NZ721622B2 - Steroid compound for use in the treatment of hepatic encephalopathy - Google Patents

Abstract

The present invention provides the steroidal compound 3-ethynyl-3-hydroxyandrostan-17-one oxime, or a pharmaceutically acceptable salt thereof, for use in treatment of hepatic encephalopathy.

Description

D COMPOUND FOR USE IN THE TREATMENT OF HEPATIC ALOPATHY Field of the ion The present invention concerns a steroid nd for use in treatment of hepatic encephalopathy.

Background of the invention Hepatic encephalopathy (HE) is a serious sychiatric and neurocognitive complication in acute and chronic liver disease. HE is a significant and increasing health care problem due to the large and increasing prevalence of c liver disease. HE is characterized by impairments of the sleep-wake cycle, cognition, memory, learning, motor coordination, consciousness, decreased energy levels and personality change, ranging from minimal HE (MHE) to overt HE (OHE). MHE is manifested with cognitive impairment and has detrimental effects on health related quality of life and the ability to perform complex tasks such as driving. In addition, OHE is clinically sted with mental and motor disorders and the symptoms ranges from disorientation through on and coma.

Naturally occurring steroids are subject to intense metabolism and are typically not suitable for oral stration. The metabolites of the endogenous steroid hormones pregnenolone, progesterone, deoxycorticosterone, cortisone and cortisol, known as pregnanolones as well as the lites of testosterone, androstenedione and dehydroepiandrosterone, have all been the subject of various studies, at least partially elucidating their role in the neurological signal system in mammals. The steroid metabolites induce CNS symptoms and disorders and steroids act as ve modulators on the gamma-aminobutyric acid receptor- chloride ionophore (GABAA-R) complex and are therefore called GABAA receptor modulating steroids (GAMS). n steroids have been shown to be specific GABAA or enhancers.

Examples of these steroids can inter alia be found in . Some of these ds are potent and have e.g. been shown to have an ability to induce amnesia, sedation and anesthesia in pharmacological dose. WO 99/45931 and WO 03/059357 disclose antagonistic effects of steroids. Wang et al. 2000 (Acta Physiol Scand 169, 333-341) and Wang et al. 2002 (J Neurosci 22(9):3366-75) disclose antagonistic effects of 36-OH-5d-pregnanone and other 5G/B pregnan ds. WO2006/056794 and WO2010/144498 discloses use of nds for treatment of liver decompensation, hepatic encephalopathy and portal hypertension. There is a need to provide new and effective therapeutic treatments for hepatic encephalopathy and related disorders Description of the invention The present invention provides the compound soc-ethynyl-BB- hydroxyandrostanone oxime or a pharmaceutically acceptable salt thereof, for use in treatment of hepatic encephalopathy. soc-Ethynyl-3[3-hydroxyandrostanone oxime belongs to a class of compounds known as GABAA receptor modulating steroid antagonists (GAMSAs).

We have found that 3α-ethynyl-3β-hydroxyandrostanone oxime is able to selectively inhibit the positive modulation of the GABA A receptor by endogenous steroids such as allopregnanolone and tetrahydrodeoxycorticosterone (THDOC).

These steroids are known to induce sedation, cognitive impairment and motor disturbances, and their concentration in the brain is increased in patients with liver disease-induced hyperammonemia and HE.

However, we have also found that 3α-ethynyl-3β-hydroxyandrostanone oxime does not have an antagonistic effect towards the action of gamma-aminobutyric acid (GABA) at the GABAA receptors. This surprising selectivity is advantageous from a safety perspective as inhibition of GABA binding at the GABAA receptors can lead to side-effects, including convulsions.

Furthermore, ynyl-3β-hydroxyandrostanone oxime acts on both the α1 and α5 GABAA receptor sub-types and so is able to exert a positive effect on both the motor and cognitive impairment, and the sedative effects, that result from the over-activation of GABAA receptors. The positive effect of 3α-ethynyl-3βhydroxyandrostanone oxime on motor and cognitive impairment has been illustrated in two animal models of HE (hyperammonemia and porta-caval anastomosis in rats vide infra).

Unlike ng ents for HE, 3α-ethynyl-3β-hydroxyandrostanone oxime does not affect a levels in vivo. Therefore, there is clearly also potential for it’s mentary use in therapy.

Accordingly, there is good basis to believe that 3α-ethynyl-3β-hydroxyandrostan- 17-one oxime is particularly well-suited to the ent of HE and d disorders.

Brief description of the drawings Figure 1 shows that soc-ethynyI-SB-hydroxyandrostanone oxime does not affect blood ammonia levels. Values are the mean 1r SEM of 12 rats per group, values icantly different from ls are indicated by asterisks; ***, p< 0001. CV = control rats treated with vehicle; HAV = hyperammonemic rats treated with vehicle; HA+GAM = hyperammonemic rats treated with 3d-ethynyI-3B-hydroxyandrostan- 17-one oxime.

Figure 2 shows that soc-ethynyI-SB-hydroxyandrostanone oxime es spatial learning of hyperammonemic rats in the Radial maze. The figure shows working errors in the radial test. Working errors in block 1. Values are the mean 1r SEM of 8 rats per group. # p < 0.05 versus HAV. CV = control rats treated with e; HAV = hyperammonemic rats treated with vehicle; HA+GAM = hyperammonemic rats treated with 3d-ethynyI-3B-hydroxyandrostanone oxime.

Figure 3 shows that soc-ethynyl-SB-hydroxyandrostanone oxime, in the Morris water maze test, restores special memory of hyperammonemic rats. The figure shows the time to find the platform on the first trial of day 3. Values are the mean 1r SEM of 8 rats per group. HAV versus CV p = 0.052. CV = control rats treated with e; HAV = hyperammonemic rats treated with vehicle; HA+GAM = hyperammonemic rats d with soc-ethynyI-SB-hydroxyandrostanone oxime.

Figure 4 shows that soc-ethynyl-3[3-hydroxyandrostanone oxime restores motor coordination of hyperammonemic rats. Values are the mean 1r SEM of 15 rats per group. Values significantly different from CV are indicated by *, p<0.05, values significantly different from HAV are indicated by ###, p<0.001. CV = control rats d with vehicle; HAV = hyperammonemic rats treated with vehicle; HA+GAM = hyperammonemic rats treated with 3d-ethynyI-3B-hydroxyandrostanone oxime.

Figure 5 shows total plasma 3α-ethynyl-3β-hydroxyandrostanone oxime concentrations.Total plasma 3α-ethynyl-3β-hydroxyandrostanone oxime concentrations in control and hyperammonemic rats 4 and 23 hours after the subcutaneous injection of 3α-ethynyl-3β-hydroxyandrostanone oxime on day five and during the last week of treatment with daily injections. HA= Hyper ammonia animals.

Figure 6 provides unbound* brain 3α-ethynyl-3β-hydroxyandrostanone oxime concentrations in control and hyperammonemic rats 1-2 hours after the s.c. injection of 3α-ethynyl-3β-hydroxyandrostanone oxime after seven weeks with daily injections of 20 mg/kg.

*Unbound brain concentration = fraction of ynyl-3β-hydroxyandrostan one oxime in the brain that is not bound to r protein or brain tissue.

Figure 7 provides representative electrophysiological measurements showing tetrahydrodeoxycorticosterone (THDOC) enhanced activation of α1β2γ2L GABAA receptors. HEK-293 cells expressing human L GABAA receptors were exposed to 30 µM GABA or 30 µM GABA plus 100 nM THDOC for 40 ms. With THDOC there was a 20 s pre-incubation before application of THDOC + GABA.

Figure 8 provides representative electrophysiological measurements showing 3αethynyl-3β-hydroxyandrostanone oxime (GAMSA) antagonism of the THDOC enhanced tion of α1β2γ2L and L GABAA receptors and no inhibition of GABA. A) 1 µM 3α-ethynyl-3β-hydroxyandrostanone oxime antagonism of the 100 nM THDOC enhanced activation of 30 µM GABA with the α1β2γ2L GABAA receptor, B) 1 µM 3α-ethynyl-3β-hydroxyandrostanone oxime does not antagonize the 30 µM GABA activation of the α1β2γ2L GABAA receptor C) 1 µM 3α-ethynyl-3β-hydroxyandrostanone oxime antagonism of the 200 nM THDOC enhanced activation of 0.3 µM GABA with the α5β3γ2L GABAA receptor; indicating nism of s effect D) 1 µM 3α-ethynyl-3β-hydroxyandrostanone oxime does not antagonize the 0.3 µM GABA activation of the α5β3γ2L GABAA receptor.

Figure 9 illustrates the ability of 3α-ethynyl-3β-hydroxyandrostanone oxime to restore motor coordination in hyperammonemic and PCS rats. Motor coordination was assessed using the beam walking test. (A) shows the data for l (CV) or hyperammonemic (HAV) rats treated with vehicle and for hyperammonemic rats treated with 3 (HA3), 10 (HA10) or 20 (HA20) mg/kg of 3α-ethynyl-3βhydroxyandrostanone oxime. (B) shows the data for sham-operated controls (SM) or PCS rats treated with vehicle and for PCS rats treated with 0.7 (PCS0.7) or 2.5 (PCS2.5) mg/kg of 3α-ethynyl-3β-hydroxyandrostanone oxime. Values are the mean ± SEM of the number of rats indicated under each bar. Values significantly different from control or sham rats are indicated by asterisks. Values significantly different from hyperammonemic or PCS rats treated with vehicle are indicated by “a”. * p< 0.05; a p< 0.05; aa p< 0.01; aaa p< 0.001.

Figure 10 illustrates the ability of 3α-ethynyl-3β-hydroxyandrostanone oxime to restore spatial memory in the Morris water maze in hyperammonemic and PCS rats. l learning memory in the Morris water maze was assessed in control (CV) or hyperammonemic (HAV) rats treated with vehicle and for hyperammonemic rats d with 3 (HA3), 10 (HA10) or 20 (HA20) mg/kg of 3αethynyl-3β-hydroxyandrostanone oxime (A, B) and in sham-operated controls (SM) or PCS rats d with vehicle and for PCS rats treated with 0.7 7) or 2.5 (PCS2.5) mg/kg of 3α-ethynyl-3β-hydroxyandrostanone oxime (C,D).

(A,C) Escape latencies (in seconds) to reach the platform during the different sessions. (B,D) Time spent (%) in the correct quadrant during the memory test.

Values are the mean ± SEM of the number of rats indicated under each bar.

Values icantly different from control or sham rats are indicated by asterisks.

Values icantly ent from hyperammonemic or PCS rats treated with vehicle are indicated by “a”. * p< 0.05; a p< 0.05.

Figure 11 illustrates the ability of 3α-ethynyl-3β-hydroxyandrostanone oxime to restore spatial ng in the radial maze in hyperammonemic and PCS rats.

Spatial learning in the radial maze was assessed in control (CV) or hyperammonemic (HAV) rats treated with vehicle and for mmonemic rats treated with 3 (HA3), 10 (HA10) or 20 (HA20) mg/kg of 3α-ethynyl-3βhydroxyandrostanone oxime (A, B) and in sham-operated controls (SM) or PCS rats treated with vehicle and for PCS rats treated with 0.7 7) or 2.5 (PCS2.5) mg/kg of 3α-ethynyl-3β-hydroxyandrostanone oxime (C,D). (A,C) Working errors during the different sessions. (B,D) Working errors during days 1-2.

Values are the mean ± SEM of the number of rats indicated under each bar.

Values significantly different from control or sham rats are indicated by asterisks.

Values significantly different from hyperammonemic or PCS rats treated with vehicle are indicated by “a”. * p< 0.05; a p< 0.05; aa p<0.01.

Figure 12 illustrates the ability of 3α-ethynyl-3β-hydroxyandrostanone oxime to increase neous motor activity during the night and partially restore the circadian rhythm of PCS rats. Motor activity was ed in sham-operated controls (SM) or PCS rats treated with vehicle or with 0.7 (PCS0.7) or 2.5 (PCS2.5) mg/kg of 3α-ethynyl-3β-hydroxyandrostanone oxime. Motor activity during each hour is shown in A; the ratio of ty during the night and during the day in B and the total activity during the day or the night in C. Lights are turned off at 7:00 pm. Values are the mean ± SEM of 8 rats per group. Values significantly different from SM rats are indicated by asterisks; * p< 0.05; ** p< 0.01; *** p< 0.001. Values significantly ent from PCS rats are indicated by a; a p< 0.05.

Figure 13 illustrates the ability of ethynyl-3β-hydroxyandrostanone oxime to normalize vertical activity during the day and to partially restore the circadian rhythm of PCS rats. The ment was carried out as described for Figure 12 but vertical counts are shown. Values are the mean ± SEM of 8 rats per group. Values significantly different from SM rats are indicated by asterisks; * p< 0.05; ** p< 0.01; *** p< 0.001. Values significantly ent from PCS rats are indicated by a; a p< 0.05; aa .

Figure 14 shows 3α-ethynyl-3β-hydroxyandrostanone oxime exposure in the plasma and in the brain at time at behavioral testing, in hyperammonemic and PCS rats. In A) hyperammonemic rats and B) PCS rats, the total plasma concentrations of 3α-ethynyl-3β-hydroxyandrostanone oxime are shown in µM.

In C) hyperammonemic rats and D) PCS rats, the unbound brain concentrations of ethynyl-3β-hydroxyandrostanone oxime are shown in nmol/kg. Note the similar exposures in the different rat models with the doses used, in hyperammonemic rats 3, 10 and 20 mg/kg/day and in rats with PCS 0.7 and 2.5 mg/kg/day. Data are from the end of the study, i.e. after nine weeks of daily treatments with ethynyl-3βhydroxyandrostanone oxime in sesame oil given s.c. once daily.

Before the t invention is described in detail, it is to be understood that the terminology employed herein is used for the purpose of describing ular embodiments only and is not intended to be limiting.

It is 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.

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, diluents, constituents etc.

The terms “administration” and “mode of administration” as well as “route of administration” are also used in their widest sense.

The compound 3α-ethynyl-3β-hydroxyandrostanone oxime as used in accordance with the invention may be administered in a number of ways depending largely on r a local, topical or systemic mode of administration is most appropriate for the hepatic encephalopathy ion to be treated. These ent modes of administration are for example l (e.g., on the skin), local (including ophthalmic and to various mucous membranes, for example vaginal and rectal ry), oral, parenteral or pulmonary, including the upper and lower airways. The preparation of such compositions and ations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the composition of the t invention.

With the term “antagonist” is meant a substance that hinders another nce, an agonist, to induce its . In this application the terms antagonist and blocker are used interchangeably.

With the term “Type A hepatic encephalopathy” is typically meant hepatic encephalopathy associated with acute liver e, typically associated with cerebral oedema.

With the term “Type B hepatic encephalopathy” is typically meant hepatic encephalopathy (bypass) caused by -systemic shunting without associated intrinsic liver disease.

With the term “Type C hepatic alopathy” is typically meant hepatic encephalopathy occurring in patients with cirrhosis - this type is subdivided in episodic, persistent and minimal encephalopathy.

With the term “minimal hepatic encephalopathy” is typically meant hepatic encephalopathy that does not lead to clinically overt cognitive dysfunction, but can be demonstrated with neuropsychological studies.

With the term “overt hepatic encephalopathy” is typically meant clinically apparent hepatic encephalopathy manifested as neuropsychiatric syndrome with a large spectrum of mental and motor disorders. Overt hepatic encephalopathy may arise episodically, over a period of hours or days in patients previously stable or patients may present with persistent neuropsychiatric alities.

With the term “hyperammonemia” is typically meant a metabolic bance terized by an excess of ammonia in the blood.

With the term “liver transplantation” is lly meant a surgical procedure to remove a diseased liver as a consequence of e.g. acute liver e or cirrhosis, and replace it with a healthy liverfrom a donor. Most liver transplant operations use livers from deceased donors but a liver may also come from a living donor (a portion of a healthy person’s liver). Patients with e.g. cirrhosis commonly experience hepatic encephalopathy and preoperative hepatic encephalopathy is a significant predictor of post-transplant neurologic complications.

With the term “acute-on-chronic liver failure” is typically meant acute decompensation of cirrhosis, at least one organ failure, or belongs to a subgroup with high short-term mortality rate.

With the term nsated cirrhosis” is lly meant liver cirrhosis without any clinical evidence but may include asymptotic esophageal or gastric varices and early ms such as fatigue and loss of energy, loss of appetite and weight loss, nausea or abdominal pain.

With the term “decom pensated cirrhosis” is typically meant advanced liver cirrhosis with a range of clinical evidence such as ce, ascites, oedema, hepatic alopathy, gastrointestinal haemorrhage, portal ension, bacterial ions, or any combination.

With the term l hypertension” is typically meant a hepatic venous pressure gradient following liver cirrhosis, with or without associated transjugular intrahepatic portsystemic shunt (TIPS).

With the term “prevention” within this disclosure, is typically meant prevention of disease or disorder hepatic encephalopathy to occur.

With the term “alleviation” within this disclosure, is typically meant reduction of or freedom from the disease or disorder hepatic encephalopathy. ts suffering from hepatic encephalopathy may show symptoms including, but not limited to, impairments of the sleep-wake cycle, cognition, , learning, motor coordination, consciousness, decreased energy levels and personality change, cognitive impairment, disorientation and coma.

The present inventors have singly shown that soc-ethynyl-BB- hydroxyandrostanone oxime may be useful for the treatment of c encephalopathy.

In a first aspect of the invention, there is provided the compound soc-ethynyl-BB- hydroxyandrostanone oxime or a pharmaceutically acceptable salt thereof, for use in treatment of hepatic encephalopathy.

In one ment of the invention, said hepatic encephalopathy is type A hepatic encephalopathy.

In another embodiment of the invention, said hepatic encephalopathy is type B hepatic encephalopathy.

In another embodiment of the invention, said hepatic encephalopathy is type C c encephalopathy.

In another embodiment of the ion, said hepatic encephalopathy is minimal hepatic encephalopathy.

In r embodiment of the invention, said hepatic encephalopathy is overt hepatic encephalopathy.

In another embodiment of the invention, said compound for use is where said c encephalopathy is treated in a patient with acute liver failure.

In another embodiment of the invention, said compound for use is where said hepatic encephalopathy is treated in a patient with chronic liver disease with or without acute-on-chronic liver failure.

In another embodiment of the ion, said compound for use is for prevention or alleviation of hepatic encephalopathy, such as type A hepatic encephalopathy, type B hepatic encephalopathy, type C hepatic encephalopathy, minimal hepatic encephalopathy, overt hepatic encephalopathy, in a patient with acute liver failure, or in a t with chronic liver disease with or without acute-on-chronic liver failure.

In another embodiment of the invention, said compound for use is ed before, during or after a liver transplantation.

In another embodiment of the invention, there is provided a pharmaceutical composition sing soc-ethynyl-SB-hydroxyandrostanone oxime or a pharmaceutically acceptable salt thereof, for use in treatment of hepatic encephalopathy, together with one or more pharmaceutically acceptable carriers, excipients and/or diluents.

In r aspect of the invention, there is provided a method of treating c encephalopathy, comprising administering a pharmaceutically effective amount of soc-ethynyl-SB-hydroxyandrostanone oxime 2015/050060 or a ceutically able salt thereof, to a patient in need thereof.

In one embodiment of the ion, said hepatic encephalopathy is type A hepatic encephalopathy.

In another embodiment of the invention, said hepatic encephalopathy is type B hepatic encephalopathy.

In another embodiment of the invention, said hepatic encephalopathy is type C hepatic encephalopathy.

In another embodiment of the invention, said hepatic encephalopathy is minimal hepatic encephalopathy.

In another embodiment of the invention, said c encephalopathy is overt hepatic encephalopathy.

In another embodiment of the invention, said patient suffers from acute liverfailure.

In another embodiment of the invention, said patient suffers from chronic liver disease with or without acute-on-chronic liver failure.

In another embodiment of the invention, said compound is provided before, during or after a liver transplantation.

In another embodiment of the invention, there is provided a method of preventing or alleviating hepatic encephalopathy, comprising administering a pharmaceutically effective amount of soc-ethynyl-SB-hydroxyandrostanone oxime or a pharmaceutically able salt f, to a patient in need thereof. Said hepatic encephalopathy may be type A hepatic encephalopathy, type B hepatic encephalopathy, type C c encephalopathy, minimal hepatic encephalopathy or overt hepatic encephalopathy. r, said prevention may be in a patient with acute liver failure, or in a patient with chronic liver disease with or without acute- on-chronic liver failure.

In a another aspect of the invention, there is provided the compound soc-ethynyl-BB- hydroxyandrostanone oxime or a pharmaceutically able salt thereof, for use in treatment of portal hypertension. Said use may also be prevention or alleviation of portal hypertension.

The patient with portal hypertension typically suffers from a liver disease, such as a chronic liver disease, cirrhosis or acute liver failure.

In another aspect of the ion, there is provided a method of treating portal ension, comprising administering a pharmaceutically effective amount of soc-ethynyl-SB-hydroxyandrostanone oxime or a pharmaceutically able salt thereof, to a t in need thereof. Said method may also be in prevention or alleviation of portal hypertension. The patient with portal hypertension typically suffers from a liver disease, such as a chronic liver disease, cirrhosis or acute liver failure.

In a another aspect of the invention, there is provided the compound soc-ethynyl-BB- hydroxyandrostanone oxime or a pharmaceutically acceptable salt thereof, for use in treatment of liver decompensation. Said use may also be tion or alleviation of liver decompensation. The patient with liver decompensation typically s from a liver disease, such as a chronic liver disease or may be suspected of having a precipitating event, such as gastrointestinal bleeding, infection, portal vein thrombosis or dehydration.

In another aspect of the invention, there is provided a method of treating liver decompensation, comprising administering a pharmaceutically effective amount of soc-ethynyl-SB-hydroxyandrostanone oxime or a pharmaceutically acceptable salt thereof, to a patient in need f. Said method may also be in prevention or alleviation of liver decompensation. The patient with liver decompensation typically suffers from a liver disease, such as a chronic liver disease or may be suspected of having a precipitating event, such as gastrointestinal bleeding, infection, portal vein osis or dehydration.

In a another aspect of the invention, there is provided use of the nd soc-ethynyl-SB-hydroxyandrostanone oxime or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating hepatic encephalopathy.

In one embodiment of the invention, said hepatic encephalopathy is type A hepatic encephalopathy.

In another embodiment of the invention, said hepatic encephalopathy is type B c encephalopathy.

In another embodiment of the invention, said c encephalopathy is type C hepatic encephalopathy.

In another embodiment of the invention, said hepatic encephalopathy is minimal hepatic encephalopathy.

In r embodiment of the invention, said hepatic encephalopathy is overt hepatic encephalopathy.

In another embodiment of the invention, said use is where said c encephalopathy is treated in a patient with acute liver failure.

In another embodiment of the invention, said use is where said hepatic encephalopathy is treated in a patient with c liver disease with or without acute-on-chronic liver failure.

In another embodiment of the invention, said use is provided before, during or after a liver transplantation.

In another embodiment of the invention, said use of the compound ynyl-3—B-hydroxyandrostanone oxime, or a pharmaceutically acceptable salt thereof, in the manufacture of a ment, may be for prevention or alleviation of hepatic encephalopathy, such as type A hepatic encephalopathy, type B hepatic encephalopathy, type C hepatic encephalopathy, minimal hepatic encephalopathy, overt hepatic encephalopathy, in a patient with acute liver failure, or in a patient with chronic liver disease with or without acute-on-chronic liver failure.

In r embodiment of this , there is ed a pharmaceutical composition comprising soc-ethynyl-SB-hydroxyandrostanone oxime or a pharmaceutically acceptable salt thereof, for use in ent of hepatic encephalopathy, together with pharmaceutically acceptable carriers, excipients and or ts. Said use may also be in prevention or alleviation of hepatic encephalopathy.

In further aspect of the invention, is the compound soc-ethynyl-BB- hydroxyandrostanone oxime WO 14308 or a pharmaceutically acceptable salt thereof for use in inhibiting or treating symptoms caused by hyperammonemia.

A further embodiment of the invention is the compound soc-ethynyl-BB- hydroxyandrostanone oxime for use in the treatment or tion of hepatic encephalopathy, such as type A hepatic encephalopathy, type B hepatic encephalopathy, type C hepatic encephalopathy, minimal hepatic encephalopathy, overt hepatic encephalopathy, in a patient with acute liver failure, or in a patient with chronic liver disease with or without acute-on-chronic liverfailure; wherein said treatment or prevention comprises the co-administration of soc-ethynyl-BB- hydroxyandrostanone oxime, or a pharmaceutically able salt f, with an ammonia-lowering compound, such as rifaximin, lactulose, ornithine phenylacetate and glycerol phenylbutyrate, ably the ammonia-lowering compound is rifaximin or lactulose, and most ably the ammonia-lowering compound is rifaximin.

A further embodiment of the invention is a method of ent or prevention of hepatic encephalopathy, such as type A hepatic alopathy, type B hepatic encephalopathy, type C c encephalopathy, minimal hepatic encephalopathy, overt hepatic encephalopathy, in a patient with acute liver failure, or in a patient with chronic liver disease with or without acute-on-chronic liverfailure; n said treatment or tion comprises the co-administration of soc-ethynyl-BB- hydroxyandrostanone oxime, or a pharmaceutically acceptable salt f, with an a-lowering compound, such as rifaximin, lactulose, ornithine phenylacetate and glycerol phenylbutyrate, preferably the ammonia-lowering compound is rifaximin or lactulose, and most preferably the ammonia-lowering compound is rifaximin.

A further embodiment of the invention is the use of the compound soc-ethynyl-BB- hydroxyandrostanone oxime in the manufacture of a medicament for the treatment or prevention of hepatic encephalopathy, such as type A hepatic encephalopathy, type B hepatic encephalopathy, type C hepatic encephalopathy, minimal hepatic encephalopathy, overt hepatic encephalopathy, in a t with acute liver failure, or in a t with chronic liver disease with or without acute- on-chronic liver failure; wherein said treatment or prevention comprises the co- administration of soc-ethynyl-SB-hydroxyandrostanone oxime, or a pharmaceutically acceptable salt thereof, with an ammonia-lowering compound, such as min, lactulose, ornithine phenylacetate and glycerol phenylbutyrate, ably the ammonia-lowering compound is rifaximin or lactulose, and most ably the ammonia-lowering compound is rifaximin.

A further aspect of the invention is the compound soc-ethynyl-BB-hydroxyandrostan- 17-one oxime, wherein one or more hydrogen atom in each possible substituent position may be substituted for deuterium or tritium, for use in the treatment of hepatic alopathy such as minimal hepatic encephalopathy or overt hepatic encephalopathy.

A further aspect of the invention is the compound soc-ethynyl-BB-hydroxyandrostan- 17-one oxime, wherein one or more en atom in each le substituent position may be substituted for deuterium or tritium, for use assays that involve determining the concentration of the compound in tissue or fluids.

According to the present invention, soc-ethynyl-SB-hydroxyandrostanone oxime may be administered through one of the following routes of administration: intravenously, nasally, per rectum, bucally, aginally, percutaneously, intramuscularly and orally. According to one embodiment, hynyl-BB- hydroxyandrostanone oxime is administered intravenously. According to another embodiment, hynyl-SB-hydroxyandrostanone oxime is administered nasally. Percutaneous administration, using soc-ethynyl-BB- hydroxyandrostanone oxime formulated as a cream, a gel, and an ointment or in the form of slow-release adhesive medicine patches, is another possible form of administration, similarly suitable for self-medication.

The ceutical composition may be adapted or adjusted ing to normal pharmacological procedures, comprising the effective pharmaceutical in a chemical form, suitable for the chosen route, together with suitable adjuvants, carriers, diluents and vehicles, tionally used and well-known to a person skilled in the art. Conventionally used adjuvants and vehicles for oral administration are for example fillers or suspending agents like titanium dioxide, e anhydride, silica, silica dalis, methylcellulose, magnesium stearate, microcrystalline ose and the like. Conventionally used adjuvants and vehicles for intravenous administration are for example sterile water for injections (WFI), e buffers (for example buffering the solution to pH 7,4) albumin solution, lipid solutions, cyclodextrins and the like. Conventionally used adjuvants and vehicles for transdermal administration are for example Vaseline, liquid paraffin, glycerol, water, MCT oil, sesame oil, vegetable oils and the like. The dose will naturally vary depending on the mode of administration, the particular condition to be treated or the effect desired, gender, age, weight and health of the patient, as well as possibly other factors, evaluated by the treating physician.

The invention will now be described by a number of rative, non-limiting examples.

Example 1.

Synthesis of hynyl-3B-hydroxyandrostanone oxime Step 1: Synthesis of 3a—ethyny/-3,8-hydroxyandrostan-17—one 3,17-androstandione (5.0 mmol) was dissolved in 50 mL dry THF at room temperature (rt) under nitrogen. Ethynyl magnesium bromide (1.1 equiv) was added dropwise at rt under stirring and the solution was left stirring overnight at rt under nitrogen flow. The on was then quenched with saturated NH4CI(aq) and the aqueous phase extracted with dichloromethane (3 x 30 mL). The ted organic phases were evaporated under reduced pressure, the resulting yellow oil dissolved in dichloromethane, washed with brine and dried over MgSO4. The solution was reduced under vacuum, and the residue purified by silica flash column chromatography (1 :4 diethylether: dichloromethane), typical yields 65 %. Eventual traces of byproducts can be eliminated by further recrystallization from diethylether. 1H NMR (400 MHz, CDCIs—de): 52.43 (s, 1H); 2.42 (m, 1H); 2.10-2.04 (m, 2H); 1.02 (m, 1H); 0.86 (s, 3H); 0.83 (s, 3H).

Step 2: Synthesis of 3a—ethynyl-3fl-hydroxyandrostanone oxime 3d-ethynyI-3i3-hydroxyandrostanone (10 mmol) was dissolved in dichloromethane 5 mL and ethanol 50 mL at room temperature and air atmosphere, in a 250 mL round bottom flask. 4 equiv. of NH20H hloride and 4 equiv. of sodium acetate were ved in 5 mL H20 and then added to the steroid solution. mL of ethanol was added and the e put on reflux overnight. The mixture was then cooled and the solvent d under reduced pressure. The white e was treated with 50 mL H20 and 50 mL dichloromethane, the aqueous phase extracted with 3 x 30 mL dichloromethane. The collected organic phases were then dried over MgSO4, filtrated and the solvent removed under reduced pressure. The final residue was purified by silica flash column chromatography dichloromethane: diethyl ether 4:1, typical yields 95-100 % (quantitative). 1H NMR (400 MHz, CDCIs—de): 47 (m, 2H); 2.43 (s, 1H); 1.00 (m, 1H); 0.80 (m, 1H); 0.90 (s, 3H), 0.83 (s, 3H).

Example 2.

Therapeutic effect of 3a-ethynyl-3B-hydroxyandrostanone oxime in animal model of hepatic encephalopathy Treatment and testing schedule In this study an animal model of chronic hyperammonemia was used that reproduces many of the cognitive and motor alterations present in hepatic encephalopathy. Rats were fed with ammonia in their food and after two weeks with ammonia enriched food they developed symptoms of hepatic alopathy.

The beam walking test was made during the 3rel and 4‘“ week of soc-ethynyI-BB- hydroxyandrostanone oxime treatment while the Morris Water maze test was made during 4‘“ -5‘“ week of 3a-ethynyI-3B-hydroxyandrostan-i7-one oxime treatment and the Radial maze test was made during 6‘“ -7‘“ week of Soc-ethynyI-3B- hydroxyandrostanone oxime treatment.

The study was divided into two series with animals (male Wistar rats), each series included the ing groups; Controls treated with vehicle (CV, n=8 per series), Controls treated with Soc-ethynyI-3B-hydroxyandrostan-i 7-one oxime (C+GAM, n=8 per series), hyperammonemic rats treated with e (HAV, n=8 per series), mmonemic rats treated with Soc-ethynyI-3B-hydroxyandrostan-i 7-one oxime (HA+GAM, n=8 per series). The once daily treatment with soc-ethynyI-BB- hydroxyandrostanone oxime at 20 mg/kg or with e was performed with subcutaneous ions of 1 ml/kg around 9 am. Treatment started one week after starting with the ammonium containing diet and continued for the whole experimental period.

Test article of soc-ethynyl-BB-hydroxyandrostanone oxime was prepared as a suspension in sesame oil at 20 mg/kg.

Spatial learning in the Radial maze The Radial maze was designed as a method to assess l ng. The apparatus is composed of a central area that gives access to eight equally-sized arms. The arms were 70 cm long and 10 cm wide and the central area was 30 cm WO 14308 in diameter. The maze was made of black Perspex and was ed 80 cm above de floor. Each arm had lateral walls with a height higher in the side proximal to the central area (30 cm) than in the distal side (5 cm). In the distal extreme of each arm, a ed cup was installed for positioning the food rewards (Hernandez- Rabaza V. et al 2010).

To habituate rats to the maze, the rats were allowed to explore the maze for 10 s on two consecutive days in the presence of distal cues (posters and objects of different sizes), which remained in place throughout ng.

Training in the radial maze was composed of five blocks of three trials each, performed on ten consecutive days. The task involved locating four pellets, each placed at the end of a different arm according to a random configuration.

Configurations were specific for each rat and were kept invariable throughout training. The number of spatial nce errors and working memory were calculated and expressed as number of reference and working errors per block. In addition, a learning index was used to te the learning of the task and was defined as number of right choices-reference errors (Hernandez-Rabaza et al.2010).

Results soc-ethynyl-SB-hydroxyandrostanone oxime restored spatial learning of hyperammonemic rats in the Radial maze. Hyperammonemic rats show reduced spatial learning and perform more working errors in the Radial maze task. Spatial memory was completely restored by soc-ethynyl-3[3-hydroxyandrostanone oxime (Figure 2).

Example 3.

Therapeutic effect of 30i-ethynyl-3B-hydroxyandrostanone oxime in animal model of hepatic encephalopathy Treatment and testing schedule The treatment and testing schedule was as set out in example 2.

Spatial memory in the Morris water maze The maze was designed as a method to assess spatial ng (Morris R. 1984).

The test was carried out using a black circular pool (160 cm diameter, 40 cm height) arbitrarily divided into four quadrants. Water y was ed by adding black paint. A arent Plexiglas platform, 10 cm in diameter, was immersed 2 cm under the water surface at the centre of one quadrant during training sessions (Monfort et al., European Journal of Neuroscience, 2007, 25, 2103-211).

The test was carried out as follows; the first day was the pre-training day, rats were put in the water two times for 30 s only to adapt to water. Then the rats were trained to learn the fixed location of the invisible platform during 3 days. Each training trial involved placing the rat into the pool facing the wall at one of the three quadrants lacking the platform. A different starting point was randomly used on each trial.

Training consisted of five swims per day. Each animal was allowed a m of 120 s to find the rm and was left for 15 s on the platform, if a rat failed to locate the platform within 120 s it was manually guided to the rm by the experimenter. The aim of this test is that the rats learn where the invisible platform is placed and reach it in the shortest time possible. The time, speed and path needed to find the hidden platform was recorded by a video tracking system provided by int Company (Viewpoint 2.5, Champagne au Mont D‘ Or, France) and used as a measure of ng of the task. After 15 training trial, the platform was removed from the pool, the rats were allowed to swim for 90 s in the pool and the time spent in the quadrant where the platform was positioned during training was recorded.

Results soc-ethynyl-SB-hydroxyandrostanone oxime completely restored l memory of hyperammonemic rats in the Morris water maze. Hyperammonemic rats showed reduced memory and needed more time than controls to find the platform (Figure 3).

Example 4.

Therapeutic effect of 30i-ethynyl-3B-hydroxyandrostanone oxime in animal model of hepatic encephalopathy Treatment and g schedule The treatment and testing schedule was as set out in example 2.

Motor coordination in the Beam walking In the beam-walking test rats are trained to traverse an ed, narrow beam to reach an enclosed escape platform. The beam is made of smooth round wood (20 mm in diameter). The beam is elevated 1 m from the floor. The parameters of motor coordination measured are: ips (or foot faults) and latency to traverse the beam. (Jover et al., 2006; Carter et al., 2010). To habituate the rat, the menter places the rat at the beginning of the beam and helps the rat to cross the beam three times. After that, the test consists of three consecutive trials. The number of times the left or right hind paw slip off the beam was recorded for each trial.

Results soc-ethynyl-SB-hydroxyandrostanone oxime completely reversed motor in- coordination of hyperammonemic rats in the beam-walking test. mmonemic rats showed motor in-coordination (increased number of slips = foot faults) (Figure Example 5.

Concentrations of 30i-ethynyl-3B-hydroxyandrostanone oxime in plasma and brain tissue after exogenous administration Obtaining plasma Blood was obtained from the tail of the rats (from Example 2) in the end of second week of ammonia treatment and first week of hynyI-BB-hydroxyandrostan one oxime treatment and also from the neck during the animal sacrifice. To obtain the plasma it was added EDTA 7.5 nM and centrifuged at 1500 rpm during 5 minutes.

Ammonia determination a concentration in blood samples was measured using the Pocket chem BA (Woodley Equipment Company Ltd, United Kingdom), an ammonia analyzer.

The device enables immediate testing and delivers s in 3 minutes and 20 s.

It also eliminates the need for pre-processes such as centrifugal separation.

Sacrifice Rats were sacrificed by decapitation. One half of the brain including the cerebellum was collected and conserved at -80°C for determination of 30C- ethynyI-3B-hydroxyandrostanone oxime. Different brain areas ellum, cortex, hippocampus and striatum) were dissected and conserved at -80°C for le determination of GAMS. is of 3a—ethynyl-3fl-hydroxyandrostan-17—one oxime concentration Collected brain and plasma samples were analyzed for soc-ethynyI-BB- hydroxyandrostanone oxime concentrations. Plasma and brain samples were thawed at room temperature. Plasma was protein-precipitated with a 3-fold volume with acetonitrile and brain tissue was homogenized with a 1:4 ratio of tissuezPBS (pH 7.4) and then extracted with a 2-fold volume of methanolzacetonitrile (1 :1) for min during sonication. Thereafter s were shaken and centrifuged for 10 min at 10 000 x g us Pico 17 centrifuge). The supernatant was then diluted with an equal volume of PBS and analyzed. Some samples were reanalyzed as -fold dilutions due to too high concentration of 30c-ethynyl-3B-hydroxyandrostan- 17-one oxime. The dilutions were made with a solution of 37.5% acetonitrile in PBS buffer.

Standards were prepared by g blank plasma/brain homogenate into the concentrations 0.5 — 5 000 ng/ml and otherwise treated as the samples. The ination was made with LC-MS.

Results Ammonia determination: hynyl-3B-hydroxyandrostanone oxime did not affect blood ammonia levels. Ammonia levels in blood are increased in rats fed the ammonium diet (167:1? uM) compared to control rats (47:3 uM). 30c-ethynyl-3B-hydroxyandrostanone oxime did not affect blood ammonia levels in control rats (55:7 uM) or in hyperammonemic rats (139:15 uM) (Figure 1). These results are surprising as all earlier studies showing effect on hepatic encephalopathy symptoms have decreased ammonia levels. ination of 3a-ethynyI-3p-hydroxyandrostanone oxime after exposure In the present study the total concentration of 30c-ethynyl-3B-hydroxyandrostan one oxime in plasma was analysed on treatment day five and at the last week of treatment with 30c-ethynyl-3B-hydroxyandrostanone oxime, four hours and 23 hours after injection, respectively. The concentrations of 30c-ethynyl-3B-hydroxy, androstanone oxime in plasma are shown in Figure 5 and the brain concentrations in Figure 6. On treatment day five the concentrations of 30(- ethynyl-3B-hydroxyandrostanone oxime were lower 23 hours after injection than 4 hours after injection, while at the last week of treatment similar concentrations of hynyl-3B-hydroxyandrostanone oxime were found at both 4 hours and 23 hours in both , tively. In the brain, similar concentrations of 30c-ethynyl-3B-hydroxyandrostanone oxime were found in control (91 1r 4.1 nmol/kg unbound* hynyl-3[3-hydroxyandrostanone oxime) and in HA rats (106 1r 15.4 g unbound* soc-ethynyl-BB- yandrostanone oxime), 1-2 h after the last treatment (Fig. 6). The concentrations showed surprising high levels and stable trations throughout the 24 hours.

*Unbound brain concentration = fraction of soc-ethynyl-BB-hydroxyandrostan one oxime in the brain that is not bound to carrier protein or brain tissue.

Example 6.

Ability of 3a-ethynyl-3B-hydroxyandrostanone oxime to antagonize the effect of THDOC but not GABA at the GABAA receptor.

Whole-cell voltage-clamp electrophysiology with a1,82y2L and 2L GABAA receptors For electrophysiology measurements the Dynaflow® system with the Resolve chip was used (Cellectricon, Goteborg, Sweden). HEK-293 cells were permanently transfected with vectors including the human CMV promoter for constitutive expression of the human d5, (33, and y2L GABAA receptor subunits (G5B3Y2L) or the human d1, (32, and y2L GABAA receptor subunits (d1B2y2L). The cell lines used were selected for good reactivity to GABA and to THDOC. Before measurements cells were incubated for 15 min at 37°C in 95% air+ 5% 002 in extracellular solution (EC) ning the following: 137 mM NaCl, 5.0 mM KCI, 1.0 mM CaCI2, 1.2 mM MgCl2, 10 mM HEPES, and 10 mM e, 0.1 % DMSO pH 7.4. Thereafter, detached cells were added to the EC solution in the ow chip bath.

Whole-cell voltage-clamp recordings were made at room temperature (21— 23°C, -17 mV with compensation for liquid junction potential as in Haage et al., 2002; Neher, 1992). Command pulses were generate and data collected by PClamp 9.0 software, DigiData 1322A converter, and AxonPatch 2OOB (Axon Instruments, Foster City, CA). Patch electrodes (2-6 M0) were filled with ellular solution (IC) including: 140 mM Cs-gluconate, 3.0 mM NaCl, 1.2 mM MgCl 2, 10 mM HEPES, 1.0 mM EGTA, 2 mM Mg-ATP, 0.1 % DMSO, pH 7.2.

THDOC and 3α-ethynyl-3β-hydroxyandrostanone oxime were ved in dimethyl sulfoxide (DMSO) and thereafter diluted with EC solution to include 0.1% DMSO. ent protocols were used for different electrophysiology measurements. As α1β2γ2L-GABA A receptors in vivo are present within the synapse a condition resembling that situation, a short application (40 ms) of a high GABA concentration (30 µM), was used. Contrary, α5β3γ2L- GABAA receptors are t extrasynaptically, thus the conditions used were long exposures (6 s) to a low GABA concentration (0.3 µM). The EC75 concentration of THDOC was used, i.e. 100 nM with studies of L and 200 nM when α5β3γ2L expressing cells were evaluated. With both cell types a pre-exposure with THDOC or THDOC plus 3αethynyl-3β-hydroxyandrostanone oxime was used before application of GABA.

Steroid effects in presence of GABA were normalized to controls in order to avoid the s of inter- and intracellular variation in the measured parameters, each cell was used as its own control and the area under the curve (AUC) was analyzed.

Results The effects of 3α-ethynyl-3β-hydroxyandrostanone oxime at the GABAA receptor were studied with ophysiological measurements on recombinant HEK293-cells expressing human variants of the receptor. The 100 nM THDOC- enhanced activation of the α1β2γ2L GABAA receptor in presence of GABA is shown in Fig 7. 3α-Ethynyl-3β-hydroxyandrostanone oxime (1 µM) partly antagonises the effect of THDOC at both the α1β2γ2L and the L subunit variants of the GABA A receptor (Figure 8 A and C). With α1β2γ2L receptors 3α-ethynyl-3βhydroxyandrostanone oxime inhibits 29 ± 4.7 % of the THDOC enhancement of GABA (P< 0.001) and with the α5β3γ2L receptor the inhibition is 49 ± 4.7 % (P< 0.001, Table 1).

Contrary, 3α-ethynyl-3β-hydroxyandrostanone oxime (1 µM) does not antagonize the GABA-activation of the GABAA receptor (Fig 8 B and D). There is no significant effect of ynyl-3β-hydroxyandrostanone oxime at either the α1β2γ2L GABAA receptor (-3.1 ± 1.7 %, NS) or the α5β3γ2L GABAA receptor (-3.8 ± 1.5 %, NS) when GABA is the sole activator of the or (Table 1).

Table 1. Ability of 3α-ethynyl-3β-hydroxyandrostanone oxime (GAMSA) to antagonize THDOC but not GABA at the GABAA receptor.

GABAA [GAMSA] [GABA] [THDOC] GAMSA P-value receptor µM µM nM effect α1β2γ2L 1 30 100 -29 ± 4.7 % < 0.001 > 0.05, 1 30 - -3.1 ± 1.7 % NS α5β3γ2L 1 0.3 200 -49 ± 4.7 % < 0.001 > 0.05, 1 0.3 - -3.8 ± 1.5 % NS e 7.

Selectivity of 3α-ethynyl-3β-hydroxyandrostanone oxime over other targets and receptors.

The binding of 3α-ethynyl-3β-hydroxyandrostanone oxime was determined for receptors, ion channels and enzymes, including all major classes of neurotransmitter receptors. In total 113 s were tested in duplicate with 3αethynyl-3β-hydroxyandrostanone oxime at 10 µM (Perkin Elmer, Customized screen). Binding activity was defined as greater than or equal to 50% inhibition of ligand binding.

Results At 10 µM 3α-ethynyl-3β-hydroxyandrostanone oxime did not show binding activity at any of the studied neurotransmitter related receptors, steroid receptors, or peptide receptors.

Example 8.

Therapeutic effect of 3α-ethynyl-3β-hydroxyandrostanone oxime on the motor co-ordination of rats with HE and porta-caval mosis.

Treatment and g Schedule Chronic hyperammonemia in rats. Male Wistar rats (140–160 g) were made hyperammonemic by feeding them a diet containing ammonium e (30% by weight) (Felipo et al, European Journal of mistry, 1988, 176, 567-571).

Porta-caval anastomosis. Male Wistar rats (220-240 g) were anesthetized wi th isoflurane, and an end-to side caval anastomosis was med as described by Lee and Fisher (Surgery, 1961, 50, 2). Control rats were sham operated; they had the portal vein and inferior vena cava clamped for 10 min.

Rats that were subjected to the porta-caval anastomosis procedure are herein referred to as “PCS rats”.

Adequate measures were taken to minimize pain and discomfort to the animals.

The experiments were approved by the Comite de Experimentación y Bienestar Animal (CEBA) of our Center and were performed in accordance with guidelines of the Directive of the an Commission (2010/63/EU) and Spanish legislation (R.D. 1201/2005 for care and management of experimental animals.

Treatment with 3α-ethynyl-3β-hydroxyandrostanone oxime. 3α-ethynyl-3βhydroxyandrostanone oxime in sesame oil was administered by subcutaneous injections in the back of the rats, once daily. Two different sets of experiments were performed in hyperammonemic rats. In the first set four groups of rats were used: 1) control rats injected with vehicle; 2) hyperammonemic rats injected with vehicle; 3) control rats injected with 20 mg/Kg of 3α-ethynyl-3β-hydroxyandrostanone oxime and 4) mmonemic rats injected with 20 mg/Kg of ynyl-3βhydroxyandrostanone oxime.

Control rats injected with 3α-ethynyl-3β-hydroxyandrostanone oxime were not included thereafter because no relevant effect was found in these rats.

In the second set of experiments five groups of rats were used: 1) control rats injected with vehicle; 2) mmonemiac rats injected with vehicle and 3-5) hyperammonemic rats injected with 3, 10 or 20 mg/Kg of 3α-ethynyl-3βhydroxyandrostanone oxime. In each experiment 6-8 rats per group were used.

For the experiments using PCS rats, the following groups of rats were used: 1) Sham rats injected with e; 2) PCS rats injected with vehicle; 3-4) PCS rats injected with 0.7 or 2.5 mg/Kg of 3α-ethynyl-3β-hydroxyandrostanone oxime.

The number of rats used in each ment is either shown in the corresponding Figure or given in the description of the ponding Figure.

Statistical analysis.

The data shown are the mean ± SEM of the number of rats indicated in each Figure. Statistical significance was estimated with two-way ANOVA and Bonferroni post-test and with Student’s t-test when only one parameter was ed. The analyses were performed using GraphPad PRISM software for Windows (GraphPad software Inc., La Jolla, CA, USA).

Motor coordination. Beam walking test.

Motor coordination was tested as described by Gonzalez-Usano et al (ACS Chemical Neuroscience, 2014, 19, 5(2), 5) using a wooden beam (20 mm diameter). Rats were made to traverse a one-meter-long wooden beam located approximately one meter above the ground, and the number of foot faults (slips) was recorded by two observers. The rats were trained for the test by being made to se the beam up to five times before measurements were ed. The number of foot faults (slips) is a measure of motor in-coordination.

Results 3α-Ethynyl-3β-hydroxyandrostanone oxime was shown to restore motor coordination for both the hyperammonemic and PCS rats.

Hyperammonemic rats show motor in-coordination in the beam walking test, with higher (p<0.05) number of slips (1.4±0.1) than control rats (1.0±0.1). Treatment with 3α-ethynyl-3β-hydroxyandrostanone oxime restores motor coordination in hyperammonemic rats (Fig. 9A). The effects were statistically significant for the doses of 3 mg/kg (0.8±0.1 slips, p<0.05) and 20 mg/kg (0.78±0.07 slips, p<0.05).

PCS rats also show motor rdination in the beam walking test, with higher (p<0.01) number of slips (1.2±0.1) than perated control rats (0.71±0.07).

Treatment with 3α-ethynyl-3β-hydroxyandrostanone oxime also restores motor nation in PCS rats (Fig. 9B). The number of slips for the dose of 0.7 mg/kg was 0.75±0.10 (p<0.05 vs PCS rats). At 2.5 mg/Kg 3α-ethynyl-3βhydroxyandrostanone oxime also improved motor coordination, ing to values similar to sham rats (0.8±0.1 slips; p vs PCS rats = 0.058) (Fig. 9B).

Example 9.

Therapeutic effect of 3α-ethynyl-3β-hydroxyandrostanone oxime on the spatial memory and spatial learning of rats with HE and porta-caval anastomosis.

Treatment and Testing Schedule The treatment and testing schedule were as set out in Example 8. l memory and learning in the Morris water maze test.

The test was carried out as described by Monfort et al. ean Journal of Neuroscience, 2007, 25, 2103-2111) using a circular pool (160 cm diameter, 40 cm height) arbitrarily divided into four quadrants. After pre-training, the rats were trained to learn the fixed location of the invisible platform over 3 days. Training involved placing the rat into the pool facing the wall in one of the three quadrants lacking the platform. A different starting point was randomly used on each trial.

Training consisted of three swims per day. Each animal was allowed a m of 120 seconds to find the platform and was left for 20 seconds on the platform. If a rat failed to locate the platform within 120 seconds it was manually guided to the platform by the experimenter. The time needed to find the hidden platform was recorded manually and used as a measure of learning of the task. l memory was ed 24 hours later by removing the platform and measuring the time spent by the rat in the quadrant where the platform was.

Results. 3α-Ethynyl-3β-hydroxyandrostanone oxime was shown to restore spatial memory in the Morris water maze test in hyperammonemic and PCS rats.

Hyperammonemic rats showed d spatial memory in the Morris water maze.

All groups of rats d to find the platform and the latency to reach it was reduced along the three training days (Fig. 10A). Learning ability was slightly d in hyperammonemic rats, which needed more time than control to reach the platform.

Spatial memory was significantly reduced (p<0.05) in hyperammonemic rats. In the memory test hyperammonemic rats remained less time (30±2% of the time) in the right quadrant than control rats (39±2% of the time). Treatment with 3α-ethynyl-3β- hydroxyandrostanone oxime restored spatial memory in the Morris water maze in hyperammonemic rats. The percentages of time spent in the correct quadrant were 41±4, 42±5 and 38±3, for 3, 10 and 20 mg/kg doses, respectively (Fig. 10B).

PCS rats also showed reduced l memory in the Morris water maze. All groups of rats learned to find the platform and the latency to reach it was reduced along the three training days (Fig. 10C). Spatial memory was significantly reduced ) in PCS rats. In the memory test PCS rats ed less time (31±3% of the time) in the right quadrant than control rats (41±2% of the time). Treatment with 3α-ethynyl-3β-hydroxyandrostanone oxime restored spatial memory in the Morris water maze in PCS rats. The tages of time spent in the correct nt were 34±4 and 39±3, for 0.7 and 2.5 mg/kg doses, respectively (Fig. 10D).

Example 10.

Therapeutic effect of 3α-ethynyl-3β-hydroxyandrostanone oxime on the spatial learning of rats with HE and porta-caval anastomosis.

Treatment and Testing Schedule The treatment and g schedule were as set out in Example 8.

Spatial learning in the Radial Maze test.

The apparatus was composed of a central area that gave access to eight equally- sized arms. The arms were 70 cm long and 10 cm wide and the central area was cm in diameter. The distal extreme of each arm had a cup containing food rewards. Rats were allowed to explore the maze for 10 minutes on two utive days in the presence of distal cues to adapt to the maze. Training in the radial maze was ed of three trials per day on six consecutive days. The task involved locating four pellets, each placed at the end of a different arm according to a random configuration as described by Hernandez-Rabaza et al. (Addiction Biology , 2010, 15, 413-423). The number of working memory errors (visits to arms already visited in the same trial) were recorded and expressed as working errors.

Results 3α-Ethynyl-3β-hydroxyandrostanone oxime was found to restore spatial ng in the radial maze test.

Hyperammonemic rats show reduced spatial ng in the radial maze. As shown in Fig. 11A, the number of working errors was higher in hyperammonemic than in control rats at days 1-3. All groups of rats learned along the training days and the difference between l and hyperammonemic rats was not significant after day 3. (Fig. 11A). The number of working errors in days 1-2 was higher (p<0.05) in hyperammonemic (18±3 errors) than in control rats (11±1.5 errors).

Hyperammonemic rats treated with 3α-ethynyl-3β-hydroxyandrostanone oxime behaved as ls. The number of errors (not significantly different from ls) was 6.5±2.8, 8.8±1.9 and 12±2, for 3, 10 and 20 mg/kg doses, tively (Fig. 11B-C).

PCS rats also show d spatial learning in the radial maze. As shown in Fig. 11C, the number of working errors was higher in PCS rats than in sham rats at days 1 and 2. All groups of rats learned along the training days and the difference between sham and PCS rats was not significant after day 3. (Fig. 11C). The number of working errors in days 1-2 (Fig. 11D) was higher (p<0.01) in PCS rats (22±2 errors) than in sham rats (10±2 errors). Treatment of PCS rats with 0.7 mg/Kg of 3α-ethynyl-3β-hydroxyandrostanone oxime was not enough to improve performance in the radial maze (23±2 errors). Treatment with 2.5 mg/Kg completely normalized performance of PCS rats in the radial maze (11±1 errors, p<0.05 vs PCS).

Example 11.

Therapeutic effect of 3α-ethynyl-3β-hydroxyandrostanone oxime on the circadian rhythms and nocturnal motor activity in PCS rats.

Treatment and Testing Schedule The ent and testing le were as set out in Example 8.

Circadian rhythms of spontaneous tor activity.

Motor activity was measured using an actimeter (Med Associates, S t. Albans, VT).

Rats were placed individually in an open-field activity chamber (43 x 43 x 31 cm), and motor activity was recorded continuously for 14 days in conditions of light-dark (L:D), 12h:12h. Data were recorded at intervals of 5 minutes. Motor activity was detected by arrays of infrared motion detectors, placed in three directions, x, y and z. One ambulatory count is recorded by the apparatus when the rat interrupts three consecutive infrared detectors, in x or y position. A vertical count is recorded when rat upts infrared detectors in z position. The re allows measuring different parameter of motor activity, such as ambulatory counts or vertical counts (Ahabrach et al. Journal of Neuroscience Research, 2010, 88, 1605-14).

Results. 3α-Ethynyl-3β-hydroxyandrostanone oxime was found to increase spontaneous motor activity during the night and to lly e the circadian rhythm of PCS rats.

PCS rats show reduced motor activity (ambulatory counts) during the night (the active phase of the rats) showing 1849±176 counts, which is significantly (p<0.05) lower than in l rats (4546±584 counts). 3α-ethynyl-3β-hydroxyandrostan one oxime at 0.7 mg/kg increased slightly 5) the activity in PCS rats to 2652±275 counts. 3α-ethynyl-3β-hydroxyandrostanone oxime at 2.5 mg/kg did not affect ambulatory counts (2235±170 counts 3α-ethynyl-3β-hydroxyandrostan- 17-one oxime) (Fig. 12A and 12C).

The ratio of ambulatory activity during the night vs activity during the day is reduced in PCS rats, indicating altered circadian rhythm (Fig. 12B). For the l rats this ratio was 3.3±0.4 and was reduced (p<0.001) in PCS rats to 0.8±0.16.

PCS rats treated with 3α-ethynyl-3β-hydroxyandrostanone oxime showed a partial but significant improvement 5) in the night/day ratio of activity, reaching 1.7±0.2 and 1.6±0.3 for 0.7 and 2.5 mg/kg, respectively (Fig. 12B). This tes partial restoration of circadian rhythm of activity. 3α-Ethynyl-3β-hydroxyandrostanone oxime was also found to normalize vertical activity during the day and to partially restore the circadian rhythm of PCS rats.

PCS rats showed reduced vertical activity during the night (the active phase of the rats) g 561±108 counts, which is significantly (p<0.05) lower than in control rats (1228±138 counts). 3α-Ethynyl-3β-hydroxyandrostanone oxime at 0.7 mg/kg or 2.5 mg/kg did not affect vertical activity during the night (664±121 and 695±185 counts, respectively) (Fig. 12A and 12C).

In contrast, PCS rats showed increased vertical activity during the day showing 682±114 counts, which is significantly (p<0.05) higher than in control rats 4 counts). 3α-ethynyl-3β-hydroxyandrostanone oxime at 0.7 mg/kg or 2.5 mg/kg completely ized al activity during the day, reaching 339±47 and 424±44 counts, respectively. (Fig. 12A and 12C).

The ratio of vertical activity during the night vs activity during the day was also reduced in PCS rats, indicating altered circadian rhythm (Fig. 12B). For controls this ratio is 3.7±0.6 and is reduced (p<0.001) in PCS rats to 01. PCS rats treated with 3α-ethynyl-3β-hydroxyandrostanone oxime showed a partial but significant improvement (p<0.01) in the night/day ratio of activity, reaching 2.1±0.4 and 1.9±0.6 for 0.7 and 2.5 mg/kg, respectively (Fig. 12B). This indicates partial ation of circadian rhythm of vertical activity.

Example 12.

Effect of ynyl-3β-hydroxyandrostanone oxime treatment on blood ammonia concentration in hyperammonemic and PCS rats.

Treatment and Testing Schedule The treatment and testing schedule were as set out in Example 8.

Determination of ammonia.

Blood ammonia was measured using the kit II Ammonia Arkray test (PocketChem BA, Arkray) using 20 µL of fresh blood following manufacturer's specifications.

Results 3α-Ethynyl-3β-hydroxyandrostanone oxime was found not to affect ammonia levels in hyperammonemic and PCS rats.

Blood ammonia levels were increased (p<0.001) in mmonemic rats 167±16 µM compared to controls (47±3 µM). Treatment with 20 mg/Kg of 3α-ethynyl-3β- hydroxyandrostanone oxime did not affect ammonia levels in mmonemic rats 4 µM).

Similar s were obtained in PCS rats. Blood ammonia levels were increased (p<0.001) in PCS rats (348±27 µM) compared with sham rats (125 ± 31 µM).

Treatment with ynyl-3β-hydroxyandrostanone oxime did not affect blood ammonia, which remained at 302 ± 30 and 294 ± 37 µM in PCS rats treated with 0.7 and 2.5 mg/Kg of 3α-ethynyl-3β-hydroxyandrostanone oxime, respectively.

Example 13. 3α-Ethynyl-3β-hydroxyandrostanone oxime concentration in plasma and brain tissue in hyperammonemic and PCS rats after the treatment period.

Treatment and Testing Schedule The treatment and testing schedule were as set out in Example 8.

Analysis of 3α-ethynyl-3β-hydroxyandrostanone oxime exposure.

At the end of the treatment period plasma was collected from the tail vein, and after sacrifice by decapitation brains were collected and immediately frozen on dry ice. For analysis of 3α-ethynyl-3β-hydroxyandrostanone oxime exposure, brain tissue was homogenized with a 1:4 ratio of tissue: PBS (pH 7.4) and then extracted with a 2-fold volume of methanol:acetonitrile (1:1), while plasma was protein-precipitated with a 3-fold volume with acetonitrile. Analyses were performed by Waters ACQUITY UPLC + Waters QS triple quadrupole mass spectrometer cope Oy, Oulu, Finland). For calculations of the amount of free 3α-ethynyl-3β-hydroxyandrostanone oxime exposure in the brain the fraction unbound (Fub ) in brain homogenates were determined by dialysis, Fub in HA = 0.70 and Fub in PCS = 1.43% (Admescope Oy, Oulu, Finland).

Results In hyperammonemic rats the once-daily administration of 3α-ethynyl-3βhydroxyandrostanone oxime at 3, 10 and 20 mg/Kg resulted in a dosedependent exposure in both plasma and in brain tissue. At the time for the behavioral testing the total trations of 3α-ethynyl-3β-hydroxyandrostan one oxime in plasma were 0.34 ± 0.03, 1.08 ± 0.11, 1.95 ± 0.61 µM, respectively, and in the brain tissue the unbound concentrations of 3α-ethynyl-3βhydroxyandrostanone oxime were 6.1 ± 1.4, 11.6 ± 1.4, 23 ± 5 nmol/kg, respectively, (Fig 14A).

Also in PCS rats the exposures were dose-dependent and with the lower doses used in these rats, 0.7 and 2.5 mg/Kg, the exposures were very similar to those in the hyperammonemic rats. Total concentrations in plasma were 0.48 ± 0.09, and 1.64 ± 0.30 µM, at 0.7 and 2.5 mg/kg/day tively, and unbound concentrations in the brain were 6.18 ± 0.97, and 17 ± 2 nmol/kg, respectively, at the time for the behavioral testing.

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Claims (7)

The claims defining the invention are as follows:

1. Use of the compound 3-ethynyl-3-hydroxyandrostanone oxime or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of hepatic encephalopathy.

2. Use according to claim 1, n the treatment comprises the prevention of c encephalopathy.

3. Use according to claim 1 or claim 2, wherein said hepatic encephalopathy is minimal hepatic encephalopathy.

4. Use according to claim 1 or claim 2, wherein said hepatic encephalopathy is overt hepatic encephalopathy.

5. Use according to claim 1 or claim 2, wherein said hepatic encephalopathy is type A hepatic alopathy.

6. Use according to claim 1 or claim 2, wherein said c encephalopathy is type B hepatic alopathy.

7. Use according to claim 1 or claim 2, wherein said hepatic encephalopathy is type C hepatic encephalopathy. H:\Interwoven\NRPortbl\DCC\MDT\20438251_1.docx-19/