RU2413516C2 - Gaba-steroid antagonists and application thereof in treating cns disturbances - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: present group of inventions refers to medicine, namely to psychiatry, and concerns the use of GABA-steroid antagonists for treating central nervous system disturbances. That is ensured by administration of compounds of the group of GABA-steroid antagonists exhibiting blocking action in relation to 3alpha-hydroxy-5alpha/beta-steroids practically without an antagonist effect on a receptor A of gamma-aminobutyric acid (GABA), and compounds having selective antagonist effect on said receptor.

EFFECT: invention provides treatment and/or prevention of CNS disturbances caused by action of 3alpha-hydroxy-5alpha/beta-steroid, as well as GABA-A overstimulation.

9 cl, 4 tbl, 2 dwg, 2 ex

Description

The invention relates to the treatment, relief of symptoms or prevention of diseases of the central nervous system (CNS) associated with and / or caused by GABA-steroids, in particular to specific steroid compounds intended for this purpose, their use for the manufacture of pharmaceutical preparations for the indicated treatment, and also methods of treatment, including their use.

BACKGROUND OF THE INVENTION

Metabolites of the sex and stress hormones pregnenolone, progesterone, deoxycortosterone, cortisone and cortisol, known as pregnanolols, as well as metabolites of testosterone, androstenedione and dehydroepiandrosterone, are objects of various studies that at least partially reveal their role in the neurological signaling system in mammals.

Steroids causing CNS symptoms and diseases of interest to the present invention have structural similarities, which are the presence of the 3alpha-hydroxy group, 5alpha or 5beta pregnan-steroid backbone and the ketone or hydroxy group at position 17, 20 or 21.

For steroids, including 3alpha-hydroxy-5alpha / beta-pregnan-20-one / ol or 3alpha-hydroxy-5alpha / beta-androstan-17-one / ol, it is shown that they are important specific amplifiers of the gamma-aminobutyric acid receptor ( A) (GABA-A). They bind to the GABA-A receptor, and their action is aimed at enhancing the effect of GABA in terms of the frequency of finding the GABA-A receptor in the open state and the duration of the open state. This effect is similar to the effect of both benzodiazepines and barbiturates. These steroid compounds, however, have a binding site different from the binding sites of these compounds. Examples of such GABA-enhancing steroids and their numbers according to Chemical Abstracts Registry (CAS) are presented in Table 1.

The nomenclature of steroids has not finally settled down, therefore, in this application the nomenclature developed by the International Union of Theoretical and Applied Chemistry (IUPAC) is used.

Table 1 GABA Steroid Nomenclature IUPAC Nomenclature CAS Number 3alpha-hydroxy-5alpha-pregnan-20-one 516-54-1 3alpha-hydroxy-5beta-pregnan-20-one 128-20-1 3alpha, 21-dihydroxy-5alpha-pregnan-20-one 567-02-2 3alpha, 21-dihydroxy-5beta-pregnan-20-one 567-03-3 3alpha, 11beta, 17alpha, 21-tetrahydroxy-5beta-pregnan-20-one 53-02-1 3alpha-11beta, 17alpha, 21-tetrahydroxy-5alpha-pregnan-20-one 302-91-0 3alpha-17alpha, 21-trihydroxy-5alpha-pregnan-11,20-dion 547-77-3 3alpha-17alpha, 21-trihydroxy-5beta-pregnan-11,20-dion 53-05-4 3alpha-hydroxy-5alpha-androstan-17beta-ol 1852-53-5 3alpha-hydroxy-5beta-androstan-17beta-ol * 3alpha-hydroxy-5alpha-androstan-17-one 53-41-8 3alpha-hydroxy-5beta-androstan-17-one 53-42-9 * CAS number not found

For some of these steroids, it has been shown that they have the ability to cause anesthesia at high pharmacological doses. They can also be used as antiepileptic agents or as sleeping pills. It has also been shown that some of these compounds have anxiolytic effects in animal experiments. However, to achieve these effects, high concentrations or high doses of the compounds are necessary. In addition, such effects are manifested in a strong form.

With regard to their direct effect on the central nervous system, as mentioned above, these compounds are similar to benzodiazepines and barbiturates. However, they also have the same adverse effects that are usually associated with benzodiazepines and barbiturates. The adverse effects of endogenous 3alpha-hydroxy-pregnan-20-on-steroids or 3alpha-hydroxy-androstane steroids underlie the negative effects on the central nervous system caused by these steroids. Since 3alpha-hydroxy-pregnan steroids and 3alpha-hydroxy-15 androstane steroids are formed in the body and are metabolites of steroid hormones and some of them are necessary for life, their production cannot be easily disrupted. These steroids are produced in large quantities for several days to weeks during the luteal phase of the menstrual cycle, i.e. after the release of an egg from a mature ovarian follicle during pregnancy and during stress. They are also produced in the brain.

Diseases caused by 3alpha-hydroxy steroids

Disorders caused by the action of endogenously formed 3alpha-hydroxy-5alpha steroids or 3alpha-hydroxy-5beta steroids on the GABA-A receptor are well studied and explained. It is also known that 3alpha-hydroxy-5alpha / beta-steroids can cause tolerance to themselves and other similar substances after exposure and that withdrawal symptoms are observed after discontinuation of 3alpha-hydroxy-5alpha / beta-steroids. In general, it is known that 3alpha-hydroxy-5alpha / beta-steroids cause CNS disorders through three possible indicated mechanisms: a) direct exposure, b) tolerance induction and c) withdrawal effect. These mechanisms are discussed in more detail below.

a) Direct exposure

It has been found that 3alpha-hydroxy-5alpha / beta-steroids can directly cause inhibition of CNS functions. Examples of symptoms caused by direct exposure to 3alpha-hydroxy-5alpha / beta-steroids are sedation, fatigue, memory impairment, learning disabilities, impaired motor function, clumsiness, increased appetite and insatiable hunger, depressed mood, irritability and depression, which are the main symptoms premenstrual dysphoric disorders, premenstrual syndrome and worsening in case of minor epilepsy. Examples of this direct effect can also be divided into sedative and anesthetic effects; impaired motor function; effects on cognitive function, memory and learning; worsening with minor epilepsy; premenstrual symptoms; mood changes; induction of anxiety in test animals; hyperphagia and increased appetite; insatiable hunger, etc.

b) Tolerance

Continuous and prolonged exposure to 3alpha-hydroxy-5alpha / beta steroids causes disturbances in the functioning of the GABA-A receptor system. Tolerance is formed, and this kind of tolerance is the initial stage of the process, which ultimately leads to the development of sensitivity to stress, difficulty concentrating, loss of control of motivation and depression. It was also found that the effect of 3alpha-hydroxy-5alpha / beta-steroids is a factor enhancing drug / drug dependence. This phenomenon has been the subject of a large number of studies. To date, the main subjects of research were the following issues: negative regulation and decreased GABA function after prolonged secretion of large amounts of 3alpha-hydroxy-5alpha / beta steroids; decreased sensitivity to benzodiazepine and 3alpha-hydroxy-5beta steroid in PMS and dependence induction.

c) withdrawal syndrome

Continuous but shorter exposure to 3alpha-hydroxy-5alpha / beta-steroids results in a withdrawal effect at the end of the exposure. This phenomenon is observed, for example, naturally during menstruation with the cessation of the production of 3alpha-hydroxy-5alpha / beta-steroids by the corpus luteum of the ovary. Such withdrawal phenomena are also observed after childbirth (post partum), with the cessation of production of 3alpha-hydroxy-5alpha / beta-steroids by the placenta. The same phenomenon was also noted at the end of the stress period, for example, at the end of work-related stress during the weekend. In response to stress, the adrenal glands produce 3alpha-hydroxy-5alpha / beta-steroids. Upon termination of their production cancellation syndrome may be observed.

Partial epilepsy, in which the patient forms an epileptic focus in the cerebral cortex and worsens during the period of withdrawal during menstruation, is an example of pathological conditions that are influenced by such a withdrawal phenomenon. This phenomenon is called "menstrual epilepsy." Other examples are menstruation-related migraines, stress-related migraines and mood changes after childbirth (post partum). Similar symptoms and pathological conditions are induced when treated with steroid hormones, such as those used in oral contraceptives, postmenopausal hormone replacement therapy, steroid treatment for inflammatory diseases, and during the administration of anabolic / androgenic steroids. The induction of direct effects, the development of tolerance and withdrawal are based on similar mechanisms.

Disturbances caused by over stimulation with gamma-aminobutyric acid (GABA)

It is known that intensive stimulation of GABA itself in certain cases inhibits the function of learning and memory. GABA overproduction impairs the function of short-term learning and memory. This phenomenon has particular clinical significance in the elderly. In this case, the antagonism of the GABA effect could be useful and potentially applicable for the treatment of memory impairment. However, if the GABA antagonism is too strong, it can be dangerous for the patient, for example, causing convulsions. Therefore, a search is underway for specific antagonists that selectively block pathological conditions induced by GABA steroids, and antagonists that also affect the GABA-activated chloride ion flow through the GABA-A receptor, but which are essentially not capable of completely blocking the GABA-A receptor.

The present inventors have unexpectedly discovered substances that mainly have an antagonistic effect on the action of GABA, as well as substances that mainly affect the effect of GABA-steroids on the GABA-A receptor. It is important to distinguish between these effects, since the antagonism of the action of GABA-steroids is manifested in other cases than the direct partial antagonism of the action of GABA itself.

Currently, the treatment of Alzheimer's disease (AD) and mild cognitive impairment (LKN) for the most part is not carried out in the absence of preventive or therapeutic therapy. The fact that GABA-A receptor agonists, such as GABA-steroids, benzodiazepines and ethanol, is associated with memory loss and that AD affects the functioning of most GABA-A receptors in brain regions, makes GABA-A receptors a potential therapeutic target for AB and LCN. In contrast to GABA-A receptor agonists associated with amnesia, inverse agonists or antagonists weaken the function of GABA-A receptors. GABA-steroid antagonists and GABA antagonists have been shown to improve the performance of learning and memory tasks in model animals. Unfortunately, non-selective and complete blocking ligands also cause convulsions. Thus, there is a need for partial and selective blockers (Maubach, K., GABA-A receptor subtype selective cognition enhancers, Curr. Drug Targets CNS Neurol Disord., 2003 (2) 233-239). Current market-leading perceptual enhancing drugs have significant adverse effects with minimal effectiveness, and there is a need for new substances, which is the subject of the present invention.

State of the art

As is known to the authors of the present invention, steroid substances that selectively inhibit the action of GABA-steroids or mainly inhibit the effect of GABA on the GABA-A receptor have not been described previously. In the prior art, only the general effect on all the effects of GABA steroids has been described.

Prince and Simmons (Prince and Simmons, Neuropharmacology, vol. 32, no. 1, pp. 59-63, 1993) used a model based on membrane fractions of the whole brain of male rats. In these subfractions of the whole brain homogenate, the authors used the binding of benzodiazepine, 3H-flunitrazepam as a model to evaluate the effect of steroids and change the conformation of the GABA-A receptor. This test was proposed as an indicator of allosteric modulation of the GABA-A receptor. However, the relationship between the change in the binding of flunitrazepam (FN3) and the change in the flux of chloride ions during GABA stimulation has not been precisely established, and the change in binding cannot be considered either as evidence of a change in the flux of chlorine ions through the GABA-A receptor, or as evidence of a change in the functioning of the GABA-A receptor . Changing the flow of chloride ions is an important effect.

The main question, that is, whether there is a relationship between the change in the binding of FN3 and the excitability of neurons, is even less studied, and such conclusions cannot be made only on the basis of results on the binding of FN3. A change in the FN3 binding capacity or the absence of such a change in binding ability does not mean a change or absence of a change in the activity of neurons or a GABA-mediated flow of chloride ions.

It is also well known that the GABA-A receptor contains several subunits that can be combined in various ways. Interestingly, in certain combinations there is no steroid recognition site. It is also known that the effect of the steroid on the binding of the convulsive substance TBPS (t-butylbicyclo-phosphorothionate, t-butylbicyclo-phosphorothionate, TBPT) varies in different regions of the brain. In addition, TBPS binding is known to change during the menstrual cycle in female rats, which indicates that the change in effect is associated with the production of ovarian hormones. It is clear that such changes associated with the menstrual cycle cannot be detected in male rats used in the studies of Prince and Simmons (see above).

US Patents 5,232,917 (Bolger et al.) And 5,939,545 (Upasani et al.) Describe a number of 3alpha-hydroxy steroids. Both of these patents relate to agonistic modulation of the GABA-A receptor. In other words, the subject of both patents is the benzodiazepine-like effect of 3alpha-hydroxy-5alpha / beta-steroids. A common feature of all steroids, which are modulators of the GABA-A receptor, is a single 3alpha-hydroxy structure. The 3beta-hydroxy steroids mentioned in these documents were used only as controls to show the specificity of 3alpha-hydroxy steroids. For steroids having only a 3-beta hydroxy structure, no modulating effect on the GABA-A receptor was shown. In all cases when an effective modulating effect on the GABA-A receptor was revealed, steroids contained a 3alpha-hydroxy group.

International publication WO 99/45931 (Backstrom & Wang) describes the antagonistic effect of one particular steroid, namely 3beta-hydroxy-5alpha-pregnan-20-one, but there is no evidence regarding the effects of other 3beta-hydroxy steroids.

The antagonistic effect of 3 beta-OH-5 alpha-pregnan-20-one with respect to 3 alpha-OH-5 alpha / beta-pregnan-20-one was first described by Wang et al. (Wang MD, Backstrom T. and Landgren S. (2000) The inhibitory effects of allopregnanolone and pregnanolone on the population spike, evoked in the rat hippocampal CA1 stratum pyramidaie in vitro, can be blocked selectively by epiallopregnanolone. Acta Physiol Scand 169, 333-341). In this work, a dose-dependent antagonistic effect of 3beta-OH-5alpha-pregnan-20-one on two 3alpha-OH-5alpha / beta steroid was shown.

International publication WO 03/059357 (Backstrom et al.) Describes the use of pregnan steroids in the treatment of CNS disorders. This source does not mention the possibility of selective inhibition of the effects of GABA steroids or the effect on GABA itself.

The fact that 3-beta steroids can also affect the effect of GABA itself has been described by Wang et al (Mingde Wang, Yejun He, Lawrence N. Eisenman, Christopher Fields, Chun-Min Zeng, Jose Mathews, Ann Benz, Tao Fu, Erik Zorumski, Joe Henry Steinbach, Douglas F. Covey, Charles F. Zorumski, and Steven Mennerick. 3beta-Hydroxypregnane Steroids Are Pregnenolone Sulfate-Like GABAA Receptor Antagonists. The Journal of Neuroscience, May 1, 2002, 22 (9): 3366- 3375). In this work, the authors show that, with maximum stimulation of GABA-A receptors using GABA, certain 3-beta-hydroxy pregnan steroids can inhibit the action of GABA itself. But such 3-beta-hydroxy steroids mainly inhibit the action of GABA steroids on the GABA-controlled flow of chloride ions, and other steroids that would inhibit the action of GABA itself were not detected.

The unresolved problem is the identification of specific antagonists of the 3 alpha-hydroxy action of pregnan steroids with a weak inhibitory effect on GABA and the identification of partial antagonists of the action of GABA, the compounds of which are physiologically safe and suitable for pharmaceutical use and which, moreover, are applicable in physiologically acceptable doses.

Thus, one of the objectives of the present invention is the identification of such specific blockers, which are antagonists of GABA-steroids and partial antagonists of GABA itself, as well as the creation of affordable new pharmaceutical preparations and methods for treating, alleviating symptoms or preventing CNS disorders associated with steroids and / or caused by them.

Other tasks, related solutions and their advantages will become apparent to a person skilled in the art upon familiarization with the description of the invention, examples and claims.

SUMMARY OF THE INVENTION

According to the present invention, it was found that steroid compounds containing a hydrogen donor at the 3-beta position in the form of a hydroxy group and, in addition, having a hydroxy group at the 20 position, unexpectedly function as effective blockers of the action of 3alpha-hydroxy pregnan- steroids and have minimal activity directed against the GABA itself. Thus, they are useful as therapeutic agents for preventing and / or treating CNS disorders associated with or caused by steroids.

In addition, the inventors of the present invention unexpectedly found that pregnan steroids with an acetate group in the 3 position or in the 3, 20 and 21 positions are able to partially inhibit the effect of GABA itself on the GABA-mediated chloride ion flux. Therefore, they are useful as therapeutic agents, for example, for preventing and / or treating disorders in which the GABA function itself is overstimulated, as in the case of Alzheimer's disease.

The present invention provides four compounds exhibiting a weak antagonistic effect on the action of GABA, but having a significant effect against the action of 3alpha-hydroxy-5alpha / beta-steroids (GABA-steroids) for use to prevent and / or treat central nervous system disorders associated with steroids or caused by them, and for the production of pharmaceuticals for these purposes, namely: 5alpha-PREGNAN-3beta, 20beta-DIOL, 5beta-PREGNAN-3beta, 20alpha-DIOL, 5alpha-PREGNAN-3beta, 20alpha-DIOL, 5beta-PREG -3b the one, 20beta-diol (see. Table 2).

table 2 List of 3beta-hydroxy-5alpha / beta-pregnan steroids that antagonize the enhanced 3alpha-hydroxy-5alpha-pregnan-20-GABA-mediated flow of chloride ions, but which have a weak antagonistic effect on GABA, tested in the present invention Substance* The product's name 1) 5alpha-PREGNAN-3beta, 20beta-DIOL UC1011 2) 5 beta-PREGNAN-3 beta, 20 alpha-DIOL UC1013 3) 5alpha-PREGNAN-3beta, 20alpha-DIOL UC1019 4) 5beta-PREGNAN-3beta, 20beta-DIOL UC1020 * Substances obtained from Steraloids Inc., USA, and from Sigma Chemicals Co.

In addition, the present invention provides ten compounds exhibiting a partial antagonistic effect on the action of GABA for use in the prevention and / or treatment of disorders resulting from over-stimulation of GABA and thus causing CNS disorders, and for the preparation of pharmaceutical preparations for these purposes, namely: 5alpha-PREGNAN-3beta-OL-20-ONE ACETATE; 5beta-PREGNAN-3beta-OL-20-OH; 5beta-PREGNAN-3beta, 21-DIOL-20-OH; 5alpha-PREGNAN-3beta, 20beta-DIOLA 3-ACETATE; 5beta-PREGNAN-3beta, 20beta-DIOLA 3-ACETATE; 5alpha-PREGNAN-3beta, 20alpha-DIOLA 3-ACETATE; 5alpha-PREGNAN-3beta, 20beta-DIOLA DIACETATE; 5beta-PREGNAN-3beta, 20alpha-DIOL DIACETATE; 5beta-PREGNAN-3beta, 20beta-DIOLA DIACETATE; 5beta-PREGNAN-3beta, 21-DIOL-20-ONA 21-ACETATE (see table 3).

One aspect of the present invention is the therapeutic use of the above compounds as blocking agents against the action of 3alpha-hydroxy-pregnan-steroids and the action of GABA. In addition, the present invention provides the use of these substances for the manufacture of pharmaceutical preparations for the treatment of many specific CNS disorders associated with or caused by steroids, and for use in methods of treatment according to the appended claims, which are incorporated herein by reference.

Table 3 List of 3beta-hydroxy-5alpha / beta-pregnan-steroids with strong antagonistic effect on GABA tested in the present invention Substance* The product's name 1) 5alpha-PREGNAN-3beta-OL-20-OH ACETATE UC1012 2) 5beta- PREGNAN-3beta-OL-20-OH UC1014 3) 5beta-PREGNAN-3beta, 21-DIOL-20-OH UC1015 4) 5alpha-PREGNAN-3beta, 20beta-DIOLA 3-ACETATE UC1021 5) 5beta-PREGNAN-3beta, 20beta-DIOLA 3-ACETATE UC1022 6) 5alpha-PREGNAN-3bet 20alpha-DIOLA 3-ACETATE UC1023 7) 5alpha-PREGNAN-3beta, 20beta-DIOLA DIACETATE UC1024 8) 5beta-PREGNAN-3beta, 20alpha-DIOLA DIACETATE UC1025 9) 5beta-PREGNAN-3beta, 20beta-DIOLA DIACETATE UC1026 10) 5beta-PREGNAN-3beta, 21-DIOL-20-ONA 21-ACETATE UC1030 * Substances obtained from Steraloids Inc., USA, and from Sigma Chemicals Co.

Brief Description of the Drawings

The invention is described in more detail in the following description and examples with reference to the tables included in the description and the accompanying graphic materials, in which Fig. 1 shows the relationship between the inhibition of the maximum GABA-mediated flow of chloride ions (30 μM GABA) and the inhibition of amplified 3alpha hydroxy-5alpha-pregnan-20-one GABA-mediated flux of chloride ions through recombinant GABA-A receptors (alpha1, beta2, gamma2L) expressed in Xenopus laevis oocytes. The 3 values correspond to the product numbers, and the numbering, consistent with the full names, is presented in Tables 2 and 3. The black cross (X) denotes a steroid antagonist that inhibits the amplified 3alpha-hydroxy-5alpha-pregnan-20-one (3 μM) GABA response ( 3 μM). Black dots (•) indicate a steroid antagonist that inhibits both the direct GABA response (30 μM) and the enhanced GALA 3alpha-hydroxy-5alpha-pregnan-20-one response.

Figure 2 shows the relationship between the inhibition of the activation of 3alpha-hydroxy-5alpha-pregnan-20-one GABA-mediated flow of chloride ions through recombinant GABA-A receptors (alpha1, beta2, gamma2L) expressed in mXenopus laevis oocytes, and inhibition of activated 3alpha-hydroxy-5alpha-pregnan-20-one of the GABA-mediated flow of chloride ions into cortical microbags from the cortex of male rats. The values correspond to the product numbers, and the full names are presented in tables 2 and 3.

Description of the invention

Before describing the present invention, it should be noted that the terminology used in this application is used only to describe specific methods of implementing the invention and is not limiting, since the scope of the present invention is defined only by the attached formula and its equivalents.

In particular, it should be noted that when used in this description and the attached formula, the words in the singular form also include the corresponding plural forms, unless otherwise follows from the context.

The following description assumes that the terms “steroid-related” and “steroid-induced”, for example, “steroid-related disorders”, encompass three possible mechanisms by which steroids act in the central nervous system: a) direct action, b) induction of tolerance and c) the effect of cancellation. Examples of such violations are given above, but they are presented to illustrate each mechanism and do not limit the scope of the invention.

The term “blocking” is used to mean an effect in which the action of 3alpha-hydroxy-5alpha / beta-steroids on the GABA-A receptor is disrupted.

It is clear that “blocking” is completely different from the effect, denoted by the terms “modulation” or “repression” or similar terms, which mean that the action still occurs, but to a lesser extent or at a slower speed.

The term "antagonist" refers to a substance that prevents another substance, an agonist, from causing its effect. In this application, the terms antagonist and blocker are used in parallel.

The term “pharmaceutical composition” is used in a broad sense, encompassing all pharmaceutically applicable compositions containing at least one active substance and additional carriers, adjuvants, components, and the like. The term "pharmaceutical composition" also encompasses a composition that contains the active substance in the form of a derivative or prodrug, for example, pharmaceutically acceptable salts, sulfates and esters. The manufacture of pharmaceutical compositions for various routes of administration is within the competence of a specialist in the field of galenic chemistry.

The terms “introduction” and “route of administration” as well as “route of administration” are also used in a broad sense. The pharmaceutical composition according to the present invention can be administered in various ways, depending mainly on which route of administration, local, local or systemic, is most suitable for the pathological condition to be treated. Such various routes of administration are, for example, local administration (e.g., to the skin), local (including ophthalmic and administration through various mucous membranes, e.g., vaginal and rectal delivery), oral or parenteral and pulmonary routes of administration, including to the upper and lower respiratory tract.

Methods for preparing such compositions are known to those skilled in the art of developing pharmaceutical preparations and compositions and can be used to develop the composition of compositions according to the present invention.

During the development of the present invention, it was unexpectedly found that pharmaceutically acceptable and practically applicable doses of steroids having a hydrogen donor group in the 3 beta position and a hydroxy group in the 20 position can inhibit the effect of 3alpha-hydroxy-5alpha / beta-steroids in vitro and have very slight antagonistic effect on the action of GABA itself. Thus, they block the development of the negative effects of 3alpha-hydroxy-5alpha / beta-steroids, but have minimal effect on the effect of GABA itself. Both mechanisms of action of 3alpha-hydroxy-5alpha / beta-steroids on the development of central nervous system disorders and the mechanism of action of 3beta-hydroxy-5alpha / beta-pregnan or 3beta-sulfate-5alpha / delta5-pregnan / pregneno steroids on their interaction with 3alpha-hydroxy -5 alpha / beta steroids. Examples of such compounds are presented in table 2.

In addition, according to the present invention, it was unexpectedly discovered that pharmaceutically acceptable and practically applicable doses of steroids that contain an acetate group at the 3 position and / or at 3, 20 and 21 positions are able to partially inhibit the inherent effect of GABA on the GABA-mediated chloride ion flux . Therefore, such steroids are used as therapeutic substances for the prevention and / or treatment of disorders in which the function of GABA itself is over-stimulated, which is observed in Alzheimer's disease. Examples of such compounds are presented in table 3.

The present invention relates to all steroids which contain a hydrogen donor group in the 3b position, in particular a hydroxy or sulfate group, and in addition a hydroxy group in the 20 position. In this description, 3 beta-hydroxy-pregnan-20-ol steroids are shown in Table 2 as examples. The authors of the present invention have shown that such steroids are antagonists and are able to antagonize the effect of 3alpha-hydroxy-5alpha / beta-pregnan steroids , but have little effect on the effect of GABA itself in the central nervous system (CNS). Surprisingly, the simultaneous treatment with 3alpha-hydroxy pregnan steroids and 3beta-hydroxy pregnan steroids mentioned in Table 2 inhibits the transport of chloride ions through the GABA recombinant receptor expressed in Xenopus laevis oocytes induced by 3alpha-hydroxy pregnan steroid but has a negligible effect on the chloride ion flux induced by only one GABA.

One of the advantages of this invention is that 3beta-hydroxy-5alpha / beta-pregnan, 20-ol-steroids, in particular 5alpha-PREGNAN-3beta, 20beta-DIOL, 5beta-PREGNAN-3beta, 20alpha-DIOL, 5alpha-PREGNAN -3beta, 20alpha-DIOL, 5beta-PREGNAN-3beta, 20beta-DIOL, have an effective antagonistic effect on the modulation of the GABA-A receptor 3alpha-hydroxy-5alpha / beta-pregnan steroids, but to a small extent affect the activation of the GABA-A receptor under the influence of GABA. A particular advantage is that this blocking is achieved in pharmacologically and physiologically acceptable concentrations.

A new and unexpected effect underlying the present invention, therefore, is that it is possible to selectively block the amplification of the GABA-activated stream by 3alpha-hydroxy-5alpha / beta-pregnan steroids by the administration of 3beta-hydroxy-5alpha / beta- pregnan-20-ol-steroids in pharmaceutically and physiologically acceptable amounts with no or little effect on the effect of GABA.

Another new and surprising effect discovered during the creation of the present invention is that 3beta-hydroxy-5alpha / beta-pregnan-steroids with an acetate group or groups at 3, 20 and 21 positions, in particular 5alpha-PREGNAN-3beta-OL -20-SHEA ACETATE; 5alpha-PREGNAN-3beta, 20beta-DIOLA 3-ACETATE; 5beta-PREGNAN-3beta, 20beta-DIOLA 3-ACETATE; 5alpha-PREGNAN-3beta, 20alpha-DIOL 3-ACETATE; 5alpha-PREGNAN-3beta, 20beta-DIOLA DIACETATE; 5beta-PREGNAN-3beta, 20alpha-DIOL DIACETATE; 5beta-PREGNAN-3beta, 20beta-DIOLA DIACETATE; 5beta-PREGNAN-3beta, 21-DIOL-20-ONA 21-ACETATE; and some 3beta-5beta-steroids, in particular 5beta-PREGNAN-3beta-OL-20-OH and 5beta-PREGNAN-3beta, 21-DIOL-20-OH, can partially inhibit the direct response of GABA to the GABA-A receptor. A particular advantage is that such blocking is achieved at pharmacologically and physiologically acceptable concentrations.

The GABA-A receptor is a chloride ion channel, and the GABA-A receptor exerts its effect by changing the flow of chloride ions through the channel. It is well known in the art that the activity of neurons in the brain decreases when the GABA-A receptor is open and an intense influx of chloride ions into the cell occurs. It is also well known that there is a relationship between the amount of chloride ions moving inside the cell and the clinical effect of drugs that are active against the GABA-A receptor.

Benzodiazepines, barbiturates and, to some extent, alcohol act through this mechanism. However, it is also considered the adverse effects of these drugs. The problem with the GABA-A receptor is that it acts in most areas of the brain. Therefore, total GABA blockers are dangerous and can cause psychotic symptoms and convulsions. If it is necessary to suppress the action of 3alpha-hydroxy-5alpha / beta-pregnan-steroids, it is desirable to use substances that have a specific antagonistic effect on the action of 3alpha-hydroxy-5alpha / beta-pregnan-steroids and in the absence of such an effect on the effect of GABA itself. However, in cases where the cause of the violation is a general over-stimulation of the GABA system due to the action of the GABA itself, it is preferable to use a more general antagonist that counteracts the action of the GABA itself. But if such common antagonists completely block the GABA-A receptor, they are dangerous.

Therefore, partial antagonists, such as those described in the present invention, are preferred.

As discussed above, there are a number of symptoms and pathological conditions that are associated with 3alpha-hydroxy-5alpha / beta-pregnan steroids, and treatment of these conditions can be carried out by blocking the action of 3alpha-hydroxy-5alpha / beta-pregnan steroids. The inventors of the present invention provide substances and methods for this kind of blocking.

In addition, there are conditions that are due to general overstimulation of the GABA-A system, and the use of partial blockers of the action of GABA would be a way to treat such conditions. The inventors of the present invention provide substances and methods for such blocking the action of GABA.

In the experimental work described by Prince and Simmons (see above), an indirect ligand binding assay was used. However, studying the binding of FN3 only, it cannot be assumed that the same effect will be observed with neuronal activity in vivo. Prince and Simmons in their work also did not claim to be. They only discuss the role of steroid binding sites and whether these sites are the same or not. This consideration is not new, and the possibility of the existence of several steroid binding sites has been reported in earlier publications. The only conclusion they made, and the possible conclusions that can be drawn on the basis of their experimental idea and model, is that the steroid changes the binding of FN3 in different ways and in some way interacts with FN3 binding. Based on the results of Prince and Simmons, it is impossible to draw any conclusion about the effect on neuronal activity or clinical significance.

In the course of the present invention, it was unexpectedly discovered that pharmaceutically acceptable and applicable doses of 3 beta-hydroxy-5alpha / beta-pregnan-20-ol-steroids, in particular 5alpha-PREGNAN-3beta, 20beta-DIOLA, 5beta-PREGNAN-3beta, 20alpha -DIOLA, 5alpha-PREGNAN-3beta, 20alpha-DIOLA; and 5beta-PREGNAN-3beta, 20beta-DIOLA, are selective blockers of the action of 3alpha-hydroxy-5alpha / beta-steroids and, thus, block the development of negative effects of 3alpha-hydroxy-5alpha / beta-steroids, but have little effect on the inhibition of GABA . The inventors of the present invention also unexpectedly found that such pharmaceutically acceptable and practically applicable doses of 3beta-hydroxy-5alpha / beta-pregnan-steroids with acetate group (s) at 3, 20 or 21 positions, in particular 5alpha-PREGNAN-3beta-OL- 20-SHEET ACETATE; 5alpha-PREGNAN-3beta, 20beta-DIOL 3-ACETATE; 5beta-PREGNAN-3beta, 20beta-DIOL 3-ACETATE; 5alpha-PREGNAN-3beta, 20alpha-DIOL 3-ACETATE; 5alpha-PREGNAN-3beta, 20beta-DIOL DIACETATE; 5beta-PREGNAN-3beta, 20alpha-DIOL DIACETATE; 5beta-Pregnan-3beta, 20beta-DIOL DIACETATE; 5beta-PREGNAN-3beta, 21-DIOL-20-ONA 21-ACETATE, and some 3beta-5 beta-steroids, in particular 5beta-PREGNAN-3beta-OL-20-ONA and 5beta-PREGNAN-3beta, 21-DIOL- 20-SHE are partial blockers of the action of GABA.

The authors of the present invention have established both the mechanisms underlying the disorders caused by 3alpha-hydroxy-5alpha / beta-steroids, and GABA itself. It has been established in the technical field that 3alpha-hydroxy-5alpha / beta-steroids cause CNS disorders through three possible mechanisms: a) direct action, b) induction of tolerance and c) withdrawal effect. In addition, it has been established that global over-stimulation of GABA ultimately disrupts the function of the GABA-A system, and disturbed functions include memory and learning, which is known to occur, for example, in Alzheimer's disease.

For comparison, in the previously described inventions, for example, Bolger et al., US Patent No. 5,232,917, and Upasani et al., US Patent No. 5,939,545, did not describe any 3-beta-hydroxy-5alpha / beta-pregnan-steroids as receptor modulators GABA-A, neither in patents, nor in corresponding scientific publications (Lan NC, Gee KW, Bolger MB & Chen JS (1991), Differential responses of expressed recombinant human gamma-aminobutyric acidA receptors to neurosteroids. Journal of Neurochemistry, 57 ( 5): 1818-1821).

Therefore, in its broadest aspect, the present invention provides chemicals capable of blocking the action of 3alpha-hydroxy-pregnan-steroids on the GABA-A receptor, but not the action of GABA itself, as well as partial antagonists of the action of GABA.

In another aspect, the present invention provides a pharmaceutical composition which comprises a compound capable of blocking the action of 3alpha-hydroxy-pregnane-steroids on the human GABA-A receptor or which is a partial blocker of the action of GABA. Preferably, said compound (Tables 2, 3) is present in the form of an applicable and pharmaceutically acceptable salt, most preferably a sodium salt.

In another aspect, the present invention provides a general method for treating and / or preventing steroid-related CNS disorders in patients, wherein the patient is administered at least one compound that is capable of blocking the effect of 3alpha-hydroxy-pregnan-steroids on the GABA-A receptor person. Preferably, said compound contains a hydrogen donor group at the 3 beta position, which is a group selected from a hydroxy and sulfate group, and a hydroxy group at the 20 position. Most preferably, said compound is selected from the substances shown in Table 2. In addition, the present invention provides a general method for treating and / or preventing a patient from having central nervous system disorders associated with general over-stimulation of GABA-A receptors, according to which at least one is administered to said patient , one compound capable of partially blocking the effect of GABA on the GABA-A receptor. Most preferred compounds are listed in Table 3. Preferably, the compound is used in the form of a suitable and pharmaceutically acceptable salt, most preferably the sodium salt.

According to the present invention, at least one of said compounds is administered by one of the following routes of administration: intravenously, nasally, rectally, intravaginally, through the skin and orally.

According to one preferred embodiment of the invention, at least one steroid is administered intravenously. According to another preferred embodiment of the invention, at least one of said steroid is administered nasally.

Nasal administration, in particular, is a promising choice, as it offers advantages associated with the convenience and possibility of self-administration by the patient. Self-administration has the advantage of allowing the patient to adjust the dose or frequency of taking the drug in accordance with either a subjective assessment of his condition, or in accordance with the schedule prescribed by the attending physician. The term “schedule prescribed by the attending physician” includes the possibility in which the patient subjectively assesses his condition either with the help of a questionnaire or rating scale, or without it, or using an algorithm or computer program indicating the next suitable dose.

Administration through the skin using substances that are part of the preparations in the form of a cream, gel, ointment or in the form of a slow-release adhesive medical patch is another possible form of administration that is also suitable for self-treatment. The advantages of the independent methods of administration of the drug mentioned above also apply to the methods of administration through the skin with the additional advantage that, if desired or necessary, administration can be easily prevented, for example by removing the medical patch.

In any of the listed or other routes of administration of the drug, the composition of the composition can be adapted or adjusted according to standard pharmacological procedures, including effective pharmaceutical preparations in a chemical form suitable for the chosen route of administration, along with applicable adjuvants and carriers used in ordinary practice and known to those skilled in the art.

Commonly used oral adjuvants and carriers are, for example, excipients or suspending agents, such as titanium dioxide, lactose anhydride, silicon dioxide, colloidal silicon dioxide, methyl cellulose, magnesium stearate, microcrystalline cellulose and the like.

Commonly used adjuvants and carriers for intravenous administration are, for example, sterile water for injection (VDI), sterile buffers (for example, buffer solutions with a pH of 7.4), albumin solutions, lipid solutions, and the like.

Common adjuvants and carriers for transdermal administration, for example, are petrolatum, liquid paraffin, glycerin, water, and the like.

Doses can be easily varied depending on the route of administration, the particular pathological condition to be treated, or the desired effect, gender, age, weight and health status of the patient, as well as, possibly, other indicators evaluated by the attending physician.

According to the present invention, with the intravenous administration of at least one of the indicated steroid, a suitable dosage range is from about 0.2 to 200 mg per kg of body weight, preferably from 0.2 to 2; from 2 to 10; from 10 to 20; from 20 to 40; from 40 to 80; from 80 to 120; 120-160 and from 160 to 200 mg per kg of body weight. Preliminary animal studies have shown that the preferred interval for intravenous administration is from about 20 to 100 mg per kg of body weight.

Appropriate dosage ranges for other routes of administration can easily be calculated by a person skilled in the art and, if necessary, tested using non-animal tests or preclinical or clinical studies.

One of the ways of implementing the present invention is a method of treating steroids associated with or caused by CNS disorders, in particular premenstrual syndrome, according to which the development of tolerance and down regulation of the GABA-A receptor is prevented by the administration of at least one substance according to the present invention. Such treatment should preserve the sensitivity of the GABA-A system and inhibit the development of a condition with less sensitivity during the luteal phase. In experiments on rats, it was shown that during prolonged treatment with progesterone, a change in the GABA-A receptor is observed. Treatment with 3beta-hydroxy-5alpha / beta-pregnan-steroids or 3beta-sulfate-5alpha / delta5-pregnan / pregneno-steroids according to the present invention is prescribed to prevent the development of tolerance and, thus, to prevent the withdrawal effect when you stop taking 3alpha-hydroxy -5 alpha / beta steroids.

The development of tolerance reduces sensitivity to endogenously formed GABA-A enhancing substances, such as 3alpha-hydroxy-5alpha-pregnan-20-one or benzodiazepines. With the rapid cessation of medication, as well as at the end of the luteal phase of the menstrual cycle, after the abolition of 3alpha-hydroxy-5alpha-pregnan-steroids, a recession occurs. This situation is observed in people with increased migraines and epileptic seizures during menstruation shortly after cessation of production and withdrawal of steroid access.

Thus, another method of implementing the present invention is a method of treating or preventing the development of tolerance and / or withdrawal symptoms by administering at least one substance according to the present invention.

The present invention further relates to a method for treating and / or preventing steroid-related or related mood disorders in patients described above, wherein at least one 3-beta-hydroxy-5alpha / beta-pregnan-20 is administered to said patient ol steroid.

Preferably, said steroid compound is selected from steroids shown in Table 2. Most preferably, said compound is used in the form of a suitable and pharmaceutically acceptable salt, most preferably a sodium salt.

Suitable routes of administration are, for example, the following: intravenous, nasal, rectal, intravaginal, skin administration and oral routes of administration.

However, for the treatment of most CNS disorders and for use as an anesthetizing agent, intravenous administration of an effective compound or compounds is preferred.

Suitable doses according to the invention for intravenous administration are doses ranging from about 0.2 to about 200 mg per kg body weight, preferably from 0.2 to 2; from 2 to 10; from 10 to 20; from 20 to 40; from 40 to 80; from 80 to 120; 120-160 and 160 to 200 mg per kg body weight, most preferably about 20 to 100 mg / kg.

As described above, the composition may be adapted or adjusted in accordance with standard pharmacological procedures. In addition, the dose can be easily varied depending on the route of administration, the particular pathological condition to be treated or the desired effect, gender, age, weight and health status of the patient, as well as, possibly, other indicators estimated by the attending physician.

In addition, the present invention encompasses methods for treating and / or preventing steroid-related or related CNS disorders in a patient, according to which one or more 3-beta-hydroxy-5alpha / beta-pregnan-20-ol-steroids is administered to the indicated patient (see table 2) in a pharmaceutically and physiologically acceptable dose. Preferably, said steroid compound is selected from steroids shown in Table 2. Most preferably, said compound is used in the form of a suitable and pharmaceutically acceptable salt, most preferably a sodium salt.

Examples of symptoms and pathological conditions caused by the action of 3alpha-hydroxy-5alpha / beta-steroids are sedation, fatigue, memory impairment, impaired learning, impaired motor function, slowness, increased appetite and craving for food, depressed mood, irritability and depression, which are the main symptoms of premenstrual syndrome and worsening with minor epilepsy.

Conditions and symptoms caused by the development of tolerance after prolonged (days) exposure to 3alpha-hydroxy-5alpha / beta-steroids are, for example, stress sensitivity, difficulty concentrating, stress or the menstrual cycle, difficulty concentrating, sleep disturbances, fatigue, loss control motivation and depression. 3alpha-hydroxy-5alpha / beta-steroids also increase drug / drug dependence. According to the present invention, such conditions or symptoms can be prevented, alleviated, or treated by administering at least one 3beta-hydroxy-5alpha / beta-pregnan-20-ol-steroid to a patient. Preferably, said steroid compound is selected from steroids shown in Table 2. Most preferably, said compound is used in the form of a suitable and pharmaceutically acceptable salt, most preferably a sodium salt.

Continuous but shorter exposure to 3alpha-hydroxy-5alpha / beta-steroids results in a withdrawal effect upon cessation of exposure. This phenomenon is observed during menstruation when the production of 3alpha-hydroxy-5alpha / beta-steroids by the corpus luteum ceases. This withdrawal phenomenon is also observed after childbirth (post partum) when the production of 3alpha-hydroxy-5alpha / beta-steroids by the placenta ceases. The same phenomenon was also noted at the end of the stress period and the cessation of production of 3alpha-hydroxy-5alpha / beta-steroids by the adrenal glands observed during stress. Examples of pathological conditions that are influenced by this phenomenon of withdrawal / withdrawal are partial epilepsy, in which the patient forms an epileptic focus in the cerebral cortex and deterioration occurs during the period of withdrawal during menstruation. This phenomenon is called "menstrual epilepsy." Other examples are menstruation-related migraine, stress-related migraine, and mood changes after childbirth (post partum). Withdrawal syndrome is a sign of previously formed tolerance.

Examples of disorders that are believed to be related to or caused by steroids include the following: epilepsy, menstrual cycle epilepsy, depression, stress-related depression, migraine, fatigue and in particular stress-related fatigue, prementsrual syndrome, premenstrual dysphoric disorder associated with the menstrual cycle of mood changes associated with the menstrual cycle of memory changes related to stress memory changes associated with the menstrual cycle are difficult the concentration associated with the menstrual cycle, sleep disturbance and fatigue. There are clear indications that obesity and increased appetite, as well as some forms of imbalance, abuse of alcohol and certain substances are also disorders associated with or caused by steroids. The present invention thus provides substances and methods for treating, alleviating symptoms, or preventing such pathological conditions.

One of the preferred ways of implementing the present invention, aimed at the problem that many women face, is a method of treating and / or preventing side effects of postmenopausal therapy in patients, according to which at least one 3-beta-hydroxy-5alpha / beta is administered to said patient pregnan-20-ol steroid. Preferably, said steroid compound is selected from steroids shown in Table 2. Most preferably, said compound is used in the form of a suitable and pharmaceutically acceptable salt, most preferably a sodium salt.

Another preferred embodiment of the invention is a method of treating and / or preventing the side effect of oral contraceptives in patients, with such a treatment method, at least one 3-beta-hydroxy-5alpha / beta-pregnan-20-ol-steroid is administered to said patient. Preferably, said steroid compound is selected from the steroids listed in Table 2. Most preferably, said compound is used in the form of a suitable and pharmaceutically acceptable salt, most preferably a sodium salt.

In this application, it is preferable to administer to the patient an effective composition that contains at least one of the above 3beta-hydroxy-5alpha / beta-pregnan-steroids or 3beta-sulfate-5alpha / delta5-pregnan / pregneno-steroids, together with oral the contraceptive used by the patient. Nasal administration and administration through the skin are also suitable routes of administration.

Thus, one of the private ways of implementing the present invention is a pharmaceutical composition that contains an oral contraceptive and a therapeutically suitable dose of at least one chemical compound capable of blocking the action of 3alpha-hydroxy-pregnan-steroids on the human GABA-A receptor, said compound (s) comprise a hydrogen donor group at the 3 beta position in the form of a group selected from hydroxy and sulfate groups, and a hydroxy group at the 20 position. Preferably, said compound is selected from steroids listed in Table 2. Most preferably, said compound is used in the form of a suitable and pharmaceutically acceptable salt, most preferably a sodium salt.

In addition, in the indicated method of implementing the present invention, the dose of a chemical compound capable of blocking the action of 3alpha-hydroxy-pregnan-steroids on the human GABA-A receptor is preferably consistent with the level of endogenous steroids during stress or during the menstrual period. According to the present invention, a chemical compound capable of blocking the action of 3alpha-hydroxy-pregnan-steroids on the human GABA-A receptor can be included in an oral contraceptive in order to both alleviate and eliminate the side effects of oral contraceptives, or in order to to alleviate or remove any undesirable effect of periodic changes in the level of endogenous steroids.

In general, the present invention encompasses the use of 3-beta-hydroxy-5alpha / beta-pregnan-20-ol-steroids, in particular the steroids listed in Table 2, either individually or in combination for the manufacture of a pharmaceutical preparation for treating or preventing any of the disorders associated with or caused by 3alpha-hydroxy-5alpha / beta-pregnan steroids described in the invention, and in particular one or more of the following disorders: epilepsy, menstrual cycle epilepsy, depression associated with stress depression, migraine, fatigue, in particular stress-related fatigue, premenstrual syndrome, premenstrual dysphoric disorder, mood changes associated with the menstrual cycle, memory changes associated with the menstrual cycle, stress related memory changes, concentration difficulties associated with the menstrual cycle associated with the menstrual cycle of sleep disorders and fatigue. Preferably, said compound is used in the form of a suitable and pharmaceutically acceptable salt, most preferably the sodium salt.

Another preferred embodiment of the invention is a method for the treatment and / or prevention of disorders caused by over-stimulation with gamma-aminobutyric acid (GABA) using 3-beta-hydroxy-5alpha / beta-pregnan-steroids which contain an acetate group or groups in 3, 20 and 21 positions , and some 3beta-hydroxy-5beta-pregnan steroids.

It is known that intensive stimulation of the GABA-A receptor of GABA itself in certain cases inhibits the learning function and memory. Overstimulation of the GABA-A receptor system impairs the function of short-term learning and memory, which is of particular clinical importance in the elderly. In this case, the antagonistic effect on the action of GABA should be useful and can be used as a treatment for memory impairment. However, if the antagonistic effect on GABA is too intense, it will be dangerous and cause convulsions. Therefore, antagonists are preferred that have a partial antagonistic effect on GABA-induced changes in the GABA-A receptor, but do not completely block the GABA-A receptor. In addition, the present invention encompasses a method of treating and / or preventing CNS disorders in patients caused by general overstimulation of the GABA-A receptor system, according to which one or more 3-beta-hydroxy-5alpha / beta-pregnan- is administered to a specified patient in a pharmaceutically and physiologically acceptable dose a steroid that contains an acetate group or groups in 3, 20 and 21 positions, and some 3beta-hydroxy-5beta-pregnan-steroids (see table 3). Preferably, said steroid compound is selected from the group consisting of the following steroids: 5alpha-PREGNAN-3beta-OL-20-OH ACETATE; 5alpha-PREGNAN-3beta, 20beta-DIOLA 3-ACETATE; 5beta-PREGNAN-3beta, 20beta-DIOLA 3-ACETATE; 5alpha-PREGNAN-3beta, 20alpha-DIOLA 3-ACETATE; 5alpha-PREGNAN-3beta, 20beta-DIOLA DIACETATE; 5beta-PREGNAN-3beta, 20alpha-DIOL DIACETATE; 5beta-PREGNAN-3beta, 20beta-DIOLA DIACETATE; 5beta-PREGNAN-3beta, 21-DIOL-20-ONA21-ACETATE, 5beta-PREGNAN-3beta-OL-20-OH and 5beta-PREGNAN-3beta, 21-DIOL-20-OH, as shown in Table 3. Most preferably indicated the compound is used in the form of a suitable and pharmaceutically acceptable salt, most preferably the sodium salt. A particular advantage is that blocking is carried out in pharmacologically and physiologically acceptable concentrations.

Examples of such disorders that are thought to be associated with overstimulation of the GABA system or caused by it are Alzheimer's disease (AD) and mild cognitive impairment (LCI), especially in the elderly. The present invention thus provides substances and methods for treating, alleviating symptoms, or preventing such pathological conditions.

Examples

The present invention is based on in vitro experiments performed to confirm the specificity of a separate action on the blocking of a reinforced 3alpha-hydroxy-5alpha / beta-pregnan-steroids GABA-mediated flow of chloride ions and a blocking effect on the response of the native action of GABA. The results obtained in vitro tests with recombinant GABA-A receptors expressed on the surface of Xenopus laevis oocytes are confirmed in tests using cortical tissue of male rats performed by the authors of the present invention.

Study of changes in the flux of chloride ions through GABA-A recombinant receptors (alpha1, beta2, gamma2L) expressed in Xenopus laevis oocytes using voltage-fixing methods

The GABA-A recombinant receptor used to test the steroids of the present invention was expressed in Xenopus laevis oocytes. Oocytes have the ability to synthesize proteins located on the membrane surface from an mRNA molecule injected into an oocyte. The receptor contains five proteins of three different types, and in the conditions of the present test, mRNA was obtained for subtypes of the receptor alpha1, beta2, gamma2L. These subtypes of the GABA-A receptor are the most common in the central nervous system. Such GABA-A receptors contain two alpha1 proteins, two beta2 and one gamma protein. After 48 hours of incubation, GABA-A receptors inserted into the membrane and behaved in vitro in the same way as the in situ receptor. Therefore, this model system is considered by experts as a suitable system for testing in vitro drugs that are active against the GABA-A receptor. The flow of chloride ions through the chlorine channel is the result of the action of GABA on the GABA-A receptor, and GABA-A receptor modulators, such as 3alpha-hydroxy-5alpha / beta-pregnan steroids, can increase the effectiveness of GABA. The flux of chloride ions through the receptor is measured as the electric current induced by the movement of ions using the voltage fixing method. Dose response curves can be obtained for agonists and antagonists, and the current depends on the activation of the GABA-A receptor observed after interaction between different doses of agonists and antagonists. By normalizing the data for a specific current value, in the present analysis of 2 μA, it is possible to compare the results for different oocytes, and normalizing the change in current with respect to a specific dose of 10 μM in this analysis, various test substances can be compared. The amplification of the 3alpha-hydroxy-5alpha-pregnan-20-one amplification of the GABA-mediated chloride ion flux was taken as 1 and was obtained by treating oocytes expressing the GABA-A receptor, 3 μM GABA + 3 μM 3alpha-hydroxy-5alpha-pregnan -20-she in an in vitro thermostat. The flux of chloride ions was estimated as the current induced by the transport of chloride ions through GABA-A receptors. The GABA-induced chloride ion flux was taken as 1 and was obtained by processing oocytes expressing the GABA-A receptor in vitro with 30 μM GABA. The inhibitory effect on 3alpha-hydroxy-5alpha-pregnan-20-one-induced enhancement of the GABA-mediated chloride ion flux of the tested UC steroids was compared with the inhibitory effect of the same UC steroids on the activation of the GABA receptor GABA-A. The results are shown in FIG. 1 and Table 4. In FIG. 1, UC steroids with a low level of GABA antagonism are indicated by X, and UC steroids with a partial GABA antagonism are indicated by a black dot (•).

Figure 1 shows the relationship between the inhibition of the maximum GABA-mediated flow of chloride ions (30 μM GABA), the X-axis, and the inhibition of the enhanced 3alpha-hydroxy-5alpha-pregnan-20-one GABA-mediated flow of chloride ions, Y- axis, in recombinant GABA-A receptors (alpha1, beta2, gamma2L) expressed in Xenopus laevis oocytes. In the drawing, the results are normalized relative to the control values of the current activated by 30 M GABA or 3 μM GABA + 3 μM 3alpha-hydroxy-5alpha-pregnan-20-one. The control current value in the absence of steroid antagonists is normalized to 0. Each steroid antagonist was tested at 10 μM. Black crosses (X) indicate steroids that show a slight decrease in GABA inhibition, but which significantly inhibit the enhanced 3alpha-hydroxy-5alpha-pregnan-20-one GABA-mediated response. The black dot (•) denotes steroid antagonists that inhibit both the direct GABA response (30 μM) and the enhanced GALA 3alpha-hydroxy-5alpha-pregnan-20-one response. The values correspond to the product numbers, and the numbers with the corresponding full names of the various substances are presented in tables 2 and 3.

Table 4 presents the results of the analyzed antagonistic effect of UC steroids on a 3 μM GABA + 3 μM 3alpha-hydroxy-5alpha-pregnan-20-one chloride ion flux and also on a chloride ion flux stimulated with 30 μM GABA. 3beta-hydroxy-5alpha / beta-pregnan-20-hydroxy steroids and 3beta-hydroxy-5alpha / beta-pregnan steroids with an acetate group or groups in 3, 20 and 21 positions and some 3beta-hydroxy-5beta were tested for antagonism β-pregnan steroids shown in Tables 2 and 3. Some of the steroids (5alpha ALPHA-PREGNAN-3beta-OL-20-OH, UC1010; 5alpha-PREGNAN-3beta, 20beta-DIOL; UC1011; 5alpha-PREGNAN-3b -20-OH ACETATE, UC1012; 5beta-PREGNAN-3beta, 20alpha-DIOL, UC1013; 5beta-PREGNAN-3beta-OL-20-OH, UC1014; 5beta-PREGNAN-3beta, 21-DIOL-201 O1) were previously described in patent applications WO 99/45931 and WO 03/059357 and are included in this application only for completeness of the data presented. For some steroids described in WO 03/059357, the present study showed the presence of additional effects, namely a weak antagonistic effect on GABA itself (5alpha-PRGNAN-3beta, 20beta-DIOL, UC1011; 5beta-PREGNAN-3beta, 20alpha-DIOL , UC1013), while for others in the present study, the presence of a partial antagonistic effect on the effect of GABA (5alpha-PREGNAN-3beta-OL-20-ONE ACETATE, UC1012; 5beta-PREGNAN-3beta-OL-20-OH, UC1014; 5beta-PREGNAN-3beta, 21-DIOL-20-OH, UС1015).

Table 4 The average normalized-1 value of the antagonistic effect of UC substances, 10 μM, with respect to the stimulated 3alpha-hydroxy-5alpha-pregnan-20-one + GABA or only one GABA chloride ion flow through rat GABA-A receptor expressed in Xenopus laevis oocytes as alpha1, beta2, gamma2L UC code Average normalized-1 3alpha-hydroxy-5alpha-pregnan-20-one inhibition The average normalized-1 GABA inhibition only GABA , 00 3alpha-hydroxy-5alpha-pregnan-20-one , 00 UC1010 -, 37 - 26 UC1011 -, 21 -, 17 UC1012 -, 42 -, 37 UC1013 -,thirty -, 23 UC1014 -, 45 -, 39 UC1015 -, 43 -, 40 UC1019 -, 36 -, 17 UC1020 -, 41 -,10 UC1021 -, 34 -, 44 UC1022 -, 40 -, 27 UC1023 -, 42 -, 32 UC1024 -, 36 -, 59 UC1025 -, 43 -, 41 UC1026 -,thirty -, 45 UC1030 -, 45 -, 31

Studies on the inhibition of 3alpha-hydroxy-5alpha-pregnan-20-one were performed in the presence of 3 μM GABA + 3 μM 3alpha-hydroxy-5alpha-pregnan-20-one. Antagonism studies for GABA were performed at a concentration of 100 μM GABA. The obtained current values were normalized with respect to stimulation of 2 microamperes, the average values of the effect presented in the table are normalized to 1 with respect to 3 μM 3alpha-hydroxy-5alpha-pregnan-20-one and 100 μM GABA, respectively. The number of analyzed cells n = 3-10.

Study of cortical tissue homogenate

Some of the steroids (see FIG. 2) analyzed on the Xenopus oocyte model were also tested by the present inventors on their ability to inhibit the effect of 3alpha-hydroxy-5alpha / beta-pregnan-steroids on the model of the chloride ion current GABA-A receptor. GABA-A receptors used in experiments on the flow of chloride ions were obtained from rat cortical tissue, which is a representative part of the brain to evaluate the effects of GABA-A receptors. Since the results obtained using the two methods are similar, it can be assumed with a high degree of probability that the result obtained in this model can be confirmed in higher mammals and humans.

In these assay methods, cortical tissue from mature male rats was homogenized in buffer. As a result of this procedure, a suspension of small vesicles was obtained, while these vesicles had GABA-A receptors on their surface.

When radioactive chloride ions were added to this suspension, a certain amount fell into the vesicles. This amount depends on how open the GABA-A receptor is. Drugs that open the receptor increase the amount of chloride ions in the vesicles relative to the dose of the drug. Similarly, drugs that close the GABA-A receptor reduce the amount of chloride ions that enter the vesicles. Labeled chloride ions outside the vesicles are washed off and separated from chloride inones inside the vesicles. The amount of radioactively labeled chloride ions in the vesicles was then measured in a liquid scintillator and a curve was obtained. In each experiment, a control with increasing doses of GABA was used to test the operation of tissue samples. As the second control used, the upper dose response curve was constructed with 3alpha-hydroxy-5alpha-pregnano-20-one + 10 μm GABA.

Figure 2 shows the relationship between the inhibition of enhanced 3alpha-hydroxy-5alpha-pregnan-20-one GABA-mediated chloride ion flux through recombinant GABA-A receptors (alpha1, beta2, gamma2L) expressed in Xenopus laevis oocytes, and inhibition of enhanced 3alpha-hydroxy-5alpha-pregnan-20-one of the GABA-mediated current of chloride ions into cortical microbags from the cortex of male rats. The data on the X and Y axes are presented as normalized values of -1 to demonstrate the inhibitory effect of individual substances. The effect of 3 μM GABA + 3 μM 3alpha-hydroxy-5alpha-pregnan-20-one was taken as 0 for these oocytes. Each steroid was tested at a concentration of 10 μM. The data obtained in the analysis of the chloride ion flux were normalized against the effect of 10 μM GABA + 1 μM 3alpha-hydroxy-5alpha-pregnan-20-one, which was taken as 0. Each steroid was tested at a concentration of 30 μM. 3 values correspond to product numbers, and full names of various substances are presented in Tables 2 and 3. The regression line for the dependence gives a normalized-1 value of inhibition of 3alpha-hydroxy-5alpha-pregnan-20-one in oocytes = -0.04 + 0.96 * .

Normalized-1 value of the flow of chloride ions into cortical microbags. Pearson correlation coefficient r = 0.90; p <0.002. Two different methods for detecting the inhibitory properties of UC steroids give well consistent results. The results show that there are differences in the effects of steroids.

The GABA-A recombinant receptor used to test the steroids of the present invention was expressed in Xenopus laevies oocytes. Oocytes have great potential for the expression of membrane proteins based on mRNA injected into the oocyte. The receptor contains three different proteins, and in the conditions of the present experiment, mRNA was obtained for the receptor subtypes alpha1, beta2, gamma2L. These subtypes of GABA-A receptor are the most common in the central nervous system. Such GABA-A receptors behave in vitro in the same way as the in situ receptor, and therefore are considered by experts as a suitable system for testing in vitro drugs that are active against the GABA-A receptor. The flow of chloride ions through the chlorine channel is the result of GABA acting on the GABA-A receptor, and GABA-A receptor modulators such as 3alpha-hydroxy-5alpha / beta-pregnan steroids can increase the efficiency of GABA. The electric current induced by the flow of chloride ions through the receptor is measured using a voltage fix method. You can obtain dose-response curves, and normalizing data for a specific current value, in the present analysis of 2 μA, you can compare the results for different oocytes, and normalizing the change in current with respect to a specific dose of 10 μM in this analysis, you can compare different test substances. The inhibitory effect of the tested UC-steroids on the induced 3alpha-hydroxy-5alpha-pregnan-20-amplification of the GABA-mediated chloride ion flux was compared with the inhibitory effect of the same UC-steroids on the activation of the GABA-GABA-A receptor. The results are presented with various notations in FIG. 1. For some of the steroids (see Figure 2) studied on the Xenopus oocyte model, the present inventors also showed that they inhibit the effect of 3alpha-hydroxy-5alpha / beta-pregnan-steroids on the model of chloride ion transport through the GABA-A receptor . GABA-A receptors used in experiments on the absorption of chloride ions were obtained from rat cortical tissue, which is a representative part of the brain for measuring the effects of the GABA-A receptor. With a high degree of probability, it can be assumed that the results obtained in this model can be confirmed on higher mammals and humans.

Although the invention is described in relation to preferred methods for its implementation, which represent the best method currently known to the inventors, it should be understood that various changes and modifications, as is obvious to specialists in this field, can be carried out without going beyond the scope of the invention, which is set forth in the formula attached to this document.

Claims (9)

1. A method of treating and / or alleviating the symptoms of a CNS disorder in which gamma-aminobutyric acid receptor A (GABA-A) is involved, comprising administering an effective amount of a substance selected from: 5alpha-pregnan-3beta, 20alpha-diol; 5beta-pregnan-3beta, 20beta-diol; 5alpha-pregnan-3beta, 20beta-diol 3-acetate; 5beta-pregnan-3beta, 20beta-diol 3-acetate; 5alpha-pregnan-3beta, 20alpha-diol 3-acetate; 5-alpha-pregnan-3beta, 20beta-diol diacetate; 5beta-pregnan-3beta, 20alpha-diol diacetate; 5beta-pregnan-3beta, 20beta-diol diacetate; and 5beta-pregnan-3beta, 21-diol-20-one 21-acetate. 2. A substance selected from the group consisting of: 5alpha-pregnan-3beta, 20alpha-diol and 5beta-pregnan-3beta, 20beta-diol for use as an active component of a drug in an amount effective to treat and / or alleviate the symptoms of a CNS disorder in which gamma-aminobutyric acid receptor A (GABA-A) is involved. 3. A substance selected from the group comprising: 5alpha-pregnan-3beta, 20alpha-diol and 5beta-pregnan-3beta, 20beta-diol for use as an active component of a drug in an amount effective to prevent, treat and / or alleviate the symptoms of the disorder CNS caused by the action of 3alpha-hydroxy-5alpha / beta-steroid, moreover, this treatment, essentially, does not have an antagonistic effect on the GABA-A receptor. 4. A substance selected from the group consisting of: 5alpha-pregnan-3beta, 20beta-diol 3-acetate; 5beta-pregnan-3beta, 20beta-diol 3-acetate; 5alpha-pregnan-3beta, 20alpha-diol 3-acetate; 5alpha-pregnan-3beta, 20beta-diol diacetate; 5beta-pregnan-3beta, 20alpha-diol diacetate; 5beta-pregnan-3beta, 20beta-diol diacetate; and 5beta-pregnan-3beta, 21-diol-20-one 21-acetate for use in an effective amount as an active component of the drug. 5. A substance selected from the group including: 5alpha-pregnan-3beta, 20beta-diol 3-acetate; 5beta-pregnan-3beta, 20beta-diol 3-acetate; 5alpha-pregnan-3beta, 20alpha-diol 3-acetate; 5alpha-pregnan-3beta, 20beta-diol diacetate; 5beta-pregnan-3beta, 20alpha-diol diacetate; 5beta-pregnan-3beta, 20beta-diol-diacetate and 5beta-pregnan-3beta, 21-diol-20-one 21 acetate, for use as an active component of a drug in an amount effective to prevent, treat and / or alleviate central nervous system disorders, caused by overstimulation of the GABA-A receptor. 6. The use of a substance selected from the group including: 5alpha-pregnan-3beta, 20alpha-diol; and 5beta-pregnan-3beta, 20beta-diol for the manufacture of a medicament, wherein said substance is contained in the medicament in an amount effective to prevent, treat and / or alleviate central nervous system disorder caused by exposure to 3alpha-hydroxy-5alpha / beta-steroid, moreover, the specified treatment, essentially, does not have an antagonistic effect on the GABA-A receptor. 7. The use according to claim 6, characterized in that said substance is 5alpha-pregnan-3beta, 20alpha-diol. 8. The use according to claim 6, characterized in that said substance is 5beta-pregnan-3beta, 20beta-diol. 9. The use of a substance selected from the group including: 5alpha-pregnan-3beta, 20beta-diol 3-acetate; 5beta-pregnan-3beta, 20beta-diol 3-acetate; 5alpha-pregnan-3beta, 20-alpha-diol 3-acetate; 5alpha-pregnan-3beta, 20beta-diol diacetate; 5beta-pregnan-3beta, 20alpha-diol diacetate; 5beta-pregnan-3beta, 20beta-diol diacetate; and 3beta-pregnan-3beta, 21-diol-20-one 21-acetate for the manufacture of a medicinal product, wherein the substance is contained in the drug in an amount effective to prevent, treat and / or alleviate CNS disorders caused by over-stimulation of the GABA-β receptor BUT.

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