obesity in female rats over a period of time of 20 days. Oophorectomy leads to an increase in the percentage of total fat (fat mass) and body mass retroperitoneal adipose tissue by 20% and 68%, respectively. These increases to be prevented, and even body fat and retroperitoneal adipose tissue is reduced with the introduction of EAT-652.l, estradiol, DHEA, or a combination of EM-652.l and DHEA or EAT-652.l and estradiol: for body fat on 46%, 46%, 59%, 56% and 45%, respectively, and 59%, 78%, 75%, 69% and 68% respectively for retroperitoneal adipose tissue. A similar experiment described in example 7. In this case, the prevention of obesity can be traced for 34 weeks, and then the percentage of body fat and retroperitoneal adipose tissue is approximately 60% increase in the oophorectomy. This increase is prevented by the introduction of ethinyl estradiol, raloxifene (EAT-1105), EAT-652.l (EM-1538), DHEA, or a combination of EM-652.l and DHEA.

In examples 8A (male) and 8B (female) evidence of the effectiveness of the invention in the prevention of obesity, as well as in the treatment of obesity. Lean and fatty rats-males and rats-females line Zucker was administered 2.5 mg/kg/day-652.l for 20 days. The model for the prevention (prophylaxis) is illustrated in lean rats, whereas the model for the treatment of obesity illustrated in already obese rats. Data on weight gain, lipoprotein lipase activity in white retroperitoneal fatty tissue and soleus muscle, and plasma concentrations of insulin, glucose, total cholesterol and triglycerides are presented in table 7 for males and in table 8 for females (see example 3; stat. the significance of these parameters).

The antiestrogen EM-652.l greatly reduced weight gain: 38% for lean rats-males and 35% for fatty rats (males), whereas the activity of lipoprotein lipase in retroperitoneal white adipose tissue and soleus muscle does not change in both sexes. Insulin in the plasma is reduced by 35%, 57% and 48% for skinny males, fat males and skinny females, respectively, while the fatty females who demonstrate the highest level of insulin, EAT-652.l no significant effect on insulin levels. Cholesterol in plasma, which is higher in the fatty group, also decreases with the introduction of EAT-652.l. Treatment of EATING 652.l no effect on serum glucose.

Example 9 illustrates the effect of EAT-652.l, DHEA or a combination of both on intact or ovariectomized the female-female rats treated with enriched sucrose and fat diet, the total weight is at, mass and activity of lipoprotein lipase in white adipose inguinal and retroperitoneal tissue, brown adipose tissue, soleus muscle and wide lateral hip muscles (VLM). In addition, data of insulin, glucose, total cholesterol, triglyceride and leptin in plasma, as well as data of cholesterol and triglycerides in the liver.

Example 10 illustrates the effect of the treatment of various selective modulators of estrogen receptor described in the literature (TSE424, Lasofoxifene, LY 353381 and Raloxifene), and EAT-652.l, the preferred compound of the invention, the weight gain and lepidopterology metabolism in rats. The test compounds were injected ovariectomised rats-females with oral catheter for 20 days (0.5 mg/rat for each connection) in 0.4% methylcellulose. As a comparison, the use of intact control, ovariectomized (OVX) control and OVX rats subjected to treatment with 17β-estradiol (E2). Body weight and feed consumption, which increase with oophorectomy, were significantly decreased in the treatment of test compounds, approaching the level observed for the intact controls. The mass and activity of lipoprotein lipase in white adipose inguinal and retroperitoneal adipose tissue was decreased by the above-mentioned treatment (except TSE 424 and LY 353381 for white adipose tissue and TSE 424 and Lasofoxifene for lipoprotein lipase activity inguinal tissue), while brown adipose tissue and muscle, apparently, does not give significant impact. You can see that all the tested compounds reduce significantly the concentration of plasma cholesterol and triglycerides, but have no effect on the glucose levels. Insulin in plasma decreased in the treatment of OVX rats EAT-652.l, TSE 424, Lasofoxifene, LY 353381 and Raloxifene.

In examples 11 and 12 is illustratedin vitrothe efficiency of known antiestrogens and compounds of this invention in estrogenzawisimy cell lines. The effect of antiestrogens on the activity of alkaline phosphatase in Ishikawa cells endometrial adenocarcinoma man presented in table 11 of example 10. Antiestrogenic activity presented in the last column in the form of the IC50expressed in nm inhibition of alkaline phosphatase stimulated by 1 nm E2while the characteristic estrogenic activity of the test compounds are given in the penultimate column as a percentage of the activity of alkaline phosphatase stimulated by 1 nm E2. You can see that the most active antiestrogens are EATING 652.l, LY 353381, Raloxifene, Lasofoxifene and TSE 424. Other at least ten times less active. However, among the most active compounds show some significant residual unwanted estroge the percent activity: LY 353381 (16%), Lasofoxifene (18%) and Raloxifene (13%). Thus, the above data indicate that the compounds of EM-652.HCl and TSE 424 not demonstrate significant estrogenic activity in Ishikawa cells. In example 12 comparison of EM-652.l offered as the preferred connection, and TSE 424 in the cells of MCF-7 breast cancer man demonstrates the advantage of the EM-652.l. Thus, this connection is more than four times more active than TSE 424 as an antiestrogen (EC50inhibition of 0.8 nm compared with 3.7 nm) (table 12).

In example 13 evaluation of the 20-day treatment, EAT-652.l, TSE 424 or Lasofoxifene on body weight, retroperitoneal adipose tissue, uterine weight and cholesterol levels ovariectomised female rats fed a commercial diet for rodents. Oophorectomy induces 17% and 19% increase in total body mass and the mass of retroperitoneal adipose tissue, respectively, while administration of 0.5 mg EAT-652.l, TSE 424 or Lasofoxifene prevents weight gain by 64%, 59% and 127%, respectively, and leads to lower values of the mass of adipose tissue than observed in intact control animals for all investigated compounds. Introduction EM-652.l and TSE 424 no effect on uterine weight compared to the OVX control animals. However, administration of 0.5 mg Lasofoxifene caused a 62% increase (p&g; 0,01) uterine weight, which completely transformed the simultaneous introduction of 2.5 mg EAT-652.l, thereby confirming the estrogenic activity of Lasofoxifene. Finally, induced by oophorectomy (OVX) increase in total serum cholesterol levels completely prevented the introduction of EAT-652.l, TSE 424 and Lasofoxifene and leads to significantly lower cholesterol values than observed in intact control animals.

Example 5

Action EM-652.l and DHEA, administered separately or in combination, on the accumulation of fat in the intact female rats, treated or not treated with LHRH-A

URMA-r-04-99

The purpose of this study is to determine the effect on the fat processing EM-652.HCl and dehydroepiandrosterone (DHEA) intact female rats, treated or not treated with the agonist of luteinizing hormone-releasing hormone (LHRH-A, the LHRH ethylamide diacetate). For this purpose intact rats-females, treated or not treated with LHRH-A (1 µg/rat)was administered daily EAT-652.l (2.5 mg/kg) and DHEA (100 mg/kg individually or in combination for 6 months. EM-652.HCl is administered orally, DHEA was applied topically, while LHRH-A were injected with subcutaneous. All processing was performed once a day.

Experimental animal:

Species: Rattus norvegicus

Line: rat Sprague-Dawley (Crl:CD®(SD) BR VAF/Plus™)

P is l: female

Age: at the beginning of dosing rats have an age of approximately 10-12 weeks.

Content & care

a) Content:

During the acclimatization period and the period of the study, rats were kept in cages made of stainless steel, each separately.

b) Temperature and humidity:

Environmental conditions (temperature, humidity) in the premises of the rats were under continuous control using automatic feature computing system. Specified conditions were temperature: 22±3°C and relative humidity: 50±20%.

C)The alternating cycle of light and darkness:

Photoperiodic cycle was 12 hours light and 12 hours of darkness. These parameters are continuously recorded using a certified auto equipped computing system. The lighting was from 07:15 until 19:15.

d)Diet:

Certified forage for rodents (Lab Diet # 5002, pellets) and tap water were providedad libitum.Rats were fasted (with access only to water) the night before opening.

Randomization:

Rats were divided into groups statistically during the acclimatization period:

Methods and experiment plan

Group test:

One hundred and twenty rats were divided into 8 groups of 15 animals each for the study described in General terms below.

ProcessingSuspension for dosing, administered orallySolutions for dosing applied topicallyLHRH-A (Injectable P.K.)Dose
(mg/kg)About./rat
(ml)Dose (mg/kg)About./rat (ml)Dose
1 µg/ratThe intact-0,5-0,5-EM-652.HCl2,50,5-0,5-DHEA-0,51000,5DHEA +
EM-652.HCl2,50,51000,5-LHRH-A-0,5 -0,50.5 mlLHRH-A +
EM-652.HCl2,50,5-0,50.5 mlLHRH-A + DHEA-0,51000,50.5 mlLHRH-A + DHEA + EM-652.HCl2,50,51000,50.5 ml

Preparation of samples for testing

LHRH-A is available in solution at a concentration of 1.0 mg/ml of the concentrated solution is diluted to obtain a final concentration of 0.002 mg/ml)using the following filling: 0.3% sodium chloride and 0.25% monobasic sodium phosphate, monohydrate in water. To bring the pH values of 5.6 to 6.2 used sodium hydroxide (water).

Suspensions/solutions for dispensing prepared every two weeks, taking into account the most recent values of body weight (average for group), in addition to LHRH-A solution. For the EM-652.HCl, administered orally to test the connection, pre-weighed in a glass bottle, was added filler (0.4% of the ethylcellulose), at least 48 hours prior to the first day of dosing. To ensure homogeneity of the suspension for dosing, it is stirred for at least 48 hours at a temperature of from 2 to 8°C. the Suspension for dispensing stored at 2-8°C. For a solution of DHEA applied topically to DHEA add the ethanol and the mixture is stirred until dissolution of DHEA. Then add polypropylenglycol and the solution is stirred to achieve its homogeneity. Solution for dispensing stored at a temperature of from 2 to 8°C (part of the solution can be stored at room temperature in order to facilitate topical application on the host). To a solution of LHRH-A, injectable subcutaneously every month carry out the dilution of the concentrated solution, using the appropriate filler. Solution for dispensing stored at 2-8°C.

Preparation of animals:

Until the day the first dose and as needed throughout the study area dorsal skin (approximately 5×5 cm) shave for topical application of DHEA.

Dosing:

The introduction of the test compounds or filler begin on day 1 of the study Protocol. Test compounds were given in the form of a suspension in 4% methylcellulose using oral catheter (0.5 ml/catheter/rat) once a day, or applied topically in a mixture of 50% ethanol-50% about jinglian (0.5 ml/application/rat) once a day. In addition, rats of groups 5-8 were usually given LHRH-A once daily subcutaneous injection (0.5 ml/injection/rat). Rats not treated with the test compound orally and/or topically, usually received only appropriate filler.

Measurement of body fat:

Body composition was determined by evaluating the complete structure of the body and the right thigh of the animal under anesthesia under the action of izoflurana using a two-level x-ray absorbtiometry (DEXA; QDR 4500A, Hologic),during the acclimatization period and 6 months after treatment.

Results

Table 4
ProcessingFAT (%)
The intact23,1±1,6
EM-652.HCl19,1±1,4
DHEA20,2±2,3
DHEA + EM-652.HCl18,8±1,3
LHRH-A36,4±2,2
LHRH-A + EM-652.HCl31,2±2,0
LHRH-A + DHEA27,4±2,1
LHRH-A + DHEA + EM-652.HClExample 6

The effect of the 20-day exposure to EM-652.HCl, estradiol and DHEA on the parameters of body fat and body mass in ovariectomized female rats

URMA-r-27-00

The purpose of this study was to assess the effect on the parameters of body fat and body mass EM-652.HCl, estradiol, DHEA, administered orally, individually or in combination, ovariectomized (OVX) rats-females receiving enriched carbohydrate diet.

Experimental animal:

Species: Rattus norvegicus

Line: rat Sprague-Dawley (Crl:CD®(SD) BR VAF/Plus™)

Gender: female

Body weight: at the beginning of the dosing body weight was approximately 200-250 g

Content & care

a)Content:

Rats were kept in cages made of stainless steel, each separately, during the acclimatization period and the study period.

b) Temperature and humidity:

Environmental conditions (temperature, humidity) in the premises of rats was continuously recorded using an automatic-equipped computing system. Specified conditions were temperature: 22±3°C and relative humidity: 50±20%.

c)The alternating cycle of light and darkness:

Photoperiodic cycle was 12 hours light and 12 hours of darkness. These parameters are continuously recorded using a certified auto ocasinojuegosonline1 technique system. The lighting was from 07:15 until 19:15.

d)Diet:

Certified forage for rodents (Lab Diet # 5002, pellets) and tap water were providedad libitumduring the acclimatization period. During the study period, rats received enriched carbohydrate dietad libitum.Enriched carbohydrate diet (diet #3) consisted of (g/100 g): corn starch 31,2; dextrose 31,2; casein 20,0; corn oil 6,4; dl-methionine, 0.3; the vitamin mixture 1,0; mineral mix AIN-76 4,9; fiber 5,0.

The day before necropsy, rats fasted (with access only to water) to late afternoon (around 16 : 00).

Randomization:

Rats were divided into groups statistically on day 0 of the study.

Methods and experiment plan

Group test:

80 rats were divided into 8 groups of 10 rats each for the study described in General terms below.

ProcessingSuspension for dosing
Dose (mg/rat)About./rat
(ml)
The intact00.5 ml
OVX00.5 ml
OVX + EM-652.l0,50.5 ml
OVX+E20,50.5 ml
OVX + DHEA1000.5 ml
OVX + EM-652.l
+ DHEA
0,5
+100
0.5 ml
+0.5 ml
OVX + EM-652.l
+ E2
0,5
+0,5
0.5 ml
+0,5 ml

Preparation of animals:

On day 0 of the study, rats of groups 2-8 were subjected oophorectomy using bilateral lateral incision under conditions of anesthesia induced by isoflurane. Rats of group 1 were subjected to sham operation.

Dosing:

The introduction of the test products or filler started on day 1 of the study (day after oophorectomy). Test compounds were given in the form of a suspension in 0.4% methylcellulose using oral catheter (0.5 ml/catheter) for 20 days.

Measurement of body composition:

To evaluate the effect of treatment on the percentage of fat, on day 21 of the study (prior to autopsy)was determined body composition, evaluating the full structure of the bodies of animals under anesthesia under the action of izoflurana using two who runaway x-ray absorbtiometry (DEXA; QDR 4500A, Hologic).

Retrieved organs and tissues

Were removed and weighed right and left retroperitoneal adipose tissue.

Results

Table 5
ProcessingFat, %Retroperitoneal adipose tissue, g
The intact15,7±0,82,20±0,20
OVX18,9±1,53,70±0,41
OVX + EM-652.HCl10,2±1,21,52±0,18
OVX + E210,3±0,60,81±0,17
OVX + DHEA7,8±0,80,92±0,17
OVX + EM-652.HCl
+DHEA
8,4±0,91,15±0,17
OVX EM-652.HCl
+E2
10,4±0,71,17±0,19

Example 7

The effect of a 34-week exposure to ethinyl estradiol, EM-652.HCl, raloxifene and DHEA on the parameters of body fat and body mass ovariectomised female rats

p> URMA-r-42-98

The purpose of this study was evaluating the effects of combined treatment with EM-652.HCl and DHEA on the parameters of body fat and body mass ovariectomised rats. For this purpose ovariectomised rats were daily injected EM-652.HCl (1 mg/kg) and DHEA (80 mg/kg), separately or in combination, for 34 weeks. EM-652.HCl is administered orally, while DHEA was applied topically to the dorsal skin. One group of animals was treated with oral 17α-ethinyl estradiol (0.1 mg/kg) and Raloxifene (EM-1105; 1 mg/kg) for comparison.

MATERIALS AND METHODS

Animals and treatment

Were used rat-female line Sprague-Dawley (Crl:CD(SD)Br) age from ten to twelve weeks, weighing approximately 235-250 g at the beginning of the treatment. Eighty rats were distributed statistically in 7 groups 11-12 animals per group as follows: 1) intact control; 2) OVX control; 3) OVX + ITS2; (0.1 mg/kg); 4) OVX + Raloxifene (1 mg/kg); 5) OVX + EM-652.HCl (1 mg/kg); 6) OVX + DHEA (80 mg/kg); 7) OVX + EM-652.HCl + DHEA. On day 1 of the study animals of the respective groups were subjected to bilateral oophorectomy (OVX) under anesthetic action izoflurana. EM-652.HCl, Raloxifene and EE2(Sterility) was administered once daily via oral catheter in the form of a suspension in 0.4% methylcellulose (0.5 ml/rat) for 34 weeks, whereas DHEA was applied topically once a day naturalnew the skin in the form of a solution in a mixture of 50% ethanol-50% propylene glycol (0.5 ml/rat) for the same period of time. After approximately 24 hours after the last dosing deprived of food during the night animals were scored by exsanguination via the abdominal aorta under conditions of anesthesia under the action izoflurana. Removed left and right retroperitoneal adipose tissue and weighed.

Measurement of body composition

After 34 weeks of treatment in individual rats anesthetized with isoflurane by scanning evaluated the full structure of the body, using a two-level x-ray absorbtiometry (DEXA; QDR 4500A, Hologic, Waltham, MA) software scanning area of the body with high resolution. Determined the structure of the body (including the body fat percentage).

Statistical analysis

Data are presented as mean ±SEM.

Results

Table 6
ProcessingRight retroperitoneal adipose tissue, gLeft retroperitoneal adipose tissue, gFat,%
Intact control1,43±0,121,80±0,2625,7±2,5
OVX control2,53±0,242,64±0,1842,1 is 1,5
OVX + EE2
(0.1 mg/kg)
1,29±0,221,15±0,1819,8±1,9
OVX-Raloxifene
(1 mg/kg)
1,2±0,121,41±0,1322,8±1,6
OVX + EM-652.HCl
(1 mg/kg)
2,07±0,182,19±0,2827,7±1,8
OVX + DHEA
(80 mg/kg)
2,16±0,25l,98±0,l726,8±2,3
OVX + EM-652.HCl
+ DHEA
l,62±0,181,47±0,1825,1±1,8

Example 8A

Action EM-652.HCl on energy balance, lipid-urine metabolism of lean and obese rats-males line Zucker

URMA-r-61-99

The purpose of this study was to detect the actions of EAT-652.l on energy balance and lipid-urine metabolism skinny and fat (fatty) rats male line Zucker. For this purpose, EAT-652.l (2.5 mg/kg) was administered orally (through a catheter) once a day for 14 days in healthy lean and obese rats-male Zucker.

Experimental animal:

Appearance: Ruttus norvegicus

Line: Zucker

Gender: male

Body weight: at the beginning of dosing body weight was approximately:

skinny rat: 175±15 g; fat rat: 235±15 g

Acclimatization

Rats acclimatized to laboratory conditions for at least five days before the start of the experiment.

Content & care

a) Content:

Rats were placed individually in cages stainless steel standard design on the acclimatization period and the period of the study.

b) Temperature:

Room temperature for rats were recorded daily. The set temperature was 22±3°C.

c)The alternating cycle of light and darkness:

Photoperiodic cycle was 10 hours light and 14 hours of darkness. Lighting started at 07:00 and ended at 17:00.

d)Diet:

During the acclimatization period, the rats received a commercial diet for rodents (Purina, # 5075) and tap waterad libitum. During the period of the study, rats were given the following diet (diet #3)consisting of (g/100 g): starch 31,2; dextrose 31,2; casein 20,0; corn oil 6,4; dl-methionine, 0.3; the vitamin mixture 1,0; mineral mix AIN-76 4,9; fiber 5,0.

Rats were fasted (with access only to water) until about 07 : 00 in the morning on the day of their opening.

Randomization:

The day before the first day of dosing, rats were weighed and divided into groups so that the floor is icy groups of animals with equivalent average body weight.

Methods and experiment plan

Group test:

Sixteen skinny and sixteen fat intact rats were divided into 4 groups of 8 rats each for the study described in General terms below:

The number of rats/groupThe state of the ratThe treatmentDose
(mg/kg)
8SkinnyEM-652.HCl2,5
8FattyEM-652.HCl2,5
8SkinnyCONTROL0
8FattyCONTROL0

Dosing:

The introduction of the test compound or filler started on day 1 of the study. Test the connection EM-652.HCl was given in the form of a suspension in 0.4% methylcellulose using oral catheter (0.5 ml/catheter/rat) once a day for 14 days. Rats of the control groups received only the filler (0.5 ml/catheter/rat) in order and during the same period of time.

Body mass index:

During the acclimatization period, the animals were weighed the day before dosing for randomization. Then the rats were weighed on the first day of dosing and every 2 days thereafter, and on the day of opening. Body mass was recorded with an accuracy of 1 g

Consumption (consumption) feed:

Feed intake was assessed every 2 days during the study period.

Method of killing:

After approximately 6-hour period of fasting, animals were subjected to autopsy (approximately 30 hours after the last dosing). They were anestesiologi ketamine-xylazine and the blood was collected cardiac puncture.

Blood samples:

Lipids (total cholesterol (HOL, CHOL) and triglyceride (TG, TG)and blood glucose was measured in frozen plasma samples using Boehringer Mannheim diagnostics Hitachi 911 Analyzer (Boehringer Mannheim Laboratory diagnostics systems). Circulating hormones and substrates was determined in frozen plasma samples using the following sets:

Leptin: a set for RIA(RIA) Linco

Insulin: set for RIA(RIA) Linco

Retrieved organs and tissues

A piece of liver (~1 g) were frozen in liquid nitrogen for later determination of the content of triglycerides and HALL (method of Folch (Folch's)). Took retroperitoneal adipose tissue and soleus muscle was frozen in liquid nitrogen for later determination of activity lipop is atinlay (LPL, LPL). Remove ventral prostate, testes, and seminal vesicles. Weigh the prostate, testicles (left and right together) and right seminal vesicles (without liquid). The right seminal vesicles drop.

Table 7
Rats-males line Zucker
GROUPWeightThe activity of lipo-proteinopathy
White to Sabry-tire adipose tissue
The activity of lipo-protein lipase
the CAMBA-lovenow
muscles
Insulin plasmaGlucose
plasma
General content the content of holes-terinaTrigly-potassium
plasma
pheno-typeprocessinggthe MCA/g proteinthe MCA/g proteinnmol/lmmol/lmmol/lmmol/l
skinnypin
role
73,0±4,22066±
353
71,7±7,9 a 10.74±0,872,10±0,151,47±0,20
skinnyEAT-652.l
2,5
mg/kg
44,9±3,51701±
348
69,5±8,40,062±0,0059,35±0,511,18±0,091,52±0,13
fatcont-
role
110,5±6,77233±
511
51,9±8,81,092±0,36911,06±0,843,07±0,284,21±0,78
fatEAT-652.l
2,5
mg/kg
71,6±3,37046±
1185
58,1±4,6value (0.475)±0,08711,84±0,622,28±0,257,16±1,06

Example 8B

Action EM-652.HCl on energy balance and lipid-urine metabolism of lean and obese rats-females line Zucker

LRMA-r-47-99

The purpose of this study was to determine the validity of EM-652.HCl on energy balance and whether the ID-urine metabolism skinny and fat (fatty) rats-females line Zucker. For this purpose intact skinny and fat rats-females line Zucker was injected EM-652.l (2.5 mg/kg) oral (catheter) once a day for 14 days.

Specification of experimental animals:

Appearance: Ruttus norvegicus

Line: Zucker

Gender: female

Body weight: at the beginning of dosing body weight was approximately:

skinny rat: 114±2 g; fat rat: 182±6 g

Acclimatization:

Rats acclimatized to laboratory conditions for at least five days before the start of the experiment.

Content & care

a) Content:

Rats were placed individually in cages stainless steel standard design on the acclimatization period and the period of the study.

b) Temperature:

Room temperature for rats was recorded once a day. The set temperature was 22±3°C.

c)The alternating cycle of light and darkness:

Photoperiodic cycle was 10 hours light and 14 hours of darkness. Lighting started at 07:00 and ended at 17:00.

d)Diet:

During the acclimatization period, the rats received a commercial diet for rodents (Purina, # 5075) and tap waterad libitum. During the period of the study, rats were given the following diet (diet #3)consisting of (g/100 g): starch 31,2; dextrose 31,2; casein 20,0; corn oil 6,4; dl-methionine, 0.3; the vitamin SMEs,0; mineral mix AIN-76 4,9; fiber 5,0. Rats were fasted (with access only to water) of approximately 07 : 00 on the morning of the opening.

Randomization:

Two days before the first day of dosing, rats were weighed and divided into groups so as to obtain a group of animals with an equivalent average body weight.

Methods and experiment plan

Group test:

Sixteen skinny and sixteen fat intact female rats were divided into 4 groups of 8 rats each for the study described in General terms below:

The number of rats/groupThe state of the ratProcessingDose
(mg/kg)
8SkinnyEM-652.HCl2,5
8FattyEM-652.HCl2,5
8SkinnyCONTROL0
8FattyCONTROL0

Dosing:

Introduction experience with the organisations or filler started on day 1 of the study. Test the connection EM-652.HCl was given in the form of a suspension in 0.4% methylcellulose using oral catheter (0.5 ml/catheter/rat) once a day for 14 days. Rats of the control groups received only the filler (0.5 ml/catheter/rat) during the same period of time.

Body mass index:

During the acclimatization period, the animals were suspended for two days prior to dosing for randomization. Then the rats were weighed on the first day of the study and every 2 days during the study period, as well as on the day of opening. Body mass was recorded with an accuracy of 1 g

Consumption (consumption) feed:

Feed intake was assessed every 2 days during the study period.

Method of killing:

After approximately 6-hour period of fasting, animals were subjected to autopsy (approximately 30 hours after the last dosing). They were anestesiologi ketamine-xylazine and removed the blood cardiac puncture.

Blood samples

Lipids (total cholesterol (HOL, CHOL) and triglyceride (TG, TG)and blood glucose was measured in frozen plasma samples using Boehringer Mannheim diagnostics Hitachi 911 Analyzer (Boehringer Mannheim Laboratory diagnostics systems). Circulating hormones and substrates (leptin, insulin, corticosterone) were determined in frozen plasma samples using the following sets:

LEP is in: set for RIA(RIA) Linco

Insulin: set for RIA(RIA) Linco

Retrieved organs and tissues

The liver was removed and weighed. A piece of liver (~1 g) were frozen in liquid nitrogen for later determination of the content of triglycerides and HALL (method of Folch). Removed the adrenal glands were weighed (left and right together) and discard. Removed the uterus and testes were weighed and preserved in 10% buffered formalin for further histological examination. The left and right ovaries (without fallopian tubes) were weighed together.

Example 9

Action EM-652.HCl, DHEA, administered separately or in combination, on energy balance and lipid-urine metabolism in rats

URMA-r-03-99

The purpose of this study was to detect the actions of EM-652.HCl, DHEA, administered separately or in combination, on energy balance and lipid-urine metabolism in rats. The effect has been evaluated by many parameters after the 20-day exposure to EM-652.HCl (0.5 mg/rat ~2.5 mg/kg, per os) and DHEA (20 mg/rat, ~100 mg/kg, local application), administered separately or in combination, are intact (INT, (INT)) and ovariectomized (OVX) rats-females fed the diet with a high content of sucrose and high fat content. For comparison, one intact and ovariectomized control group receives enriched diet, while the other control animals (intact and OVX) usually get a commercial diet for rodents.

Animal testing:

Species: Rattus norvegicus

Line: rat Sprague-Dawley (Crl:CD®(SD) BR VAF/Plus™)

Gender: female

Body weight: at the beginning of dosing body weight was approximately 190-220,

Acclimatization:

Rats acclimatized to laboratory conditions for at least five days before the start of the experiment.

Content & care

a) Content:

Rats p. who was memali individually in cages stainless steel standard design on the acclimatization period and the period of the study.

b) Temperature:

The temperature inside rats were recorded once a day. The set temperature was 22±3°C.

c)The alternating cycle of light and darkness:

Photoperiodic cycle was 10 hours light and 14 hours of darkness. Lighting started from 06:00 and stopped at 16:00.

d)Diet:

During the acclimatization period, the rats received a commercial diet for rodents (Purina, # 5075) and tap waterad libitum. During the study period group 1 and 2 generally continued to receive commercial diet, while group 3-10 usually received a diet with a high content of sucrose and high fat content, consisting of (g/100 g)sucrose 45; corn oil 10; melted pork fat 10; casein 22,5; dl-methionine, 0.3; the vitamin mixture 1,2; mineral mix AIN-76 5,5; fiber 5,5.

Rats were fasted (with access only to water) approximately22 : 00 the night before opening.

Randomization:

A few days after their arrival, rats were weighed and divided into groups so as to obtain a group of animals with an equivalent average body weight.

Methods and experiment plan

Group test:

80 rats were divided into 10 groups of 8 rats each, for the research described in General terms below:

Table 8
Rat-female strain Zucker
GROUPWeightThe asset of lipo-protein lipase White to Sabry-tire adipose tissueThe activity of lipoprotein lipase
Kambala prominent muscles
Insulin
plasma
Glucose
plasma
Total cholesterolTriglycerides
plasma
phenotypeeduca-mentgthe MCA/g proteinthe MCA/g
protein
nmol/lmmol/l mmol/lmmol/l
skinnycontrol175±41586±
155
55,8±8,10,122±
0,022
of 9.45±0,682,33±0,101,25±0,16
skinnyEAT-652.l
2.5 mg/kg
154±51349±
246
60,8±11,10,063±
0,012
a 9.60±0,411,57±0,140,83±0,07
fatcontrol313±93980±
327
36,9±8,60,410±
0,033
10,63±0,55a 4.83±0,2712,51±3,25
fatEAT-652.l
2.5 mg/kg
291±93819±
485
36,6±7,10,515±
0,091
11,7±0,472,37±0,9722,20±of 4.44
aceon ProcessingDose (mg/rat)
Per osLocally
Food1INT00
OVX
INT
0
0
0
0
INT + EM-652.HCl0,50
Special diet2INT + DHEA020
INT + EM-652.HCl + DHEA
OVX
OVX + EM-652.HCl
0,5
0
20
0
0,50
OVX + DHEA020
OVX + EM-652.HCl + DHEA0,520

1Commercial feed for rodents.

2A diet with a high content of sucrose and high fat content.

Preparation of animals:

On day 0 of the study rats from the respective groups of adversariality (bilateral lateral incision) under anesthetic action izoflurana. Intact rats were subjected to sham operation.

Dosing:

The introduction of the test compounds or fillers started the day after oophorectomy (Day 1). Test the connection EM-652.HCl was given in the form of a suspension in 0.4% methylcellulose using oral catheter (0.5 ml/catheter/rat) once a day for 20 days, while DHEA was applied topically to the dorsal skin (0.5 ml/application/rat) once per day during the same time period. Rats of the control groups received only the filler (per os or local). Rats were scored on day 21 of the study after approximately 24 hours after the last dosing.

Body mass index:

Rats were weighed on day 0 (surgery), every 2 days during the study period and on the day of opening. Body mass was recorded with an accuracy of 1.0,

Consumption (consumption) feed:

Feed intake was assessed every 2 days during the study period.

Method of killing:

On day 21 of the study starving during the night the rats were scored after approximately 24 hours after the last dosing. They were anestesiologi ketamine-xylazine and removed the blood by puncture of the heart.

Blood samples:

Lipids (total cholesterol and triglycerides in the blood was determined in frozen serum samples, using Boehringer annheim diagnostics Hitachi 911 Analyzer (Boehringer Mannheim Laboratory diagnostics systems). Circulating hormones and substrates (insulin, leptin, glucose) were determined in serum samples, as follows:

Insulin: set for RIA(RIA) Linco

Glucose: Automatic glucose analyzer Beckmann

Leptin: a set for RIA(RIA) Linco

Retrieved organs and tissues

All animals were removed and weighed the following fabrics:

Muscle (soleus and lateral vastus (VLM)), fat (retroperitoneal and inguinal), heart, brown adipose tissue. In addition, usually extract the marrow.

A piece of liver was frozen for subsequent determination of the content of TG and HALL (method of Folch), and other tissues were subjected to the processing for determining the activity of LPL(LPL). Remove the uterus (and the ovaries in the intact groups), stripped the rest of the fat, weighed and preserved in 10% buffered formalin.

Results

Table 9
GROUPThe total massWhite adipose tissue
inguinal
White adipose tissue retroperitonealBrown adipose tissue
weightthe activity of lipo-Proteus the lipase weightthe activity of lipo-protein lipaseweightthe activity of lipo-protein lipase
dietprocessingggthe MCA/g proteingthe MCA/g proteingthe MCA/g protein
foodfact. control239±60,410±
0,043
252±550,983±0.104 g1526±2170,329±0,0351053±138
foodOVX control294±60,888±
0,146
570±1001,472±0,1992415±1470,466±0,041688±88
Sucrose+fatfact. control257±100,716±
0,107
344±73 to 1.765±0,2921470±2420,376±0,0331073±152
Sucrose+fatFact.+EAT-652.l238±50,435±
0,043
160±231,058±rate £ 0.1621277±560,306±0,0441196±83
Sucrose+fatFact.+DHEA252±50,659±
0,075
194±451,067±0,1491177±1880,373±being 0.036982±72
Sucrose+fatFact.+EAT-652.l + DHEA249±40,489±
0,083
224±450,909±0,1241349±1330,322±0,026885±78
Sucrose+fatOVX control316±81,800±
0,251
725±1063,058±0,2972503±215 0,519±0,064867±99
Sucrose+fatOVX + EM-652.l268±50,709±
0,085
310±631,370±0,2111612±1860,408±0,025678±65
Sucrose+fatOVX + DHEA274±90,885±
0,156
583±1341,699±0,1511905±1120,342±0,0491069±96
Sucrose+fatOVX +EM-652.l + DHEA263±50,658±
0,088
306±421,436±rate £ 0.1621542±1180,373±0,040876±75

GROUPSoleusLateral vastusLiver
weightthe activity of lipo-protein lipaseweightthe activity of lipo-protein lipasetotal cholesterol-Rintrigly-potassium
dietthe treatmentgthe MCA/g proteingthe MCA/g proteinmmol/lmmol/l
FoodFichtel0,106±
0,002
62,7±3,00,872±
0,027
11,7±3,50,076±
0,004
0,134±
0,019
FoodOVX control0,116±
0,005
43,8±3,31,000±
0,042
13,1±3,70,082±
0,004
0,169±
0,027
Sucrose+fatFichtel0,096±
0,005
55,0±5,1 0,927±
0,040
13,1±3,20,083±
0,002
0,123±
0,014
Sucrose+fatfact.+EAT-652.l0,098±
0,005
53,9±3,30,965±
0,034
16,2±3,10,096±
0,011
0,360±
is 0.102
Sucrose+fatfact.+
DHEA
0,105±
0,004
53,6±7,50,893±
0,043
11,5±2,00,068±
0,003
0,038±
0,007
Sucrose+fatfact.+652.l+
DHEA
0,098±
0,005
56,6±4,50,971±
0,029
19,5±3,50,101±
0,008
0,184±
0,033
Sucrose+fatOVX control0,110±
0,004
39,2±3,91,037±
0,026
10,9±1,820,097±
0,007
0,262±
0,052
Sucrose+fatOVX+EM-652.lin 0.104±
0,004
39,1±5,00,944±
0,030
10,8±3,20,092±
0,006
0,348±
0,078
Sucrose+fatOVX+DHEA0,103±
0,005
45,9±4,70,920±
0,049
13,1±5,40,079±
0,005
0,165±
0,047
Sucrose+fatOVX+EM-652.l+
DHEA
0,101±
0,001
48,4±4,80,929±
0,038
11,0±4,10,106±
0,008
0,273±
0,031

0,92±0,21
GROUPInsulin
plasma
Plasma glucoseTotal cholesterol
plasma
Triglycerides plasmaLeptin plasma
diet the treatmentnmol/lmmol/lmmol/lmmol/lng/ml
Foodfact. control0,071±0,0107,56±0,241,61±0,170,49±0,121,404±0,224
FoodOVX control0,146±0,0278,69±0,572,10±0,100,68±0,102,388±0,737
Sucrose+fatfact. control0,079±0,0139,07±0,481,60±0,120,66±0,173,572±0,699
Sucrose+fatfact.+EAT-652.l0,057±0,0049,03±0,491,15±0,060,60±0,122,019±0,402
Sucrose+fatfact.+
DHEA
0,048±0,012 8,05±0,511,59±0,170,61±0,111,977±0,255
Sucrose+fatfact.+EAT-652.l+
DHEA
0,049±0,0137,99±0,440,93±0,080,95±0,211,071±rate £ 0.162
Sucrose+fatOVX control0,125±0,02210,46±0,721,62±0,260,83±0,137,900±1,982
Sucrose+fatOVX +EM-652.l0,089±0,0139,06±0,301,15±0,110,97±0,161,989±0,326
Sucrose+fatOVX + DHEA0,052±0,0098,64±0,241,57±0,120,62±0,082,757±0,631
Sucrose+fatOVX +EM-652.l +DHEAto 0.060±0,0108,65±0,380,87±0,091,672±0,327

Example 10

Action EM-652.HCl, TSE 424, Lasofoxifene, LY 353381 and Raloxifene on energy balance and lipid-urine metabolism ovariectomised female rats.

URMA-r-45-00

The purpose of this study was to detect actions on energy balance and lipid-urine metabolism in rats treated different selective modulators of estrogen receptor described in the literature, and compared the obtained results with the results of the effects of EM-652.l. For this purpose, the test compounds were administered by oral catheter for 20 days (0.5 mg/rat for each connection; 0.5 ml/rat) ovariectomised rats-females. Were used for comparison of intact control, ovariectomized (OVX) control and OVX rats treated with 17β-estradiol (E2).

Experimental animal:

Species: Rattus norvegicus

Line: rat Sprague-Dawley (Crl:CD®(SD) BR VAF/Plus™)

Gender: female

Body weight: at the beginning of dosing body weight was approximately 200-225,

Content & care

a) Content:

Rats were placed individually in cages stainless steel standard design on the acclimatization period and the period of the study.

b) Temperature:

Environmental conditions (temperature, humidity the spine) in the premises of rats was continuously recorded, using automatic feature computing system. Specified conditions were: temperature 22±3°C and relative humidity 50±20%.

c) alternating Cycle of light and darkness:

Photoperiod was 10 hours light and 14 hours of darkness. Lighting started at 07:15 and was interrupted at 17:15.

d)Diet:

During the acclimatization period, the rats received a certified diet for rodents (Lab Diet, # 5002, pellets) and tap waterad libitum,while during the period of the study, rats received a diet with a high carbohydrate diet (diet #3) and tap waterad libitum.The diet consisted of (g/100 g): corn starch 31,2; dextrose 31,2; casein 20,0; corn oil 6,4; dl-methionine, 0.3; the vitamin mixture 1,0; mineral mix AIN-76 4,9; fibers of 5.0. Rats were fasted (with access only to water) of approximately 07 : 00 on the morning of the day of the opening.

Randomization:

Rats were divided into groups so that each group can have animals with equivalent average body weight.

Methods and experiment plan

Group test:

For the study described in General terms below, seventy-seven rats were divided into 8 groups of 9-10 rats each.

Suspension dosing
The number of ratsProcessing
Dose
(mg/rat)
About./rat
(ml)
9The intact00.5 ml
9OVX00.5 ml
10OVX + EM-652.l0,50.5 ml
10OVX + TSE 424 (EAT-3527)0,50.5 ml
10OVX + Lasofoxifene (EAT-3555)0,50.5 ml
10OVX+LY 353381 (EM-1665)0,50.5 ml
10OVX + Raloxifene (EAT-1105)0,50.5 ml
9OVX + E20,50.5 ml

Preparation of animals:

On day 0 of the study, rats of groups 2-8 were subjected oophorectomy (is postoronnim lateral incision) under anesthetic action izoflurana. Rats of group 1 were subjected to sham operation.

Body mass index:

Rats were weighed on day 0 (surgery) and then every 2 days during the study period, as well as on the day of autopsy.

Feed consumption:

Feed intake was assessed every 2 days.

Body composition (percentage of fat)

To determine the effect of treatment (percentage) fat content was determined body composition on day 17 of the study, using a two-level x-ray absorbtiometry (DEXA; QDR 4500A, Hologic, Waltham, MA).

The method of killing

On day 21 of the study after approximately 24 hours after the last dosing and 6 hours of fasting animals under anesthesia ketamine-xylazine, scored by exsanguination through the abdominal aorta.

Blood samples:

Lipids (total cholesterol and triglycerides) and blood glucose was determined in frozen serum samples, using Boehringer Mannheim diagnostics Hitachi 911 Analyzer (Boehringer Mannheim Laboratory diagnostics systems). Circulating hormones and substrates (insulin and leptin) were determined in the serum sample, using the following sets:

Insulin: set for RIA(RIA) Linco

Leptin: the kit Linco RIA

Retrieved organs and tissues

Were removed and weighed the following fabrics:

Muscle (right soleus and right lateral large muscle b is the DRA (VLM)), fat (right retroperitoneal and right inguinal fabric), brown adipose tissue, heart, liver, uterus and vagina.

A piece of liver (~0.5 g) were frozen in liquid nitrogen for later determination of the content of triglycerides and HALL (method of Folch). Pieces ~0.1 g in all tissues (except the uterus and vagina) were frozen in liquid nitrogen and kept at -80°C for subsequent determination of activity of lipoprotein lipase (LPL, LPL). The uterus and vagina were stored in 10% buffered formalin for subsequent histological examination. The body of the animal was placed in a metal box and kept at -20°C until measurement of the energy balance.

Results

0,244±
0,028
Table 10
ProcessingThe total massWhite adipose tissue inguinalWhite adipose tissue
retroperitoneal
Brown adipose tissue
weightthe activity of lipo-protein lipaseweightthe activity of lipo-protein-Lipa is s weightthe asset of lipo-protein lipase
ggthe MCA/g proteingthe MCA/g proteingthe MCA/g protein
The intact232±70,350±
0,037
379±691,31±
0,09
1537±
253
0,410±
0,030
973±115
OVX284±50,320±
0,048
797±971,55±
0,15
2664±
338
0,380±
0,027
880±130
OVX +EM-652.l252±4684±2161,32±
0,21
1917±
365
0,339±
0,038
900±91
OVX+
TSE424
260±50,326±
0,042
766±1731,27±
0,15
1750±
140
0,350±
0,019
1017±83
OVX+La cefoxitin210±20,187±
0,023
801±1380,867±
0,132
1417±
163
0,244±
0,022
982±98
OVX+LY 353381231±40,290±
0,038
559±1650,968±
0,120
1365±
119
0,368±
0,031
888±65
OVX+RA-moxifen230±30,245±
0,047
579±461,32±
0,24
1510±
52
0,307±
0,26
960±116
OVX+E2198±40,158±
0,019
416±740,550±
is 0.102
1442±
243
0,211±
0,021
1198±62

/tr>
ProcessingSoleusLateral vastus
weightthe activity of lipoprotein lipaseweightthe activity of lipoprotein lipase
gmceg protein gthe MCA/g
The intact0,113±0,02017,2±2,20,924±0,031not determined.
OVXto 0.108±0,00617,6±3,90,963±0,045not determined.
OVX+EM-652.lamount of 0.118±0,01814,5±4,11,00±0,047not determined.
OVX+TSE4240,100±0,00411,7±1,90,993±0,33not determined.
OVX+Lasofoxifene0,096±0,00310,9±1,50.800 to±0,048not determined.
OVX+LY 3533810,092±0,00210,1±1,00,955±0,20not determined.
OVX+Raloxifene0,099±0,00310,2±2,00,795±0,055not determined.
OVX + E20,086±0,00310,9±2,00,718±0,026not determined.

ProcessingThe total consumption of foodInsulin
plasma
Glucose
plasma
Total cholesterol
plasma
Triglycerides plasmaLeptin
plasma
gnmol/lmmol/lmmol/lmmol/lng/ml
The intact332±140,086±
0,11
13,6±0,72,41±0,160,62±0,072,46±0,37
OVX389±80,178±
0,030
15,9±1,92,58±0,110,63±0,053,48±0,40
OVX+EM-652.l0,122±
0,021
15,9±1,41,41±0,090,91±0,171,10±0,22
OVX+TSE 424347±90,113±
0,029
16,5±2,71,66±0,160,93±0,242,12±0,44
OVX+laso-foxygen283±10,095±
0,017
15,8±1,321,29±0,060,81±0,210,97±0,29
OVX+LY 353381312±7in 0.104±
0,11
17,1±0,721,15±0,081,23±0,281,22±0,21
OVX+Rahl-xifen307±80,077±
0,10
13,8±1,21,19±0,081,20±0,351,39±0,30
OVX+E2255±90,070±
0,007
11,8±1,35 2,03±0,150,43±0,040,309±
0,097

Example 11

The effect of the compounds of the invention and compounds of the prior art on the activity of alkaline phosphatase in Ishikawa cells endometrial adenocarcinoma person

Materials

Keeping a clean cell cultures

Line Ishikawa human cells extracted from thoroughly differentiated endometrial adenocarcinoma, was kindly provided by Dr. Erlio Gurpide, The Mount Sinai Medical Center, New York. NY. Ishikawa cells, as usual, support minimal support environment Needle (MEM)containing 5% (vol./about.) FBS (fetal calf serum) and supplemented with 100 u/ml penicillin, 100 μg/ml streptomycin, 0.1 mm non-supportive solution of amino acids. Cells were seeded in flasks FalconT75 at a density of 1.5×106cells at 37°C.

Experiments with cell cultures

Twenty-four hours before the start of the experiment environment close to confluently Ishikawa cells replace fresh, does not contain estrogen, basal medium (EFBM), consisting of a mixture of 1:1 (vol./vol.), not containing phenol red Ham's F-12 medium and medium Needle in the modification of Dulbecco (DMEM), supplemented with 100 u/ml penicillin, l00 mg/ml streptomycin, 2 mm glutamine and 5% FBS, double-processed-loaded dextran coal dust to remove endog is by steroids. The cells are then harvested with 0.1% Pancreatin (Sigma) and 0.25 mm HEPES, resuspended in EFBM and seeded in 96-well flat-bottomed titration microplates Falcon at a density of 2.2×104cells/well in a volume of 100 μl and give the opportunity to adhesivity to the surface of the tablets within 24 hours After the medium replaced with fresh EFBM, containing the indicated concentration of compounds in a final volume of 200 μl. Cells are incubated for five days with replacement of the medium after 48 hours.

Analysis of alkaline phosphatase

At the end of the incubation period titration microplates overturn and medium decanted. Tablets are washed with 200 μl per well of PBS (0.15 M NaCl, 10 mm sodium phosphate, pH 7.4). Then PBS (SFR) is removed from the plates, carefully leaving a small amount of residual SFR, and this washing procedure was repeated once more. Then phosphate buffered salt solution is decanted and the inverted tablets gently soak a paper towel. After replacing the coating tablets incubated at -80°C for 15 min followed by thawing at room temperature for 10 minutes Then the tablets are placed on ice and add 50 ál of ice solution containing 5 mm p-nitrophenylphosphate, 0.24 mm MgCl2and 1 M diethanolamine (pH of 9.8). Then the tablets are heated to room temperature and give the opportunity to develop yellow okra is ke, associated with obtaining p-nitrophenyl (8 min). Tablets control at 405 nm in a spectrophotometer to read the tablets enzyme-linked immunosorbent assay (BIO-RAD, model 2550 EIA Reader).

Calculations

Curves dose-response, as well as the IC50were calculated using the method of weighted iterative nonlinear quadratic regression.

Table 11
NAMEThe CODE NAMESTRUCTUREStimulation of alkaline phosphatase test connectionsInhibition of alkaline phosphatase, incentive-reach 1Nm E2test connections
% stimulation of 1 nm E2*
(number of experiments)
IC50(nm)
(number of experiments)
EM-652.HClEM-652.HCl; EM-15381,88±0,26 (22)1,52±0,22 (18)
OH-TamoxifenEM-882 32,4±2,2 (8)31,9±6,0 (5)
OH-ToremifeneEM-88029,6±2,1 (6)72,1±7,6 (3)
IdoxifeneEM-75025,1±1,5 (5)>1000 (2)
GW-5638EM-17967,75±5,5 (2)No inhibition
DroloxifeneEM-83523,8±3,1 (7)291±115 (4)
Raloxifene LY 156758EM-110512,8±1,7 (8)3,39±0,9 (6)
LY 353381EM-166515,5±0,25 (5)1,87±0,07 (2)
Lasofoxifene (free bases of the tion) EM-311417,9 (1)4,24 (1)
TSE 424EM-35270,6 (1)of 5.84 (1)

*% stimulation of 1 nm E2=

OD 405 nm connection-OD 405 nm basal/OD 405 nm 1 nm E2OP 405 nm basal. Cm. also Labric et al. EM-652 (SCH 57068), a third generation SERM acting as pure antiestrogen in the mammary gland and endometrium, J. Steroid Biochem. and Mol. Bio.69,51-84, 1999.

Example 12

Action EM-652.HCl and FCE 424 on the proliferation of cell lines MCF-7 breast cancer man

Ways

Keeping a clean cell culture

MCF-7 cancer cells human mammary gland obtained from the American type culture collection #HTB 22 when the passage 147 and, as usual, is cultivated in not containing phenol red medium Needle-ham F12 modification of Dulbecco, with the above additions and 5% FBS. Cell line MCF-7 mammary adenocarcinoma person receive from the pleural effusion of a patient, a 69-year-old female Caucasian ethnicity. Using cells MCF-7 between the passages 148 and 165 and pereceived weekly.

The study of cell proliferation

Cells in their logarithmic last phase of the OST collect 0.1% Pancreatin (Sigma) and resuspended in an appropriate environment, containing 50 ng of bovine insulin/ml and 5% (vol./about.) FBS, double-processed-loaded dextran coal dust, to remove endogenous steroids. Cells were seeded in 24-hole culture tablets Falcon plastic (2 cm2/well) at a given density and provide an opportunity to adhesivity to the surface of the tablets within 72 hours After the medium replaced with fresh medium containing the indicated concentration of compounds diluted in 1000 times relative to the initial solution in 99% double-distilled ethanol in the presence or in the absence of E2. Control cells received only ethanol filler (0,1% EtOH, vol./vol.). Cells incubated during specific time intervals with the replacement of the medium at intervals of 2 or 3 days. The number of cells was determined by measurement of DNA content.

Calculations and statistical analysis

Curves dose-response, as well as the IC50calculated using the method of weighted iterative nonlinear regression least squares. All results are presented as mean ± SEM.

Table 12
NameThe code nameStimulation of DNA test connections Inhibition
1 nm E2stimulate DNA test connections
% stimulation
1 nm E2*
IC50(nm)
EM-652.HClEM-652.HCl;
EM-1538
no stimulationcoefficient was 0.796
TSE 424EM-3527no stimulation3,68

Example 13

Comparative effect on uterine weight, fat and lipid EM-652.HCl, TSE 424 and Lasofoxifene introduced, individually or in combination with EAT-652.l, ovariectomised rats-females

URMA-r-44-00

The purpose of this study was to compare the effect on uterine and vaginal histology following exposure to EM-652.HCl, TSE 424 and Lasofoxifene entered individually or in combination with EM-652.HCl, ovariectomised rats-females. For this purpose each compound administered orally for 20 days ovariectomised rats-females and at the end of the period of treatment to remove the uterus and fat and weighed.

Specification of experimental animals:

Appearance: Ruttus norvegicus

Line: Rat Sprague-Dawley (Crl:CD®(SD) BR VAF/Plus™)

Gender: female

Body weight: At the beginning of dosing body weight was the approximately 225-250,

Content & care

a) Content:

Rats were placed individually in cages stainless steel standard design on the acclimatization period and the period of the study.

b) Temperature and humidity:

Environmental conditions (temperature, humidity) in the premises of rats was continuously recorded using an automatic-equipped computing system. Specified conditions were: temperature 22±3°C and relative humidity 50±20%.

c)The alternating cycle of light and darkness:

Photoperiodic cycle was 12 hours light and 12 hours of darkness. These parameters are continuously recorded using an approved automatic-equipped computing system. The lighting was from 07:15 until 19:15.

d)Diet:

Certified forage for rodents (Lab Diet # 5002, pellets) and tap water were providedad libitum.

Randomization:

Rats were randomized into each group randomly during oophorectomy.

Group test:

Seventy rats distributed into 7 groups 9-11 animals for each of the research described in General terms below:

-
PROCESSINGSUSPENSION FOR DOSING
Dose
(mg/rat)
The intact
OVX-
OVX + EM-652.HCl0,5
OVX + EM-35270,5
OVX + EM-652.HCl + EM-35272,5 + 0,5
OVX + EM-35550,5
OVX + EM-652.HCl + EM-35552,5 + 0,5

Preparation of animals:

Animals from groups 2 to 9 were subjected oophorectomy General anesthesia, induced by isoflurane, on day 1 of the study.

Dosing:

The filler or the test compounds were given in the form of a suspension in 0.4% methylcellulose using oral catheter (0.5 ml/catheter/rat), starting from day 2 to day 21 of the study.

Body mass index:

Rats were weighed on day 1 of the study and at necropsy (DI 22, SD 22)

Method of killing:

24 hours after the last dosing starving during the night the rats under anesthesia induced with isoflurane, scored exsanguination through the abdominal aorta.

Retrieved organs and tissues

Uterine and retroperitoneal adipose tissue is recovered and weighed.

Table 13
ProcessingFinal body weight
(g)
Retroperitoneal adipose tissue (g)The weight of the uterus
(mg)
Cholesterol
(mmol/l)
Interny222±5**1,04±0,13*485,8±28,4**1,52±0,09**
OVX266±51,42±0,24152,9±4,71,91±0,09
OVX+EM-652.l238±4**0,63±0,08**173, 0mm±7,11,00±0,11**
OVX+TSE 424248±5**0,84±0,09**162,6±5,21,20±0,10**
OVX+TSE 424+
EAT-652.l
(2.5 mg)
241±1**0,91±0,17**180,2±5,20,99±0,07**
OVX+Lasofoxifene210±3**0,52±0,05**247,4±9,1**0,95±0,09**
OX+Lasofoxifene+EAT-652.l (2.5 mg) 234±3**0,74±0,10**182,3±4,60,95±0,07**

*p <0,05; **p <0,001, the experimental group versus OVX control.

EXAMPLES of PHARMACEUTICAL COMPOSITIONS

As an example, and not limitation the following are a few of pharmaceutical compositions, uses the preferred active SERM EM-800 or EM-652.l, individually or in combination with one of the preferred active precursors of the sex steroid hormone DHEA, androst-5-ene-3b,17b-diol 3-acetate or androst-5-ene-3b,17b-diol of dikemaskini. Instead of (or in addition to) EM-800 or EM-652.HCl, DHEA, androst-5-ene-3b,17b-diol 3-acetate or androst-5-ene-3b,17b-diol of dikemaskini can be used other compounds of this invention or a combination of them. The concentration of the active component can be varied within a wide range, as discussed here. The number and types of other components that may be included, as well-known in this field.

An example of A

The preferred SMRA-s (SERMs) of the present invention is administered orally.

Tablet

Componentwt. -%
(by weight of the total composition)
EM-652.HCl 5,0
Gelatin5,0
Lactose73,5
Starch16,5

Example B

Capsules

Componentwt. -%
(by weight of the total composition)
EM-652.HCl5,0
Lactose water80,0
Starch4,8
Microcrystalline cellulose9,8
Magnesium stearate0,4

Pharmaceutical composition for combination therapy

Example C

Tablet

Componentwt. -%
(by weight of the total composition)
EM-652.HCl5,0
DHEA15,0
Gelatin5,0
Lactose 58,5
Starch16,5

Example D

Gelatin capsule

Componentwt. -%
(by weight of the total composition)
EM-652.HCl5,0
DHEA15,0
Lactose water65,0
Starch4,8
Microcrystalline cellulose9,8
Magnesium stearate0,4

Sample KITS

As an example, and not limitation are a few sets that use the preferred active SMRA EM-800 or EM-652.l and the preferred precursor to sex steroid hormone DHEA, androst-5-ene-3b,17b-diol 3-acetate or androst-5-ene-3b,17b-diol dyamically.

Instead of (or in addition to) EM-800 or EM-652.HCl, DHEA, androst-5-ene-3b,17b-diol 3-acetate or androst-5-ene-3b,17b-diol of dikemaskini can be used other compounds of this invention or a combination thereof. The concentration of the active(s) component(s) can be varied in Shiro the ohms range, as was shown here. The number and types of other components that may be included, as well-known in this field.

An example of A

SMRA (SERM) of the present invention is administered orally, while the predecessor of the sex steroid hormone is administered transdermally.

The composition is based on SMRA (SERM) for oral administration (capsules)

Componentwt. -%
(by weight of the total composition)
EM-652.HCl5,0
Lactose water80,0
Starch4,8
Microcrystalline cellulose9,8
Magnesium stearate0,4

Composition based predecessor of sex steroid hormone for local use (gel)

Componentwt. -%
(by weight of the total composition)
DHEA10,0
Triglyceride Caprylic-capric acid (Neobee M-5)Hexyleneglycol15,0
Transcutol (onomatology 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 B

SMRA (SERM) and the predecessor of the sex steroid hormone administered orally.

Composition esteroidales antiestrogen for oral administration (capsules)

Componentwt. -%
(by weight of the total composition)
EM-652.HCl5,0
Lactose water80,0
Starch4,8
Microcrystalline cellulose9,8
Magnesium stearate0,4

The composition of the precursor Polo is wow steroid hormone for oral administration (capsules)

Componentwt. -%
(by weight of the total composition)
DHEA15,0
Microcrystalline cellulose84,6
Magnesium stearate0,4

In the above compositions of EM-800 or EM-652.HCl, you can substitute other SMRA s and DHEA, androst-5-ene-3b,17b-diol 3-acetate or androst-5-ene-3b,17b-diol dyamically can be replaced by other inhibitors of sex steroid hormone. The above formulations may include more than one SMRA or more than one predecessor, and in this case, it is preferable that the combined mass percentage (active component) corresponded to the mass percent of one predecessor or one SMRA shown in the above examples.

This invention has been described in the preferred embodiments and examples and is not limited to them. For specialists in this area is evident over a wide range of applicability and scope of the present invention, which is limited to only the supplied here by the claims.

1. A method of treating or reducing the risk of Vozniknovenie what I insulin resistance, includes introduction to the subject in need of such treatment or reduction, a therapeutically effective amount of a selective modulator of estrogen receptor, which is an EM-652.HCl:

2. The method according to claim 1, further comprising introducing a therapeutically effective amount of estrogen selected from the group consisting of estradiol and premarin, or predecessor of sex steroid hormone selected from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androst-5-ene-3b,17b-diol and compounds, turninvivoin one or the other predecessor.



 

Same patents:

FIELD: chemistry, pharmacology.

SUBSTANCE: invention relates to medications, capable of inhibiting Na+/H+-exchange (NHE-exchangers, NHE inhibitors).

EFFECT: increased efficiency of inhibitors.

3 cl, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medication for treatment or prevention of disease, which developed on the basis of structural and/or functional, and/or compositional changes of lipids in cell membranes, selected from cancer, vascular diseases, inflammatory diseases, metabolic diseases, obesity and excessive body weight, neurological or neurodegenerative disorders, which represents compound of formula COOR1-CHR2-(CH2)a-(CH=CH-CH2)b-(CH2)c-CH3 (I) or its pharmaceutically acceptable salts and derivatives, selected from esters, ethers, alkyl, acyl, phosphate, sulfate, ethyl, methyl or propyl; in which a and c can have independent values from 0 to 7; b can have independent values from 2 to 7, where R1 is selected from the following radicals: H, Na, K, CH3O, CH3-CH2O and OPO(O-CH2-CH3)2, and R2 is selected from the following radicals: OH, OCH3, O-CH3COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, NOH, F, HCOO and N(OCH2CH3)2.Invention also relates to application of formula (I) compound and pharmaceutical composition, which contains it.

EFFECT: medications, based on claimed compound, are more efficient than medications of preceding level of technology.

22 cl, 7 dwg, 16 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to compounds, represented by formula (I) , where X1 and X2 independently represent CH or N; ring U represents benzene ring, pyrazole ring, 1,2,4-oxadiazole ring, 1,2,4-thiadiazole ring, isothiazole ring, oxazole ring, pyridine ring, thiazole ring or thiophene ring, m represents integer number, which has values from 0 to 1; n represents integer number, which has values from 0 to 3; R1 represents hydroxygroup or C1-6 alkyl; R2 represents any of (1)-(3): (1) halogen atom; (2) hydroxygroup; (3) C1-6 alkyl or C1-6 alkoxy, each of which can independently contain any group, selected from group of substituents α; group of substituents α includes fluorine atom and hydroxygroup, or its pharmaceutically acceptable salt. Invention also relates to pharmaceutical composition, possessing inhibiting activity with respect to xanthenes oxidase, including formula (I) compound or its pharmaceutically acceptable salt as active ingredient.

EFFECT: derivative, which contains condensed ring structure, intended as means for prevention and treatment of disease, associated with abnormal level of uric acid in serum.

15 cl, 11 tbl, 126 ex

FIELD: medicine.

SUBSTANCE: method involves a combination of peloid applications and ultrasonic exposure. Peloid is applied on anterolateral thighs and an anterior abdominal wall. The applications are alternated every second day. Peloid is silt sulphide mud of Melkovodnenskiy deposit. The mud is applied in layer 10 mm thick at temperature 34-36°C. The applications are exposed to ultrasound at frequency 880 kHz in a continuous mode. The applications of the anterior abdominal wall are exposed to ultrasound at intensity 0.2-0.4 Wt/cm2 for 5 - 10 minutes. The anterolateral thighs are exposed to ultrasound at intensity 0.4-0.6 Wt/cm2, for 6-8 minutes for each thigh. Upon the completion of the procedure, the patient is covered with a hydrophobic tissue and left for 10 minutes. The therapeutic course is 10 procedures.

EFFECT: reducing obesity effectively by exposing on the hormonal activity of fat tissue, maintains the results obtained within three months.

2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to versions of a compound of formula where R1 is a hydrogen atom; R2 is a lower alkyl group; P is H; , where P1, P2 and P3 are identical or different and are selected from a hydrogen atom, a lower alkyl group and a C14-C22 alkenyl group substituted with a lower alkyl group; or where P1 is an alkenyl group, and each of P2 and P3 is a hydrogen atom; and Y is a C14-C22 alkenyl group with at least one double bond having a Z-configuration, and having a first double bond at the third carbon-carbon bond from the omega (ω)-end of the carbon chain, capable of lowering the level of triglycerides and cholesterol, a pharmaceutical or lipid composition based on the disclosed compounds, as well as use (versions) of the disclosed compounds.

EFFECT: high efficiency of using compounds.

32 cl, 6 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics, and aims at the prevention and treatment of hypovitaminosis and the normalisation of metabolism. The drug preparation contains vitamin A, vitamin D3, vitamin E, vitamin C; a selenium compound is presented by DAFS-25 in the following ratio of the ingredients in 1 l of the solution: vitamin A - 25.0-35.0 ml, vitamin D3 - 0.03-0.05 ml, vitamin E - 55.0-65.0 g, vitamin C - 90.0-110.0 g, DAFS-25 - 0.2-0.4 g, polysorbate-80 - 190.0-210.0 ml, 2-pyrrolidone 39.0-41.0 ml, distilled water - up to one litre.

EFFECT: using the declared invention enables increasing the immune status in poultry, normalising the antioxidant and detoxifying systems, improving the livability, egg production and meat production along with reducing the feed consumption per a unit of product.

3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to cardiology, and may be used for treating and preventing arterial hypertension with metabolic syndrome. That is ensured by adding the food ration with the functional foodstuff 'Samarskiy Zdorovyak' No 61 in a min daily dose of 33.3 g per one intake - with breakfast or lunch or dinner with underlying drug-induced therapy.

EFFECT: enabled treatment and prevention of arterial hypertension with metabolic syndrome

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to medicine. A pharmaceutical formulation for the treating diseases associated with endothelial dysfunction contains an active ingredient presented by a methyl pyridine derivative - 1.0-6.0 wt %; purine - 10.0-80.0 wt % and additive agents - the rest. The active substance is presented by compounds of a group: 3 -(N,N-dimethyl carbamoyloxy)-2-ethyl-6-methylpyridinium succinate, 3-methylpyridinium succinate, 2-ethyl-6-methyl-3-hydroxypyridinium hydrochloride, 6-trichloromethyl-2-chloropyridine (nitrapyrin), 2-ethyl-6-methyl-3-hydroxypyridine succinate. Purine is presented by inosine, adenosine, hypoxanthine. The pharmaceutical formulation may be presented in the form of injections, lyophilisate, solid capsules, tablets and suppositories.

EFFECT: formulation according to the invention provides creating the stable drug dosage form which considerably exceeds the existing analogues in pharmacodynamics activity on the endothelial dysfunction and toxicological properties.

4 cl, 4 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: present compounds can be used, for example, in treating diseases of the central nervous system, peripheral nervous system, cardiovascular system, pulmonary system, gastrointestinal system and the endocrine system.

EFFECT: described compounds are useful in treating a range of diseases or conditions in which interaction with the histamine H3 receptor is beneficial.

9 cl, 216 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel phenylaminopyrimidine compounds of formula I, which are JAK kinase inhibitors. In particular, these compounds selectively act on JAK2 kinase. The compounds can be used to treat diseases such as immunological and inflammatory diseases; hyperproliferative diseases, myeloproliferative diseases; viral diseases; metabolic diseases; and vascular diseases. In the compound of formula I , Q and Z are independently selected from N and CR1; R1 is independently selected from hydrogen, halogen, R2, OR2, OH, R4, OR4, CN, CF3, (CH2)nN(R2)2, where n equals 1,2 or 3, NO2, R2R4, NR2SO2R3, COR4, NR2COR3, CO2H, CO2R2, NR2COR4, R2CN, R2OH, R2OR3 and OR5R4; or two substitutes R1 together with carbon atoms with which they are bonded form an unsaturated 5- or 6-member heterocyclic ring containing 1-4 N atoms; R2 is C1-4alkyl; R4 is R2, C2-4alkenyl or phenyl; R4 is NH2, NHR2, N(R1)2, substituted or unsubstituted morpholine, CH2morpholine, substituted or unsubstituted thiomorpholine, substituted or unsubstituted thiomorpholino-1-oxide, substituted or unsubstituted thiomorpholino-1,1-dioxide, substituted or unsubstituted piperazinyl, substituted or unsubstituted piperidinyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted imidazolyl, substituted or tetrahydrofuranyl unsubstituted and substituted or unsubstituted tetrahydropyranyl; R5 is C2-4alkylene; R6-R9 are independently selected from H, RXCN, halogen, substituted or unsubstituted C1-4alkyl, OR1, CO2R1, N(R1)2, NO2 and CON(R1)2, wherein at least one of R6-R9 is RXCN; the rest of the values of the radicals are given in the claim.

EFFECT: high efficiency of treatment.

29 cl, 7 dwg, 2 tbl, 93 ex

FIELD: medicine.

SUBSTANCE: what is presented is using oestriol for preparing a pharmaceutical dosage form for the vaginal administration of oestriol in a dose of 0.1 mg/day or less for preventing and/or treating urogenital atrophy in females prone to a high probability of the development of oestrogen-dependent tumour or the patients suffering or suffered from oestrogen-dependent tumour.

EFFECT: it is shown that oestriol has the limited absorption from the declared dosage form that reduces its systemic exposure as compared to the control preparation that is "Ovestinon®" cream containing oestriol 0,1% with the effective elimination of vaginal atrophy.

24 cl, 1 dwg, 7 tbl

FIELD: food industry.

SUBSTANCE: invention relates to a method for preparation of water dispersions of phytosterols; preparation of a water dispersion of one or multiple phytosterols includes the following stages: a) dispersing one or multiple phytosterols, water and one or multiple sodium or potassium salts of a fatty acid (10-200 mg of such salt(s) taken per 1 g of phytosterol(s)) in a disperser at a temperature within the range of 140°C - 250°C to produce a water emulsion of such phytosterol(s) and b) emulsion cooling to produce a water dispersion of such phytosterol(s). Alternatively, the method envisages the following stages. a) dispersing one or multiple phytosterols, water and one emulsifier (10-200 mg of such emulsifier taken per 1 g of phytosterol(s)) in a disperser at a temperature within the range of 140°C - 250°C to produce a water emulsion of such phytosterol(s); b) emulsion supply into a heated homogeniser at a temperature within the range of 140°C - 250°C to produced a homogenised emulsion and c) emulsion cooling to produce a water dispersion of phytosterol(s).

EFFECT: produced are highly stable dispersions with high concentration of phytosterols.

14 cl, 15 tbl, 20 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to gynaecology. The method involves the daily transcranial electrical stimulation and drug administration. The transcranial electrical stimulation is characterised by current intensity 1.0÷1.5 mA, 20 - 30 minutes for 10÷15 days. In addition, the patients intakes the phytocomposition 'Myrrasyl-1'. The phytocomposition is taken 15÷20 minutes before meals 2 tablets 2 times a day for 10÷15 days. Thereafter, the phytocomposition 'Myrrasyl-1' is administered daily for 20÷30 days, 1 tablet 2 times a day; the preparation 'Sagenit' is administered before meals 1 tablet a day.

EFFECT: method provides higher clinical effectiveness in the patients suffering the hepatobiliary system by reducing the current intensity during the transcranial electric stimulation.

2 ex, 2 cl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, veterinary science and pharmaceutical industry. The invention provides using triterpene glycosides from holothuria that is frondoside A or a complex of frondoside A and cholesterol as an agent for inhibiting the multiple drug resistance of tumour cells, and for preparing a pharmaceutical composition inhibiting the multiple drug resistance of tumour cells.

EFFECT: using the invention enables extending the range of products inhibiting the multiple drug resistance of tumour cells.

2 cl, 4 ex, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmaceutical industry and concerns a dry powder preparation containing micronised: salmeterol xinafoate and fluticasone propionate. The preparation contains a carrier consisting of lactose of average particle size 100-120 mcm and sodium benzoate. What is also described is a method for producing the preparation.

EFFECT: formulation possesses higher percentage of a respirable fraction of the active substances.

4 cl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, particularly to using phytoecdysone for preparing a composition for reducing abdominal fat in mammals. Using 20-hydroxyecdysone for preparing the composition for reducing abdominal fat in mammals with 20-hydroxyecdysone introduced as pure 20-hydroxyecdysone, or in the form of saponin-free quinoa extract.

EFFECT: presented 20-hydroxyecdysone effectively reduces abdominal fat in mammals.

8 cl, 5 dwg, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are presented: using 3-alpha-androstanediol and 5-HT1A agonist for preparing a drug for sexual dysfunction on demand, wherein above 3-alpha-androstanediol and 5-HT1A agonist substantially release at the same time approximately from one to two hours before sexual activity, thereby peak effects of 3-alpha-androstanediol and 5-HT1A agonist (particularly, of flesinoxan) coincide in time at least partially; the respective pharmaceutical composition and kit for treating sexual dysfunction on demand. The declared drug, composition and kit may additionally contain type 5 phosphodiesterase inhibitor (PDE5 - sildenafil).

EFFECT: invention provides a short (over a few hours) superphysiological peak of blood 3-alpha-androstanediol that provides calling the greatest possible attention to erotic symbols and sexual motivation, and relieving the depressed behavioral responses to the maximum.

11 cl

FIELD: chemistry.

SUBSTANCE: invention relates to steroid ligands for use in nuclear receptor-based inducible gene expression systems. The invention also relates to methods of modulating the expression of genes of interest using a system containing one or more nuclear receptor complexes and one or more steroid ligands. Other aspects include ligand compositions including therapeutic compositions.

EFFECT: improved method.

21 cl, 1 ex, 9 tbl, 20 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is presented is the use of oestriol for preparing a pharmaceutical composition for vaginal administration for preventing and/or treating urogenital atrophy due to estrogen deficiency in women, wherein said composition is administered so that the patient receives a dose of oestriol 0.3 mg/day or less with this administration being daily or once every two days, once every three days or once a week for at least 3 weeks and a related method for preventing and/or treating. It is shown that the administration of oestriol in a dose of 10 times less than the standard one; it eliminates the state of vaginal atrophy.

EFFECT: invention provides the therapeutic effectiveness similar to the responses to the modern methods of treating, but with greater safety leading to a better quality of life.

14 cl, 10 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: pharmaceutical composition contains as a first active agent, 6β, 7β; 15β, 16β-dimethylenene-oxo-17α-pregn-4-ene-21,17-carbolactone (drospirenone) in an amount according to a daily dose after the administration of the composition and making approximately 2 to approximately 4 mg, and as a second active agent, 17a-ethinylestradiol (ethinylestradiol) in an amount according to a daily dose and making approximately 0.01 mg to approximately 0.05 mg together with one or more pharmaceutically acceptable carriers or additives. The composition contains drospirenone applied on inert carrier particles. A method for preparing a pharmaceutical composition involves spraying of the drospirenone and ethinylestradiol solution on the inert carrier particles. The pharmaceutical preparation according to the invention contains a number of separately packed and individually taken daily dosage units of the described compositions in a single package used for oral administration for at least 21 days running with said daily dosage units containing the combination of drospirenone and ethinylestradiol. The composition may additionally contain 7 and less daily dosage units containing no active agent, or containing ethinylestradiol only.

EFFECT: invention provides higher oral bioavailability of drospirenone.

20 cl, 5 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of general formula I , where R1 is a hydrogen atom, a lower alkyl, CD3, -(CH2)n-CHO, -(CH2)n-O-lower alkyl, -(CH2)n-OH, -(CH2)n-cycloalkyl or is a heterocycloalkyl (where the heterocycloalkyl is a partially unsaturated ring containing up to 6 carbon atoms, at least one of which is substituted with O); R2 is a hydrogen atom, a halogen atom, hydroxy, lower alkyl, di-lower alkyl, -OCH2-O-lower alkyl or lower alkoxy; or the piperidine ring along with R2 forms a spiro-ring selected from 4-aza-spiro[2,5]oct-6-yl; Ar is an aryl or heteroaryl (where the heteroaryl is a cyclic aromatic hydrocarbon radical consisting of one ring and containing 6 ring atoms, and which contains at least one heteroatom selected from N), optionally having one, two or three substitutes selected from a halogen atom, lower alkyl, lower alkyl having as substitutes, a halogen atom, a lower alkoxy having as substitutes, a halogen atom, cycloalkyl, lower alkoxy, S-lower alkyl, heterocycloalkyl (where the heterocycloalkyl is a partially unsaturated ring containing up to 6 carbon atoms, at least one of which is substituted with N), or optionally having as substitutes, phenyl, optionally having R' as substitutes, and R' is a halogen atom, CF3, lower alkyl, lower alkoxy or a lower alkoxy having as substitutes, a halogen atom, or is a heteroaryl (where the heteroaryl is a cyclic aromatic hydrocarbon radical consisting of one ring and containing 6 ring atoms, and which contains at least one heteroatom selected from N and S); R is a lower alkyl, heterocycloalkyl (where the heterocycloalkyl is a partially unsaturated ring containing up to 6 carbon atoms, at least one of which is substituted with O), aryl or heteroaryl (where the heteroaryl is a cyclic aromatic hydrocarbon radical consisting of one ring and containing 6 ring atoms, and which contains at least one heteroatom selected from N), Where the aryl and heteroaryl optionally have as substitutes, one or two R'; n equals 0, 1, 2 or 3; or to a pharmaceutically acceptable acid addition salt, a racemic mixture or a corresponding enantiomer and/or optical isomer of said compound. The invention also relates to pharmaceutical compositions based on a glycine reuptake inhibitor of a compound of formula I.

EFFECT: obtaining novel compounds and a pharmaceutical composition based thereon, which can be used in medicine to treat neurological and psychoneurological disorders.

22 cl, 1 tbl, 128 ex

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