WO2009142594A1

WO2009142594A1 - The use of 3beta-ethenyl, methyl, 3alpha-hydroxy-steroids for the treatment of cns disorders

Info

Publication number: WO2009142594A1
Application number: PCT/SE2009/050575
Authority: WO
Inventors: Torbjörn BÄCKSTRÖM; Gianna Ragagnin; Jessica STRÖMBERG; Inga-Maj Johansson; Per Lundgren; Monica Isaksson; Magdalena Taube
Original assignee: Umecrine Ab
Priority date: 2008-05-20
Filing date: 2009-05-20
Publication date: 2009-11-26
Also published as: CN101585862B; WO2009142594A9; CN101585862A

Abstract

Compounds exhibiting 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid blocking action with no or only low antagonism against the gamma-aminobutyric acid (A) receptor (GABA-A) are presented. Methods for the prevention, treatment and/or alleviation of various CNS-disorders are suggested, as well as pharmaceutical compositions for such use.

Description

THE USE OF 3BET A-ETHENYL, METHYL, 3ALPHA-HYDROXY-STEROIDS FOR THE TREATMENT OF CNS DISORDERS

The present invention concerns the treatment, alleviation or prevention of GABA-steroid related and/or induced disorders of the central nervous system (CNS), and in particular specific steroid compounds with increased resistance against metabolism for this purpose, their use for the manufacture of pharmaceuticals for said treatment, as well as methods of treatment involving their use.

Background of the invention

The metabolites of the sex and stress hormones pregnenolone, progesterone, desoxycortico- sterone, cortisone and Cortisol, known as pregnanolones; as well as the metabolites of testosterone, androstendione and dehydroepiandrosterone, known as androstanes, have all been the subject of various studies, at least partially elucidating their role in the neurological signal system in mammals.

The harmful steroids inducing CNS symptoms and disorders of interest in the present application all have a structural similarity in comprising a 3alpha-hydroxy group, a delta4- pregnene or a δalpha or δbeta pregnane steroid body, and a ketone or hydroxy group on position 17, 20 or 21.

Steroids comprising 3alpha-hydroxy-delta 4-5, 5alpha/beta-pregna(e)n-20-one/ol or 3alpha- hydroxy-5alpha/beta-androsta(e)n-17-one/ol (3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids) have been shown to be important specific enhancers of the gamma-aminobutyric acid (A) receptor (GABA-A). They bind to the GABA-A receptor and act by enhancing the effect of

GABA in terms of prolonging the GABA-A receptors opening duration. The effect is similar to the effects of both benzodiazepines and barbiturates. Said steroid compounds however have a binding site separate from that of both these compounds. Examples of such GABA enhancing steroids and their number according to the Chemical Abstracts Registry / Chicago Academy of Science (CAS) are given in Table 1.

The steroid nomenclature is not entirely consistent, and therefore the nomenclature developed by the International Union of Pure and Applied Chemistry (IUPAC) will be used throughout this application. Table 1. Nomenclature of GABA-steroids

^* CAS Number not found

Some of these steroids have been shown to have an ability to induce anesthesia at a high pharmacological dose. They can also be used as anti-epileptic agents, or as soporific agents. Some of these compounds have also been shown to possess anxiolytic effects in animal experiments. To reach these effects, however, high concentrations or high doses are required. Additionally, they appear as acute effects.

With respect to their direct CNS effects, as mentioned above, these compounds are similar to benzodiazepins and barbiturates. However, they also have similar adverse effects as normally associated with benzodiazepins and barbiturates. The adverse effects of the endogenous 3alpha-hydroxy-pregnan-20-one-steroids or 3alpha-hydroxy-androstan-steroids are the basis for the negative CNS effects induced by these steroids. As the 3alpha-hydroxy-pregnane- steroids and 3alpha-hydroxy-androstan-steroids are endogenously produced and are metabolites of steroid hormones, some of them essential for life, their production cannot easily be interrupted. These steroids are produced in high amounts during several days to weeks during the luteal phase of the menstrual cycle, i.e. after the release of an ovum from a mature ovarian follicle, during pregnancy and during stress. They are also produced within the brain.

Diseases caused by 3alpha-hvdroxy-steroids

Disorders that are caused by the action of endogenously produced 3alpha-hydroxy-5alpha steroids or 3alpha-hydroxy-5beta steroids on the GABA-A receptor are well characterized and understood. It is also known that 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids can induce tolerance to themselves and to other similar substances after exposure, and that withdrawal symptoms occur at withdrawal of the 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids. In summary, it is generally known that 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids cause CNS disorders through the above-described three possible mechanisms: a) direct action, b) tolerance induction, and c) withdrawal effect. These mechanisms will be discussed in closer detail below. a) Direct action

It is established that 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids can directly cause inhibition of CNS functions. Examples of symptoms caused by the direct action of 3alpha- hydroxy-delta 4-5, 5alpha/beta-steroids are sedation, tiredness, memory disturbance, learning disturbance, disturbance of motor function, clumsiness, fractures, injuries, increased appetite and food cravings, obesity, increased frequency of relapse in alcohol abuse in sober alcoholics, cravings for abuse substances e.g. alcohol, negative mood as tension, irritability and depression, which are the cardinal symptoms in the premenstrual dysphoric disorder, premenstrual syndrome and the worsening of Petit MaI epilepsy. Examples of this direct action can also be divided into sedative and anesthetic effects; disturbance of motor function; effects on cognitive function, memory and learning; worsening of Petit MaI epilepsy; premenstrual symptoms; mood changes; induction of anxiety in test animals; hyperphagia and increased appetite; food cravings etc.

High stimulation of the GABA-A receptor is in certain situations known to inhibit learning and memory function and worsen short-term learning and memory function. This is clinically relevant especially in elderly people. In such a situation, an antagonism of GABA's effect could be beneficial, and potentially useful in the treatment of memory disturbance. Currently, the treatment of Alzheimer's disease (AD) and Mild Cognitive Impairment (MCI) is largely unrealized, with no preventive or curative therapies. The fact that GABA-A receptor agonists like GABA-steroids, benzodiazepines and alcohol are amnesic, and that the GABA-A receptors in brain areas most affected by AD, highlighted the GABA-A receptor as a potential therapeutic target in AD and MCI. In contrast to the amnesic GABA-A receptor agonists, antagonists will attenuate GABA-A receptors function. GABA-Steroid antagonists have been shown to improve performance in animal models of learning and memory. Unfortunately, non-selective and totally blocking ligands also induce 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). The current cognition enhancers marketed have prominent adverse effects with minimal efficacy and there is a need for new substances, which is the subject of the present invention.

b) Tolerance

Continuous and long exposure to 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids causes malfunctioning of the GABA-A receptor system. A tolerance develops and this tolerance is the initial step in a process that ultimately leads to stress sensitivity, concentration difficulties, and loss of impulse control and depression. The action of 3alpha-hydroxy-delta 4-5, 5alpha/beta- steroids has also been found to be a factor, which reinforces drug dependency. This has been the focus of extensive research. The following themes have hitherto been the main subject of research: down regulation and decreased GABA function after long-term secretion of high amounts of 3alpha-hydroxy-5alpha/beta reduced steroids; benzodiazepine and 3alpha- hydroxy-5beta steroid sensitivity in PMS; and dependence induction.

c) Withdrawal symptoms A continuous but shorter exposure to 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids results in a withdrawal effect when the exposure is ended. This phenomenon occurs for example naturally, during menstruation when the production of 3alpha-hydroxy-delta 4-5, 5alpha/beta- steroids by the corpus luteum of the ovary is interrupted. This withdrawal phenomenon also occurs after giving birth {post partum) when the 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid production by the placenta is interrupted. The same phenomenon is also noted when a period of stress is ended, e.g. when work-related stress is interrupted during weekends. As a response to stress, the adrenals have produced 3alpha-hydroxy-delta 4-5, 5alpha/beta- steroids. When this production is interrupted, withdrawal symptoms may occur.

Examples of conditions that are influenced by this withdrawal phenomenon are partial epilepsy where the patient has an epileptic focus in the cerebral cortex where a worsening occurs at the withdrawal period during menstruation. This phenomenon is called "catamenial epilepsy". Other examples are menstrual related migraine, stress related migraine, and mood changes post partum. Similar symptoms and conditions are induced during treatment with steroid hormones, such as used in oral contraceptives, postmenopausal hormone replacement therapy, steroid treatment for inflammatory diseases and during intake of anabolic/androgenic steroids. The mechanisms are similar in the induction of direct effects, tolerance development and withdrawal.

To the best knowledge of the present inventors, this is the first time a therapeutic use of 3beta- ethenyl, methyl, 3alpha-hydroxy-pregnan/pregnen-steroids as blocking substances against the 3alpha-hydroxy-pregnan-steroid action is disclosed. This action by the 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids work as antagonists to 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids is very surprising, completely new and has never been disclosed in the prior art. The general opinion among the skilled persons is that all 3alpha-hydroxy-steroids are agonists on the GABA-A receptor enhancing the effect of GABA. In the present invention 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids are shown to possess an antagonistic effect against 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids enhancing effect of GABA via the GABA-A receptor. In addition the use of these steroid substances that selectively inhibit GABA- steroid action on the GABA-A receptor have not been disclosed earlier.

One objective of the present invention is thus to identify specific blockers of 3alpha-hydroxy- delta 4-5, 5alpha/beta-steroid (GABA-steroid) action and antagonists that are resistant towards in Vivo metabolism and to make available novel pharmaceuticals and methods for the treatment, alleviation or prevention of steroid related and/or steroid induced CNS disorders. Further objectives, the associated solutions and their advantages will be obvious to a skilled person upon familiarizing himself with the description, examples and claims.

Summary of the invention The present inventors have created compounds that are protected from metabolism in the 3 position of the steroid. In addition, the present inventors have surprisingly found that pregnane steroids with an ethynyl, methyl or ethyl group in 3beta position and a hydroxy group in 3alpha position and a keto, or hydroxy group in 20 and/or 21 position are able to function as efficient blockers of the 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid action and thus have utility as therapeutic substances for the prevention and/or treatment of steroid related or steroid induced CNS disorders. They therefore have utility, as therapeutic substances, e.g. for the prevention and/or treatment of disorders were the GABA function is over stimulated by 3alpha-hydroxy- delta 4-5, 5alpha/beta-steroid action, as is the case in like in Alzheimer's disease. To the best knowledge of the present inventors, this is the first time a therapeutic use of 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan/pregnen-steroids as blocking substances against the 3alpha- hydroxy-delta 4-5, 5alpha/beta-steroid action is disclosed. In addition, these substances are now suggested for the manufacture of pharmaceuticals for the treatment of many specific steroid related or steroid induced CNS disorders and for use in methods of treatment, according to the attached claims, which are incorporated herein by reference. That 3beta- ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids work as antagonists to 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids is very surprising as the general opinion among the skilled persons is that all 3alpha-hydroxy-pregnan/pregnen-steroids are agonists on the GABA-A receptor enhancing the effect of GABA.

In summary, it is now known that 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids cause CNS disorders through the above described three possible mechanisms: a) direct action, b) tolerance induction, and c) withdrawal effect. The compounds described in the present invention belong to the pregnane-, pregnene-, androstane- androstene- series, with the addition of opportune functionalities. The compounds can be used alone or as prodrugs and/or in combination with formulations and other compositions in order to enhance and modulate the effects on CNS. Compositions within the scope of this invention include all compositions wherein the compounds of this invention are contained in an amount that is effective to achieve the intended purposes.

To the best knowledge of the present inventors, this is the first time the steroid compounds are invented with the increased resistance against metabolism and show an antagonism towards 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids. In addition, these substances are now suggested for the manufacture of pharmaceuticals for the treatment of many specific steroid related or steroid induced CNS disorders and for use in methods of treatment, according to the attached claims. More specifically a first embodiment of the present invention relates to a method for the treatment and/or alleviation of a CNS disorder, where the gamma-aminobutyric acid (A) receptor (GABA-A) is involved, wherein the effect on the GABA-A receptor is controlled through the use of a substance which inhibits 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid action substantially without antagonistic effect on the GABA action on the receptor.

In one aspect of said first embodiment said substance is chosen among 3beta-ethynyl, 3alpha- hydroxyl, 5alpha-pregnan-20-one, 3beta-ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20-one, 3beta-ethynyl, 5beta-pregnan-3alpha20(R)-diol, 3beta-ethynyl, 5alpha-pregnan-3alpha,20(R)- diol, 3beta-ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20(S)-ol.

In another aspect of said first embodiment said substance is chosen among 3beta,20-dimethyl, 5alpha-pregnan-3alpha,20-diol, 3beta,20-dimethyl, and 5beta-pregnan-3alpha,20-diol.

A second embodiment of the present invention relates to a substance chosen among 3beta- ethynyl, 3alpha-hydroxyl, 5alpha-pregnan-20-one, 3beta-ethynyl, 3alpha-hydroxyl, 5beta- pregnan-20-one, 3beta-ethynyl, 5beta-pregnan-3alpha20(R)-diol, 3beta-ethynyl, 5alpha- pregnan-3alpha,20(R)-diol, 3beta-ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20(S)-ol, 3beta,20- dimethyl, 5alpha-pregnan-3alpha,20-diol, 3beta,20-dimethyl, 5beta-pregnan-3alpha,20-diol for use as a medicament.

In one aspect of said second embodiment said substance is chosen among 3beta-ethynyl, 3alpha-hydroxyl, 5alpha-pregnan-20-one, 3beta-ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20- one, 3beta-ethynyl, 5beta-pregnan-3alpha20(R)-diol, 3beta-ethynyl, 5alpha-pregnan- 3alpha,20(R)-diol, 3beta-ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20(S)-ol, 3beta,20-dimethyl, 5alpha-pregnan-3alpha,20-diol, 3beta,20-dimethyl, 5beta-pregnan-3alpha,20-diol for use as a medicament for the prevention, treatment and/or alleviation of CNS-disorders caused by the action of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids, said treatment being substantially without antagonistic effect on the GABA-A receptor.

A third embodiment of the present invention relates to the use of a substance chosen among 3beta-ethynyl, 3alpha-hydroxyl, 5alpha-pregnan-20-one, 3beta-ethynyl, 3alpha-hydroxyl, 5beta- pregnan-20-one, 3beta-ethynyl, 5beta-pregnan-3alpha20(R)-diol, 3beta-ethynyl, 5alpha- pregnan-3alpha,20(R)-diol, 3beta-ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20(S)-ol, 3beta,20- dimethyl, 5alpha-pregnan-3alpha,20-diol, 3beta,20-dimethyl, 5beta-pregnan-3alpha,20-diol for the manufacture of a medicament for the prevention, treatment and/or alleviation of a CNS- disorder caused by the action of a 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid on the GABA- A receptor.

In one aspect of said third embodiment said CNS-disorder is chosen among tolerance development, withdrawal symptoms, sedation, memory disturbance, learning disturbance, disturbance of motor function, balance disturbances, clumsiness, obesitas, increased appetite, food cravings, stress sensitivity; concentration difficulties, specifically stress linked difficulties in concentration and menstrual cycle linked difficulties in concentration; sleep disorders, specifically menstrual cycle linked sleep disorders; loss of impulse control, mood changes post partum; epilepsy, specifically menstruation cycle dependent epilepsy, stress dependent epilepsy, catamenial epilepsy, and worsening of Petit MaI epilepsy; depression, specifically stress related depression; irritability, tension; migraine, specifically menstrual related migraine and stress related migraine; tiredness, specifically stress related tiredness and menstrual cycle linked tiredness; premenstrual syndrome, premenstrual dysphoric disorder, menstrual cycle linked mood changes, menstrual cycle linked memory changes, stress related memory changes; side effects of postmenopausal therapy in human patients; side effects of oral contraceptives in human patients; alcoholism, relapse in substance abuse, and relapse in alcohol abuse.

A forth embodiment of present invention relates to an isolated cell or cell line expressing a GABA-A receptor including the subunits alphal , beta2, and gamma2L.

In one aspect of said forth embodiment said isolated cell or cell line is HEK-293 and the GABA- A receptor is functional and permanently expressed.

A fifth embodiment of present invention relates to a method for producing an isolated cell or cell line expressing a GABA-A receptor including the subunits alphal , beta2, and gamma2L, comprising the following steps

- obtaining an isolated cell or cell line - excising the coding regions of the GABA-A receptor subunits alphal , beta2, and gamma2L

- ligating each coding region into a mammalian expression vector respectively

- transfecting said cell or cell line with said expression vectors serially, with intermediate selection and purification steps - obtaining a cell or cell line stably expressing a GABA-A receptor including the subunits alphal , beta2, and gamma2L.

In one aspect of said fifth embodiment the cell or cell line is HEK-293. In another aspect of said fifth embodiment the intermediate selection and purification steps involves the use of subunit specific antibodies.

The present inventors have found that steroid compounds possessing a 3beta-ethynyl, 3alpha- hydroxyl, 5alpha/beta-pregnan-20/21-ol/one surprisingly function as efficient blockers of the 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid action but are minimally active against GABA itself. They have thus utility as therapeutic substances for the prevention and/or treatment of steroid related or steroid induced CNS disorders.

In addition, the present inventors have surprisingly found that 3beta,20-dimethyl, 5beta- pregnan-3alpha,20-diol are able to inhibit the 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid action but are minimally active against GABA itself. They therefore have utility, as therapeutic substances, are now suggested for the manufacture of pharmaceuticals for the treatment of many specific steroid related or steroid induced CNS disorders and for use in methods of treatment, according to the attached claims which are incorporated herein by reference

The present inventors have created new compounds that are protected from metabolism in the 3 position of the steroid. They have surprisingly invented 3beta-ethenyl, methyl, 3alpha- hydroxy-pregnan/pregnen-steroids as blocking substances against the 3alpha-hydroxy-delta A- 5, 5alpha/beta-steroid action. In addition, these substances are now suggested for the manufacture of pharmaceuticals for the treatment of many specific steroid related or steroid induced CNS disorders and for use in methods of treatment, according to the attached claims which are incorporated herein by reference.

In summary, it is now known that 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids cause CNS disorders through the above described three possible mechanisms: a) direct action, b) tolerance induction, and c) withdrawal effect. The compounds described in the present invention belong to the 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan - series, with the addition of opportune functionalities. The compounds can be used alone or as prodrugs and/or in combination with formulations and other compositions in order to enhance and modulate the effects on CNS. Compositions within the scope of this invention include all compositions wherein the compounds of this invention are contained in an amount that is effective to achieve the intended purposes.

Terminology

Before the present invention is described, it is to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof. In particular, it must be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" also include plural referents unless the context clearly dictates otherwise.

In the following description, the terms "steroid related" and "steroid induced" as in "steroid related disorders" are meant to encompass the three possible mechanisms by which steroids act on the central nervous system: a) direct action, b) tolerance induction, and c) withdrawal effect. Examples of such disorders have been given above, but they are meant to illustrate each mechanism, and not to be construed as limiting the invention.

The term "blocking" is meant to define an effect where in this case the 3alpha-hydroxy-delta A- 5, 5alpha/beta-steroids are prevented from acting on the GABA-A receptor. It is understood that "blocking" is an entirely different effect than meant by "modulation" or "repression" or similar terms, which suggest that an action is still taking place, but to a lesser extent or at a slower rate.

With the term "antagonist" is meant a substance that hinders another substance, an agonist, to induce its effect. In this application the terms antagonist and blocker are used simultaneously.

As used herein the expression "substantially without antagonistic effect" is not intended to exclude antagonistic effects of such small magnitude that they have no detectable physiological significance. In a particular embodiment of the present invention the expression "substantially without antagonistic effect" means lack of detectable effect when measured by the method described below in connection to Table 3 below, that is when the antagonistic effect is measured as mean % change in chloride flow relative to the chloride flow when the GABA-A receptor is stimulated with 200 nM 3alpha-hydroxy-5alpha-pregnan-20-one-21ol (THDOC) + 30 μM GABA, which is set as 100% stimulated chloride flux.

The term "pharmaceutical composition" is used in its widest sense, encompassing all pharmaceutically applicable compositions containing at least one active substance, and optional carriers, adjuvants, constituents etc. The term "pharmaceutical composition" also encompasses a composition comprising the active substance in the form of derivate or a prodrug, such as pharmaceutically acceptable salts, sulphates and esters. The manufacture of pharmaceutical compositions for different routes of administration falls within the capabilities of a person skilled in galenical chemistry.

The terms "administration" and "mode of administration" as well as "route of administration" are also used in their widest sense. The pharmaceutical composition of the present invention may be administered in a number of ways depending largely on whether a local, topical or systemic mode of administration is most appropriate for the condition be treated. These different modes of administration are for example topical (e.g., on the skin), local (including ophthalmic and to various mucous membranes such for example vaginal and rectal delivery), oral or parenteral and pulmonary, including the upper and lower airways.

The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the composition of the present invention.

The terms "steroid related" or "steroid induced" are meant to encompass the three possible mechanisms by which steroids act on the central nervous system: a) direct action, b) tolerance induction, and c) withdrawal effect.

Detailed description of the invention

The invention concerns new compounds, with protection of metabolism and with surprising a blocking effect on 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids, and method of producing compounds with antagonistic and blocking effects of the 3alpha-hydroxy-delta 4-5, 5alpha/beta- steroid induced CNS disorders. The present invention arises out of the discovery that steroids represented by the formulae 3beta-ethenyl, methyl, 3alpha-hydroxy-steroid have effect as modulators of the GABA receptor signaling as antagonists or inverse agonists.

The presence of a tertiary alcohol moiety in position 3 has been shown to be able to prolong the half-life of a steroidal compound within the body through preventing metabolic oxidations or degradation in the body. The presence of a hydrogen-bond acceptor/donator group bonded to the D ring of a steroidal molecule influences the ability of the steroid to modulate the GABA receptor signaling.

The present invention is concerning steroids represented by the formulae 3beta-ethenyl, methyl, 3alpha-hydroxy-steroid or a pharmaceutically acceptable salt, prodrug or solvate thereof:

The present inventors have shown that steroids having a 3beta-ethenyl, methyl, 3alpha- hydroxy-steroid configuration can block the action of 3alpha-hydroxy-delta 4-5, 5alpha/beta- steroids oh the human GABA-A receptor expressed in HEK-294 cells in vitro, thus blocking the development of the negative effects of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids. Both the mechanism of action of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids in the development of CNS disorders and the mechanism of action of 3beta-ethenyl, methyl, 3alpha-hydroxy- pregnan-steroids in their interactions with 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids have been addressed. Examples of such compounds are given in Table 2. The surprising and completely new part in this invention is that a steroid 3alpha-hydroxy group can be an antagonist to the GABA-A receptor. It has generally been assumes among skilled persons that all steroids with a 3alpha-hydroxy group are agonists to the GABA-A receptor. The new invention is that when the metabolism is hindered by inducing a 3beta-ethenyl, methyl, or ethyl group to the 3alpha-hydroxy-pregnane-steroid the compound becomes an antagonist. When a 3alpha-ethenyl, methyl, or ethyl group is introduced and the hydroxy group is in 3beta position the effect disappears.

Table 2. List of 3beta-ethenyl, methyl, ethyl, 3alpha-hydroxy-preqnan-steroids tested in the present invention^*

^*AII substances were made by Umecrine AB, Umea, Sweden.

The present invention concerns all steroids having a 3beta-ethenyl, methyl, in 3beta position, here exemplified by the 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids described in table 2. The present inventors have shown that these steroids are antagonists and able to block the effect of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids effects in the central nervous system (CNS). Surprisingly, simultaneous treatment with a 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids and 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids mentioned in table 2, inhibits the uptake and chloride flux thru the GABA-A receptor induced by the 3alpha- hydroxy-delta 4-5, 5alpha/beta-steroid.

One advantage of the invention is that 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids efficiently block and antagonize the GABA-A receptor modulation effect of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids. A particular advantage is that this blocking is achieved at pharmacologically and physiologically suitable concentrations.

3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids have been shown by the present inventors to inhibit the effect of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids in a GABA-A receptor model of chloride uptake. The GABA-A receptor is a chloride channel and the GABA-A receptor exercises its action via changing the influx of chloride through the channel. The neuronal activity in the brain is decreased when the GABA-A receptor is open and a large amount of chloride flux into the cell. There is a relation between the amount of chloride moving in and the clinical effect of a GABA-A receptor active drug.

Benzodiazepines and barbiturates exercise their action via this mechanism. This however also accounts for the adverse effects of these drugs. The new and surprising finding behind the present invention is that it is possible to block the action of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids on the chloride uptake by administering 3beta-ethenyl, methyl, 3alpha- hydroxy-pregnan-steroids in pharmaceutically and physiologically acceptable amounts to HEK- 293 cells, permanently transfected with the human alpha1 beta2gamma2 GABA_A receptor expressing a functional alphal beta2gamma2L GABA_A receptor. The effect of the 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids were measured by determining the chloride flux through the GABA-A receptor using Patch-clamp technique, described in details in examples below, under the influence of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids. It can be assumed, with a high degree of confidence, that the results achieved in this model can be confirmed in human patients.

As has been discussed above, there are a number of symptoms and conditions that are related to 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids, and where blocking the 3alpha-hydroxy- delta 4-5, 5alpha/beta-steroid effect would be a treatment of the condition in question. The present inventors have made available substances and methods for such blocking.

The present inventors have shown that 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids can block the action of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids, thus blocking the development of the negative effects of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids. The inventors have determined both the mechanism of action behind disorders caused by 3alpha- hydroxy-delta 4-5, 5alpha/beta-steroids, and the mechanism of action behind the interaction of 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids with 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids. 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids cause CNS disorders through three possible mechanisms a) direct action, b) tolerance induction, and c) withdrawal effect.

The present invention concerns a new surprising effect by steroids possessing a 3beta-ethenyl, methyl, 3beta position, and a 3alpha-hydroxy group in the 3alpha position. These substances have utility as therapeutics and as components for the manufacture of therapeutics.

A possible mechanism of action of 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroid in the treatment of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid induced CNS conditions and symptoms e.g. learning disturbance, is to block the direct GABA enhancing effect by 3alpha- hydroxy-5alpha-pregnan-20-one. Similar decrease in 3alpha-hydroxy-delta 4-5, 5alpha/beta- steroid induced appetite, cravings for abuse substances like alcohol, disturbance of motor function, sleep and fertility may be blocked by the administration of 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroid.

One embodiment of the present invention is accordingly a method for treatment of steroid related or steroid induced CNS disorders, in particular premenstrual syndrome, wherein tolerance development is prevented and the down-regulation of the GABA-A receptor prevented by administration of at least one substance according to the invention. This treatment would preserve the sensitivity of the GABA-A system and inhibit the development of a less sensitive state during the luteal phase. It has been shown in rat that there is a change of the GABA-A receptor during chronic 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid treatment. The treatment with 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroid according to the present invention sets out to prevent the development of tolerance, and thus to hinder the withdrawal effect when the 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid is withdrawn. A tolerance development will decrease the sensitivity for GABA-A enhancing substances endogenously produced like the 3alpha-hydroxy-5alpha-pregnan-20-one or benzodiazepines. When the drug is rapidly removed, like at the end of the luteal phase in the menstrual cycle a rebound effect arrives after the withdrawal of the 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid. Such a situation is found in humans with increase in migraine and epileptic seizures during the menstruation shortly after the end in production and withdrawal of the steroids.

Another embodiment of the present invention is accordingly a method for treatment or prevention of tolerance development and/or withdrawal symptoms, by administration of at least one substance according to the invention.

The present invention further concerns a method for the treatment and/or prevention of steroid related or steroid induced mood disorders in human patients described above, according to which method at least one 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroid is administered to said patient. Eligible routes of administration are for example the following: intravenously, nasally, per rectum, intra vaginally, percutaneously injections or implants and orally. Nasal administration offers the benefits of ease and the possibility of self-administration by the patient. Percutaneous administration, using the substances formulated as an implant, a cream, gel, and an ointment or in the form of slow-release adhesive medicine patches, is another possible form of administration, e.g. for self-medication. In any of these or other routes of administration, the formulation of the composition may be adapted or adjusted according to normal pharmacological procedures, comprising the effective pharmaceutical in a chemical form, suitable for the chosen route, together with suitable adjuvants and vehicles, conventionally used and well-known to a person skilled in the art.

For the treatment of many CNS disorders and for the uses as anti anaesthetic the effective compound or compounds may however be administered intravenously. In one embodiment of the invention the doses, in intravenous administration, are doses in the range of 0.02 - 20 mg per kg body weight.

Further, the present invention encompasses methods for the treatment and/or prevention of steroid related or steroid induced CNS disorders in human patients, according to which method one or more 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroids (see table 2) is/are administered in a pharmaceutically and physiologically acceptable dose to said patient.

Examples of symptoms and conditions caused by the direction action of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids are sedation, tiredness, memory disturbance, learning disturbance, disturbance of motor function, clumsiness, increased appetite and food cravings, relapse in substance and alcohol abuse, negative mood as tension, irritability and depression which are the cardinal symptoms in the premenstrual syndrome and the worsening of Petit MaI epilepsy. Conditions and symptoms caused by tolerance development after long time (days) exposure to 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids are e.g. stress sensitivity, concentration difficulties, stress or menstrual cycle linked difficulties in concentration, sleep disorders, tiredness, loss of impulse control and depression. 3alpha-hydroxy-delta 4-5, 5alpha/beta- steroids also reinforce drug dependency. According to the present invention, these conditions or symptoms can be prevented, alleviated or treated by the administration of at least one 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroid to the patient.

A continuous but shorter exposure to 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids gives a withdrawal effect when the exposure is ended. This phenomenon occurs during menstruation when the production of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids by the corpus luteum of the ovary is interrupted. This withdrawal phenomenon also occurs after giving birth (post partum) when the 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid production by the placenta is interrupted. The same phenomenon is also noted when a period of stress is ended and the 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids produced by the adrenal during the stress are interrupted. Examples of conditions that are influenced by this withdrawal/ abstinence phenomenon are partial epilepsy where the patient has an epileptic focus in the cerebral cortex where a worsening occurs at the withdrawal period during menstruation. This phenomenon is called "catamenial epilepsy". Other examples are menstrual related migraine and stress related migraine and mood changes post partum. Abstinence is a sign of an earlier developed tolerance.

Examples of such disorders, believed to be steroid related or steroid induced, include the following: epilepsy, menstruation cycle dependent epilepsy, stress dependent epilepsy, depression, stress related depression, migraine, tiredness and in particular stress related tiredness, premenstrual syndrome, premenstrual dysphoric disorder, menstrual cycle linked mood changes, menstrual cycle linked memory changes, stress related memory changes, menstrual cycle linked difficulties in concentration, menstrual cycle linked sleep disorders and tiredness. There are strong indications that also obesitas and increased apetite, and relapse in substance and alcohol abuse, as well as some forms of balance disturbances are steroid related or steroid induced. The present invention thus offers substances and methods for treatment, alleviation or prevention of these conditions.

One embodiment of the invention, addressing a problem afflicting numerous women, relates to a method for the treatment and/or prevention of side effects of postmenopausal therapy in human patients, according to which method at least one 3beta-ethenyl, methyl, 3alpha- hydroxy-pregnan-steroid is administered to said patient. Another embodiment of the invention relates to treatment and/or prevention of side effects of oral contraceptives in human patients, in which treatment at least one 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroid is administered to said patient. In this application the effective composition of at least one 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroid may be administered together with the oral contraceptive, taken by the patient. Nasal and percutaneous administrations are also possible routes of administration.

In general, the present invention encompasses the use of 3beta-ethenyl, methyl, 3alpha- hydroxy-pregnan-steroid, either alone or in combination, for the manufacture of a pharmaceutical for the treatment or prevention of any one of the 3alpha-hydroxy-delta 4-5,

5alpha/beta-steroid related or -induced disorders described in the specification, and especially one or several of the following disorders: epilepsy, menstruation cycle dependent epilepsy, stress dependent epilepsy, depression, stress related depression, migraine, tiredness and in particular stress related tiredness, premenstrual syndrome, premenstrual dysphoric disorder, menstrual cycle linked mood changes, menstrual cycle linked memory changes, stress related memory changes, menstrual cycle linked difficulties in concentration, menstrual cycle linked sleep disorders, tiredness, alcoholism, relapse in substance abuse, and relapse in alcohol abuse.

Examples

Preparation of HEK-293 cells, permanently transfected with the human alpha1 beta2gamma2 GABA_A receptor expressing a functional alphal beta2gamma2L GABA_A receptor.

Inventive cell lines permanently expressing a functional the human GABA-A receptor was made in following steps. The GABA-A receptor subunits alphal (308-1727 NM_000806), beta2 (214-1679 NM_000813), and gamma2L (290-1785 NM_198904) including introduced Kozac sequences just before the start codons were subcloned into mammalian expression vectors containing Geneticin, Hygromycin B, and Zeocin resistance, respectively. The production of the plasmids used in the sub-cloning of the different subunits is published in Rahman, M, Borra, VB, Isaksson, M, Johansson, IM, Ragagnin, G, Backstrδm, T and Wang, MD Clin Exp

Pharmacol Physiol 2008, PMID 18430052, Epub ahead of print. The expression vectors - pcDNA 3.1+/Geneticin, pcDNA 3.1-/Hygromycin, and pcDNA 3.1+/Zeocin are all purchased from Invitrogen. Shortly the method used was; The coding regions of the GABA-A receptor subunits alphal , beta2, and gamma2L were with the restriction enzymes EcoR I (alphal and gamma2L), and Not I + BamH I (beta2) excised from plasmids earlier generated (Rahman et al 2008), and purified. The excised subunit coding regions were ligated into the mammalian expression vectors pcDNA 3.1+/Geneticin (alphal ), pcDNA 3.1 -/Hygromycin (beta2), and pcDNA 3.1+/Zeocin linearized with the same restriction enzymes as for the coding regions. A HEK-293 cell line stably expressing the three GABA-A receptor subunits was produced by transfection of the subunits one at a time. Transfection was done with the use of Lipofectamine and DNA-Plus reagent (both from Invitrogen). The transfection was followed by selection with the appropriate antibiotics, cell separation with the use of subunit specific antibodies (beta2 and gamma2), and production of single cell colonies. Produced cell lines were analysed with immunocytochemistry for the three GABA-A receptor subunits. Cell cytospin was used to concentrate the cells. A standard ICC protocol was used with secondary antibodies tagged with the fluorophores Texas red and Alexa flour 488. Cells are then analysed by immunofluorescence. Apparatus used in the protocol is a cell cytospin machine, and a fluorecence microscope for analyses of presence of the expected GABA-A receptor proteins. When a stable cell line was established and selected test were made to show the function of the GABA-A receptor with normal and good reactivity towards GABA (Figure 1 ) and 3alpha- hydroxy-5alpha-pregnan-20-one-21ol, (tetrahydrodesoxycorticosterone, THDOC) (Figure 2).

Short description of the drawings

Fig 1 shows the reactivity of one inventive cell line towards GABA;

Fig 2 shows the reactivity of one inventive cell line towards 3alpha-hydroxy-5alpha-pregnan-20- one-21ol, (tetrahydrodesoxycorticosterone, THDOC).

Examples, GABA(A) receptor effects of UC-steroids

Aim: To investigate their effect of 3beta-ethenyl, methyl, 3alpha-hydroxy-pregnan-steroid UC- steroids on the GABAA receptor function in absence and in presence of GABAA receptor agonist Tetrahydrodeoxycorticosterone (THDOC) by the Dynaflow™ system on HEK-293 cells. In these tests the protocol was optimized to be similar to the physiological conditions in the synaptic cleft.

Cell culture: HEK-293 cells, permanently transfected with the human alpha1 beta2gamma2 GABA_A receptor subtypes, were seeded at a density of 3 x 10⁴/ 25 cm² in cellbind culture flask. The transfected cells were used for patch-clamp experiments 3 days after seeding. When using the cells for patch-clamp experiments the cells were washed twice with O₂ bubbled EC-solution (see below). About 5 ml_ EC was then added and the cells were kept in the incubator for about 15 minutes. After 15 minutes the cells come loose from the bottom of the flask and were separated by carefully sucking couple of times with a Pasteur pipette.

Dynaflow™ system: Dynaflow™ system with DF-16 Pro Il chips was used for all patch-clamp experiments. The DF-16 Proll chips have non-sticky inserts for the wells. The channel width is 150 μm and the height 50 μm. The well volume is 280 μl_. Run time at the flow rate of 26 μL/min. is 180 min. The pump settings are as follow: Omnifix 2 ml_ syringe with inner diameter of 9.65 mm is used. The syringe pump flow rate for DF-16 Pro Il chip is 26 μL/min. Steroids and GABA: GABA was dissolved in EC-solution by ultra sound for about 40 minutes to the concentration of 10 mM in room temperature. All steroids were dissolved to the concentration of 6 mM in DMSO. The DMSO concentration was 0.1 % in all end-solutions, including the wash solution (EC) and the solution with GABA alone. End-solutions are the solutions added into the wells of the chip.

Electrophysiology: Patch electrodes were pulled from 1.5 mm O. D., 0.86 mm I. D. borosilicate capillary glass without filament. Typical electrodes had a resistance of 2-5 MΩ when filled with intracellular solutions. The intracellular solution consisted of (in mM): 140 Cs-gluconate, 3.0 NaCI, 1.2 MgCI₂, 1.0 EGTA, 10 HEPES. pH was adjusted to 7.2 with CsOH. The extracellular (EC) solution used during recordings contained (in mM): 137 NaCI, 5.0 KCI, 1.0 CaCI₂, 1.2 MgCI₂, 10 HEPES, 10 glucose. pH was adjusted with NaOH to 7.4. After compensating for the liquid junction potential a steady holding potential of -17 mV was used in all experiments. In physiological conditions the HEK-293 has a resting potential at -40 mV and a low concentration of chloride ions inside the cell. By using the holding potential of -17 mV and the intracellular solution with low chloride ion concentration the chloride ions flux into the cell when the receptors are activated. All experiments were performed at room temperature (21 to 23 ⁰C). A standard protocol was used for all experiments.

Protocol

GABA applications: By using the Dynaflow equipment it is possible to study transfected HEK- 293 during almost physiological conditions. The Dynaflow system allows application of solutions for as short as 40 ms up to minutes in time. Physiologically, in the synaptic cleft, GABA is released in mM range for about 2 ms. In these experiments we have applied GABA ± steroid for 40 ms. We found that in almost all cells, the first GABA application gave a smaller response than the second GABA application. There was no difference in response between the second and the third GABA application. Therefore the first GABA application is always repeated twice and the second response is used in the analysis.

Washout: GABA is quite solubility in water and easy to washout from the receptor. The washout time was set to 1 min. after application with GABA solely. Steroids on the other hand are difficult to dissolve in water and also difficult to washout from the receptor. In our experiments, we used THDOC as the GABA agonist. With 2 minutes washout time, 200 nM THDOC had been completely washout as shown by neither an accumulative nor a desensitization effect.

Incubation: To see the effect of the steroids and to achieve stable results it was found that the steroids have to be incubated on the receptor before application of GABA. Different incubation times were studied to achieve the optimal time for attain stable results and minimize the washout time. Incubation time of 20 s. showed to be the optimal time for washout time of 2 min. Conclusion: The optimized protocol is like follow: 20 s. incubation of steroids, 40 ms. GABA ± steroids application, 2 min. washout. The first GABA application is repeated twice with a washout time of 1 min. between the first and the second application.

Results: It was noticed that the effects can differ between the cells if they are in bad conditions. Therefore, some minor changes were made in the intracellular buffer and complementary experiments were done on the steroids.

Experiments were performed in vitro to confirm the GABA(A) receptor effect as antagonists blocking effect of 3alpha-hydroxy-steroids with 3alpha/beta-ethenyl, methyl, 3beta/alpha- hydroxyl group pregna(e)n or androstan steroid skeleton and testes were also 3alpha/beta- ethynyl, 3alpha-hydroxyl groups (Table 2, 3).

Table 3. Median effects of tested UC-steroids with a 3alpha/beta-ethenyl, methyl, ethyl,

3beta/alpha-hydroxyl group in concentration of 1 uM tested against the GABA-steroid THDOC (3alpha-hydroxy-5alpha-pregnan-20-one-21ol) in 200 nM + GABA 30 uM measured as change in chloride flow through the GABA-A receptor. (n=4-5 cells)

Table 3 gives a summary of the tested UC-steroid antagonistic effect as mean % change in chloride flow relative to the chloride flow when the GABA-A receptor is stimulated with 200 nM 3alpha-hydroxy-δalpha-pregnan-20-one-21ol (THDOC) + 30 μM GABA = 100% stimulated chloride flux. Tested for antagonism are the 3alpha/beta-ethenyl, methyl, ethyl, 3alpha/beta- hydroxy-δalpha/beta-pregnan-steroids with keto, hydroxy and methyl groups at 20 position shown in Table 2 and 3. Some of the steroids, (3alpha-ethynyl, 3beta-hydroxyl, δalpha- pregnan-20-one UC2003 and 3alpha-ethynyl, 3beta-hydroxyl, androstan-17-one, UC2028 and 3beta-ethyl, 3alpha-hydroxyl, δalpha-pregnan-20-one, UC2023) has no effect on the GABA-A receptor and are included here only for the sake of completeness to show that the antagonistic effect is specific to the 3alpha/beta-ethenyl, methyl, 3alpha/beta-hydroxy-δalpha/beta-pregnan- steroids. Although the invention has been described with regard to its certain embodiments, including the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the intention which is set forth in the claims appended hereto.

Claims

1. A method for the treatment and/or alleviation of a CNS disorder, where the gamma- aminobutyric acid (A) receptor (GABA-A) is involved, characterized in that the effect on the GABA-A receptor is controlled through the use of a substance which inhibits 3alpha-hydroxy- delta 4-5, 5alpha/beta-steroid action substantially without antagonistic effect on the GABA action on the receptor.

2. The method according to claim 1 , wherein said substance is chosen among 3beta-ethynyl, 3alpha-hydroxyl, 5alpha-pregnan-20-one, 3beta-ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20- one, 3beta-ethynyl, 5beta-pregnan-3alpha20(R)-diol, 3beta-ethynyl, δalpha-pregnan- 3alpha,20(R)-diol, 3beta-ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20(S)-ol.

3. The method according to claim 1 , wherein said substance is chosen among 3beta,20- dimethyl, 5alpha-pregnan-3alpha,20-diol, 3beta,20-dimethyl, 5beta-pregnan-3alpha,20-diol.

4. A substance chosen among 3beta-ethynyl, 3alpha-hydroxyl, 5alpha-pregnan-20-one, 3beta- ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20-one, 3beta-ethynyl, 5beta-pregnan-3alpha20(R)- diol, 3beta-ethynyl, 5alpha-pregnan-3alpha,20(R)-diol, 3beta-ethynyl, 3alpha-hydroxyl, 5beta- pregnan-20(S)-ol, 3beta,20-dimethyl, 5alpha-pregnan-3alpha,20-diol, 3beta,20-dimethyl, 5beta- pregnan-3alpha,20-diol for use as a medicament.

5. A substance chosen among 3beta-ethynyl, 3alpha-hydroxyl, 5alpha-pregnan-20-one, 3beta- ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20-one, 3beta-ethynyl, 5beta-pregnan-3alpha20(R)- diol, 3beta-ethynyl, 5alpha-pregnan-3alpha,20(R)-diol, 3beta-ethynyl, 3alpha-hydroxyl, 5beta- pregnan-20(S)-ol, 3beta,20-dimethyl, 5alpha-pregnan-3alpha,20-diol, 3beta,20-dimethyl, 5beta- pregnan-3alpha,20-diol for use as a medicament for the prevention, treatment and/or alleviation of CNS-disorders caused by the action of 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroids, said treatment being substantially without antagonistic effect on the GABA-A receptor.

6. The use of a substance chosen among 3beta-ethynyl, 3alpha-hydroxyl, 5alpha-pregnan-20- one, 3beta-ethynyl, 3alpha-hydroxyl, 5beta-pregnan-20-one, 3beta-ethynyl, 5beta-pregnan- 3alpha20(R)-diol, 3beta-ethynyl, 5alpha-pregnan-3alpha,20(R)-diol, 3beta-ethynyl, 3alpha- hydroxyl, 5beta-pregnan-20(S)-ol, 3beta,20-dimethyl, 5alpha-pregnan-3alpha,20-diol, 3beta,20- dimethyl, 5beta-pregnan-3alpha,20-diol for the manufacture of a medicament for the prevention, treatment and/or alleviation of a CNS-disorder caused by the action of a 3alpha- hydroxy-delta 4-5, 5alpha/beta-steroid on the GABA-A receptor.

7. The use according to claim 6 wherein said CNS-disorder is chosen among tolerance development, withdrawal symptoms, sedation, memory disturbance, learning disturbance, disturbance of motor function, balance disturbances, clumsiness, obesitas, increased appetite, food cravings, stress sensitivity; concentration difficulties, specifically stress linked difficulties in concentration and menstrual cycle linked difficulties in concentration; sleep disorders, specifically menstrual cycle linked sleep disorders; loss of impulse control, mood changes post partum; epilepsy, specifically menstruation cycle dependent epilepsy, stress dependent epilepsy, catamenial epilepsy, and worsening of Petit MaI epilepsy; depression, specifically stress related depression; irritability, tension; migraine, specifically menstrual related migraine and stress related migraine; tiredness, specifically stress related tiredness and menstrual cycle linked tiredness; premenstrual syndrome, premenstrual dysphoric disorder, menstrual cycle linked mood changes, menstrual cycle linked memory changes, stress related memory changes; side effects of postmenopausal therapy in human patients; side effects of oral contraceptives in human patients; alcoholism, relapse in substance abuse, and relapse in alcohol abuse.

8. An isolated cell or cell line expressing a GABA-A receptor including the subunits alphal , beta2, and gamma2L.

9. The isolated cell or cell line according to claim 8, wherein the cell or cell line is HEK-293 and the GABA-A receptor is functional and permanently expressed.

10. A method for producing isolated cell or cell line expressing a GABA-A receptor including the subunits alphal , beta2, and gamma2L, comprising the following steps

- obtaining an isolated cell or cell line

- excising the coding regions of the GABA-A receptor subunits alphal , beta2, and gamma2L - ligating each coding region into a mammalian expression vector respectively

- transfecting said cell or cell line with said expression vectors serially, with intermediate selection and purification steps

- obtaining a cell or cell line stably expressing a GABA-A receptor including the subunits alphal , beta2, and gamma2L.

11. The method according to claim 10, wherein the cell or cell line is HEK-293.

12. The method according to claim 10, wherein the intermediate selection and purification steps involves the use of subunit specific antibodies.