INTRODUCTION

The present invention relates to novel compounds having hepatoprotective and hepatotherapeutic activity, to pharmaceutical compositions containing them, and to methods of using them.

BACKGROUND OF THE INVENTION

The incidence of a number of conditions associated with elevated levels of fat in the liver is increasing. Non-alcoholic fatty liver disease (NAFLD) is a term used to embrace a range of conditions characterised by the build-up of fat in the liver of people who do not drink alcohol to excess. NAFLD includes different clinical entities: steatosis is the stage where fat is first detected in the liver. Non-alcoholic steatohepatitis (NASH), referring to a condition following on from steatosis, is associated with excess fat in the liver, together with inflammation and associated damage. The prevalence of NAFLD in unselected populations from developed countries is high and estimates vary between 2- to 30%. Current data suggest that approximately 2 to 3% of the same population have NASH. The majority of individuals with simple hepatic steatosis do not progress to NASH. However, subjects who do are at far higher risk of progression to cirrhosis with subsequent hepatocellular carcinoma and liver damage and death. A recent study evaluated the long- term clinical outcome and histological course of patients with NAFLD and elevated liver enzymes, and demonstrated that the overall survival of these patients was significantly lower than that of the reference population. Currently, no specific treatment for steatosis or NASH exists. There is a need for effective treatment of steatosis, NASH, and other conditions associated with fatty liver, including Metabolic Syndrome and certain other conditions associated with obesity.

Ursodeoxycholic acid (UDCA) is a hydrophilic dihydroxylated bile acid: 3α,7β- dihydroxy-5P-cholanoic acid. It was first identified in the bile of the Chinese black bear, and is also present in very small amounts as a secondary bile acid in humans (1-3% of the total bile acid pool) where it is formed by 7β epimerization of the primary bile acid chenodeoxycolic acid in the gut by intestinal bacteria. UDCA prolongs survival in primary biliary cirrhosis and it improves biochemical parameters of cholestasis in various cholestatic disorders. Surprisingly, we have now found that a specific derivative of UDCA has utility for the prevention and treatment of conditions associated with the pathological accumulation of fat in the liver and as a research tool useful in the research and development of other therapeutic compounds in relation to the aforementioned diseases and disorders.

SUMMARY OF THE INVENTION

The present invention provides 1 , 1 -dioxo- 1 -ursodeoxycholamino-tetrahydrothiopyran-4- carboxylic acid, also known as 2-(4-((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)- hexadecahydro-3 ,7-dihydroxy- 10, 13-dimethyl- 1 H-cyclopenta[a]phenanthren- 17- yl)pentanamido)-tetrahydro-2H-l,l-dioxo-thiopyran-4-yl) acetic acid, or

ursodeoxycholylthiopyranone alpha amide acid, (hereinafter referred to as Compound I), which has the formula:

or a salt, ester, or amide thereof. DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention are effective in the prevention and treatment of conditions associated with fatty liver, for example the reduction of steatosis. They may also be used in the prevention or treatment of excess weight, e.g. obesity. Further, they may be used as research tools - for example, they may be used as comparators during drug screening. The compounds are novel. The invention also provides a pharmaceutical composition comprising a compound according to the invention together with a pharmaceutically acceptable carrier; a compound according to the invention for use in therapy, specifically, for use in the treatment or prevention of conditions associated with fatty liver, especially non-alcoholic steatohepatitis or steatosis, but also including similar conditions associated with the use of alcohol (such as alcoholic steatohepatitis, ASH), and other indications such as primary biliary cirrhosis (PBC), cholestasis, prevention or treatment of drug induced liver injury (DILI), and the metabolic syndrome (which is generally defined by the presence of 2 or more of the following: abdominal obesity (waist circumference > 102 cm for men, >88 cm for women); triglycerides > 150 mg/dL; high density lipoprotein < 40 mg/dL for men, 50 mg/dL for women; blood pressure > 130/85 mmHg; fasting blood glucose > 110 mg/dL); the use of a compound of the invention for the manufacture of a medicament for the treatment, prevention or amelioration of conditions as mentioned above; and a method of treating, preventing or ameliorating a condition as mentioned above in a subject, which comprises administering a

therapeutically effective amount of a compound or a composition according to the invention to said subject. The subject to be treated according to the present invention is typically a mammal. The mammal is generally a human but may for example be a commercially reared animal or a companion animal. Further, the invention provides the use of a compound according to the invention as a research tool. In some embodiments, the compound is useful as a comparator in screening assays to assist in identifying and/or profiling a compound with similar or superior activity to the compound of the invention in the test conditions applied. For example, compounds of the invention may be used to identify and/or profile compounds having therapeutic activity (e.g. superior therapeutic activity) in the same or similar disease or disorders as compounds of the invention, e.g. NAFLD and/or NASH and/or other hepatic diseases or disorders. In other embodiments, compounds of the invention may be used as a positive control in cell based, in vitro and/or in vivo test assays, particularly in models of NAFLD and/or NASH and/or other hepatic diseases or disorders.

Compound I may be used as such, or in the form of a salt, ester, or amide thereof. The compound may also be used in the form of a solvate, including a solvate of such a salt, ester or amide. Accordingly the invention also provides 1,1-dioxo-l- ursodeoxycholamino-tetrahydrothiopyran-4-carboxylic acid, or a salt, ester, amide or solvate thereof, including a solvate of such a salt, ester or amide. Preferably a salt, ester, amide or solvate is one which is pharmaceutically acceptable.

Suitable salts of Compound I are, for example, metal salts, for example alkali metal or alkaline earth metal salts, for example sodium, potassium, calcium and magnesium salts; or salts with ammonia, primary, secondary or tertiary amines, or amino acids, for example mono-, di- or tri-alkylamines, hydroxyalkylamines, and nitrogen-containing heterocyclic compounds, for example isopropylamine, trimethylamine, diethylamine, tri(i- propyl)amine, tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, lysine, histidine, arginine, choline, caffeine, glucamine, procaine, hydrabamine, betaine, ethylenediamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, morpholine, n-alkyl piperidines, etc. The sodium salt of Compound I forms one preferred embodiment of the invention. When the compound of the invention is an ester, it may be an ester of the -C0 ₂ H group with any suitable alcohol, for example an alkanol. Alternatively, it may be an ester of any of the -OH groups present in the molecule with any suitable acid, for example any carboxylic or sulfonic acid. When the compound of the invention is an amide, this may be an amide derived from any suitable amine, for example a mono-, di- or tri-alkylamine, or any of the amines mentioned above.

Except where otherwise stated, throughout this specification and claims, any alkyl moiety present in a compound of the invention or in an intermediate used for the preparation of a compound of the invention, may be straight chain or branched, and may for example contain from 1 to 6, especially from 1 to 4, carbon atoms such as methyl.

Thus, a compound of the invention may for example have the formula:

in which R is H or one equivalent of a pharmaceutically acceptable metal, for example an alkali metal, for example sodium, or an alkyl group, for example a C _1-6 alkyl group, for example a methyl group.

Many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as solvates. For example, a complex with water is known as a hydrate. Such solvates form part of the invention.

The present invention also provides a process for the preparation of a compound of the invention, which comprises reacting a compound of the general formula

or a protected derivative thereof with a compound of the general formula or a protected derivative thereof, in which Y and Z represent groups capable of reacting together to form an amide link -CO.NH-, under conditions such that an amide link is formed.

Any groups Y and Z capable of forming an amide link may be present. For example, Y may represent a -C0 ₂ H group (in which case the starting material (II) is ursodeoxycholic acid (UDCA)), or an active derivative thereof, and Z may represent an -NH ₂ group. Reaction conditions conventional for the formation of amide linkages may be used; for example, the reaction may be carried out in the presence of an amide coupling agent, of which many are known in the art, for example EEDQ (2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline), DMTMM ((4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmoφholinium chloride), NMM (N- methylmorpholine), DCC (Ν,Ν'-dicyclohexylcarbodiimide), HBTU (O-benzotriazole- Ν,Ν,Ν',Ν'-tetramethyl-uronium-hexafluoro-phosphate) and HOOBt (1-hydroxy- benzotriazole). Any suitable solvent, for example an organic solvent such as a halogenated hydrocarbon for example chloroform, may be used. Reaction temperatures can vary over a wide range, but the use of elevated temperatures may be preferred.

During synthesis of a compound of the invention, protecting groups may be used and removed as desired. For example, the -C0 ₂ H group of the compound of formula HI may be present in the form of an ester, thus:

(Ilia)

in which R ¹ represents an alkyl group, especially a group, for example a methyl group, leading to an ester of Compound I. Compounds of the formula III are known, or can be prepared by methods analogous to known methods.

A resulting compound of the invention may be converted into any other compound of the invention by methods analogous to known methods, for example an ester wherein R = alkyl may be converted to the corresponding acid by hydrolysis (e.g. using an aqueous hydroxide such as NaOH) or an acid maybe converted to a corresponding metal salt (e.g. using an aqueous metal hydroxide, such as NaOH to produce the sodium salt). The amount of the compound of the invention which is required to achieve a therapeutic effect will, of course, depend upon whether the effect is prophylactic or curative, and will vary with the route of administration, the subject under treatment, and the form of disease being treated. It is generally preferable to use the lowest dose that achieves the desired effect. The compound of the invention may generally be administered at a dose of, for example, from 0.1 to 500 mg/kg per day, typically from 50 to 150 mg/kg day, for example about 100 mg/kg day. Unit dose forms may conveniently contain an amount of compound of the invention which is effective at such dosage or as a multiple of the same, for example units containing 5 mg to 500 mg, usually around 10 mg to 200 mg. The compound of the present invention may be administered one or more times per day, for example, two or three times per day, or even more often, for example, four or five times per day.

While it is possible for the active ingredient to be administered alone, it is preferable for it to be present in a pharmaceutical formulation or composition. Suitable pharmaceutical formulations according to the invention include those suitable for oral (including sub-lingual), parenteral (including subcutaneous, intradermal, intramuscular, intravenous, and

intraarticular), nasal, inhalation (including fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators), topical (including dermal, buccal, and sublingual) and rectal administration. The most suitable route may depend upon, for example, the nature and stage of the condition and disorder of the recipient. Forms suitable for oral administration include for example tablets, capsules, pills, granulates, dragees, wafers, solutions or suspensions.

Forms suitable for nasal administration include for example drops, sprays and aerosols.

Forms suitable for topical administration to the skin include, for example, gels, creams, ointments, pastes, foams or adhesive patches.

Forms suitable for rectal administration include suppositories, rectal capsules and enema solutions.

Forms suitable for transdermal administration generally comprise an adjuvant that enhances the transdermal delivery of the compound of the invention. Suitable adjuvants are known in the art.

A pharmaceutical composition of the present invention may be in unit dosage form. Suitable oral unit dosage forms include those mentioned above. For administration by injection or infusion unit dosage forms include, for example, vials and ampoules. Unit dosage forms for topical administration to the skin include blister packs or sachets, each blister or sachet containing a unit dose of, for example, a gel, cream or ointment, for example, as described above. A metered dosing device may be provided, for example, a pump device, for dosing a predetermined volume of a topical composition, for example, a cream, ointment or gel. A preparation may provide delayed or sustained release, for example a depot preparation or an adhesive patch.

Preferred formulations are those suitable for oral administration, for example in the form of tablets, capsules, pills or the like, or in the form of solutions suitable for injection such as in water for injections BP or aqueous sodium chloride.

Pharmaceutical compositions as described above may also comprise one or more further active ingredients in addition to the compound of the invention, for example, a further active ingredient with efficacy in the treatment or prevention of conditions associated with fatty liver, or with efficacy in the treatment or prevention of excess weight.

The following Examples illustrate the invention. The reaction scheme used for Examples 1 to 3 is shown in Figure 1 of the attached drawings.

Example 1: Synthesis of the methyl ester of 1,1-dioxo-l-ursodeoxycholamino- tetrahydrorhiopyran-4-carboxylic acid (compound 4 of Figure 1, the methyl ester of Compound I)

(a) Preparation of Intermediate 2 of Figure 1.

To a suspension of amino acid 1 (264.3 g) in methanol (2.6 L) at 0°C was added thionyl chloride (350 mL). The mixture was heated to reflux overnight and then cooled to room temperature. A small amount of precipitate formed that was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was washed with Et ₂ 0 and filtered to afford intermediate 2 as white solid (258.0 g, 92%).

(b) To a suspension of intermediate 2 (258.0 g) in DMF (2.4 L) at room temperature was added Et ₃ N (738 mL). The resulting mixture was stirred at room temperature for 30 min and ursodeoxycholic acid (346.2 g) was added followed by 2-ethoxy-l-ethoxycarbonyl-l,2- dihydroquinoline (1,2-EEDQ, 261.9 g). This mixture was heated to 95°C for 8h and cooled to room temperature. Insolubles were removed by filtration and the filtrate was concentrated under reduced pressure. The residue was acidified with 1.5 N HC1, and the resulting solid was filtered, washed with water, Et ₂ 0, and air-dried overnight to give the desired product, compound 4 of Figure 1. The material was used as such in the next step (HPLC purity 92%, Evaporative Light Scattering Detector).

Example 2: Synthesis of l,l-dioxo-l-ursodeoxycholamino-tetrahydrothiopyran-4- carboxylic acid (compound 5 of Figure 1, Compound I).

To a suspension of compound 4 (as prepared in Example 1) in MeOH (1.5 L) was added 2N NaOH (2.0 L) and the resulting mixture was heated to reflux for 3h. The mixture was cooled to room temperature and most of the volatiles were removed under reduced pressure. The residue was diluted with water, acidified with 6N HC1, and the resulting solid was removed by filtration, washed with IN HC1, water, Et ₂ 0, and dried in a vacuum over 40°C to afford the desired product, compound 5 of Figure 1 : >98% HPLC purity, Evaporative Light Scattering Detector.

Example 3: Synthesis of the sodium salt of 1,1-dioxo-l-ursodeoxycholamino- tetrahydrothiopyran-4-carboxylic acid

To a solution of compound 5 (427.0 g) (prepared as in Example 2) in MeOH (2 L) was added a solution of NaOH (30.1 g) in water (70 mL). The mixture was stirred for lh and concentrated under reduced pressure. The residue was taken into MeOH (2 L) and concentrated to dryness. This process was repeated. The resulting semi-solid was triturated with Et ₂ 0 (4 L) and the solid was filtered and dried to afford the desired product as a white solid (445.0 g), quantitative yield).

Example 4 - Prophylactic effect of the compound of Example 3 in an ethanol-induced model of steatohepatitis The purpose of this study was to investigate the potential prophylactic effects of the compound of Example 3 (100 mg/kg/day) by a four week continuous intravenous infusion in rats fed a liquid diet known to induce liver damage by a model of steatohepatitis as described by Lieber et al (Liquid diet technique of ethanol administration: 1989 update; Lieber CS, DeCarli LM; Alcohol & Alcoholism 1989; 24 (3): 197-211). The required amount of the test compound, corrected for strength (purity) (83.7% w/w), was dissolved in the vehicle (0.9% sodium chloride injection) and sterilised by filtration using a 0.22 μιη filter. The formulation was prepared fresh daily (concentration of 2.78 mg/mL of the test compound).

During an acclimatisation period, Wistar Hannover rats were fed ad libitum a standard liquid diet formulated according to the composition described in the reference article "Model of nonalcoholic steatohepatitis"; Lieber et al; Am. J. Clin. Nutr., 2004; 79: 502-509. On the day of surgery (1 1-15 days after animal arrival), the animals were anaesthetised and implanted with a urethane cannula PhysioCath™ into the vena cava via the left femoral vein. The cannula was tunnelled subcutaneously up to the dorsal site of the tail and connected to a syringe pump by means of a swivel joint. Following surgery the animals received an analgesic treatment and an antibiotic for a total of four days. At the end of the post-surgery therapy the animals were maintained by continuous intravenous infusion using physiological saline at a rate of 0.5 mlVhour.

Administration of the compound of Example 3 started 8-13 days after the last antibiotic treatment. All study animals were carefully examined during the pre-test period to verify possible reactions to the surgery. The animals were then placed on continuous infusion.

On the day of allocation (eight days prior to the start of treatment) all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The animals were allocated to groups to give approximately equal initial group mean body weights.

Each group comprised six male rats. The treatments are summarised below: Group Dose level

Diet

Number (mg/kg/hour)

1 Standard Vehicle

2 Fat + Ethanol Vehicle

3 Fat + Ethanol Test compound (4.17*)

^♦ calculated as pure compound; equivalent to 5.0mg/kg/hr test compound before correction for strength (83.7%w.w). From the day of allocation up to the end of the study, relevant diets were provided ad libitum. Group 1 animals were fed with a standard liquid diet. Groups 2 and 3 animals were fed with a high-fat liquid diet supplemented with 5 g/dL of ethanol.

The solution of the compound of Example 3 was administered by continuous intravenous infusion at an approximate rate of 1.5 mL/kg/hour over an infusion period of 24 hours

(Control Group animals received the vehicle, isotonic saline, at the same volume and flow rate). The dose volume was calculated on the basis of the most recently recorded group mean body weight. All animals were dosed continuously for a minimum of 28 consecutive days up until necropsy.

The results obtained were as follows:

Mortality

There was no drug related mortality subsequent to treatment.

Clinical signs

There were no drug related clinical signs subsequent to treatment.

Body weight

A slight transient decrease in body weight was observed in animals of Groups 2 and 3 when compared to mean control values (Group 1) during the first week of treatment. This effect was not considered to be drug related since at initiation of treatment control animals were slightly heavier and the body weight gain over 4 weeks was comparable between all the groups. Terminal body weight and organ weights

At the end of the treatment period, no difference in terminal body weight was observed in treated rats, when compared with controls. When compared to control values an increase in absolute and relative liver weight was noted in all animals in which liver damage was induced with no apparent difference between the groups.

Macroscopic observations

Detailed macroscopic observations were reported for each animal in the study.

Pale coloration and/or swelling of the liver were noted in all Group 2 animals and in 2/6

Group 3 animals with an apparent increase in incidence of enlarged liver amongst treated animals when compared with the control animals (Group 1). Some control and treated animals showed abrasions or scabs or swelling on the implant site, pale firm masses involving the caudal vena cava and swollen and/or enlarged spleen. In addition, enlarged liver lobes with pale/creamy material were noted in one Group 3 animal. These liver changes could have been a consequence of the mechanical trauma caused by the misplacement of the catheter in the portal vein. However, these changes were considered as an expression of inflammatory reactions, due to the route of administration.

Microscopic observations

Microscopic evaluation was performed on H&E stained liver sections from animals that were sacrificed at the end of treatment the treatment period.

In order to characterise the presence of lipid or glycogen accumulation in the hepatocytes, histochemical stains, such as "Oil red O" specific for lipid and "PAS" (Periodic Acid Schiff) specific for glycogen, were performed on frozen liver sections and paraffin liver sections, respectively. The results of the histochemical stains, "Oil red O" and "PAS", were scored by a semi-quantitative scale:

negative, weakly positive, positive, strongly positive.

Representative photomicrographs were taken for normal and treatment-related lesions in the liver stained with H&E (Haematoxylin and eosin), "Oil red O" or "PAS" in each group. Hepatocytic steatosis, described as macrovesicular and/or microvesicular forms, is morphologically defined by the presence of a single large fat vacuole that displaces the nucleus to the periphery (macro vesicular) or by numerous small lipid vacuoles, often giving a foamy appearance with nuclei located in the centre of hepatocytes (microvesicular).

Slight to moderate microvesicular steatosis, mainly localised in the periportal/midzonal area, histochemically confirmed with positive staining to Oil red O stain, was noted in rats dosed with the compound of Example 3 (Group 3).

Negative or weakly positive PAS staining, indicative of glycogen depletion, was noted in the fat diet plus ethanol fed rats, either receiving the vehicle alone or the test compound, in comparison to the controls receiving only the standard diet. However, decreased severity and distribution in the hepatic lobule of steatosis were noted in treated animals (Group 3), when compared with Group 2 animals, fed with the fat diet plus ethanol and receiving the vehicle.

The experimental results confirmed the induction of a marked steatohepatitis in all control animals fed with fat diet plus ethanol. An evident reduction of hepatic steatosis, defined as slight microvesicular steatosis with a decreased severity and distribution in the hepatic lobule, was noted in animals receiving the test compound, when compared with controls fed with the fat diet plus ethanol. The study results indicate that four weeks of continuous intravenous infusion of the compound of Example 3 (100 mg/kg/day) showed a prophylactic activity in rats in which hepatic steatosis had been induced.

Example 5 - Therapeutic effect of the compound of Example 3 in an ethanol-induced model of steatohepatitis. The purpose of this study was to investigate the potential therapeutic effects of the compound of Example 3 (100 mg/kg/day) following a four week intravenous infusion in rats in which liver damage had previously been induced by a model of steatohepatitis as described by Lieber et al (see Example 4 above).

Animal management, diet, and preparation including surgery was as described in Example 4. Each group comprised six male rats. The group identification and animal numbers assigned to each treatment are summarised below:

*calculated as pure compound; equivalent to 5i0mg/kg/hr test compound before correction for purity (83.7%w.w).

From day 1 up to the end of the study, relevant diets were offered ad libitum. Group 1 animals were fed with a standard liquid diet. Groups 2 and 3 animals were fed with a high-fat liquid diet supplemented with 5 g/dL of ethanol.

The solution of the compound of Example 3 was administered by continuous intravenous infusion at an approximate rate of 1.5 mL/kg/hour over an infusion period of 24 hours (Control Group animals received the vehicle, isotonic saline, at the same flow rate).

The dose volume was calculated on the basis of the most recently recorded group mean body weight. All animals were dosed continuously for a minimum of four consecutive weeks (Weeks 5 - 8) up until necropsy.

The results obtained were as follows:

Mortality

Three animals of Group 3, treated with the test compound, were found dead on days 47, 51 and 43 of the study. The most relevant changes detected during necropsy and

histopathological examinations seemed to be related to inflammatory reactions caused by mechanical trauma of the infusion system. No other deaths occurred during the study. Clinical signs

Individual animals of groups 1 and 2 showed ulcerated tail (from slight to moderate), whereas only one animal of Group 3 showed this sign. Scabbed tail was observed in individual animals of groups 2 and 3. These clinical signs were related to the infusion system

implantation.

Body weight

From day 8 of the study up to the end of treatment period, animals of Groups 2 and 3 showed a slight decrease in body weight when compared to animals of Group 1 receiving the standard diet. This slight decrease in body weight was related to ethanol content in the fat diet. No differences in body weight were observed between animals of Group 2 receiving saline and animals of Group 3 treated with the test compound by continuous intravenous infusion.

Food consumption

After 6 days of administration of the fat diet plus ethanol until the end of the treatment period, animals of Groups 2 and 3 showed a statistically significant decrease in food consumption when compared to control animals of Group 1. As known from literature data, the decrease in food intake was related to the presence of ethanol in the fat diet. Animals treated with the test item did not show any significant difference in food consumption when compared to those of Group 2 receiving the same diet regimen. Therefore, administration of the test compound did not affect food intake in rats.

Terminal body weight and organ weights

At the end of the treatment period, animals of Groups 2 and 3 showed a slight decrease in terminal body weight when compared to the control group (Group 1 ). As expected, an increase in absolute liver weight and relative liver weight to body weight was observed in animals administered the fat diet and ethanol (Groups 2 and 3) when compared to the control animals fed with the standard diet (Group 1). The increase in absolute liver weight was statistically significant for Groups 2 and 3 when compared to Group 1 and the increase in relative liver weight to body weight was statistically significant for animals of Groups 2 and 3. Macroscopic observations

Detailed macroscopic observations were reported for individual animals in the study.

Unscheduled Deaths Three Male rats, group 3, receiving the test compound (dying on days 47, 51 and 43 of the study respectively) showed changes related to the infusion-system implantation.

Final sacrifice

Pale coloration and/or swelling and/or increased size of the liver were reported in the majority of males of group 2, fed with the fat diet plus ethanol and receiving the vehicle, and in Group 3, dosed with the test compound. A proportion of control and treated animals showed either abrasions, scabs or swelling on the implant site, pale firm masses involving the caudal vena cava and swollen and/or enlarged spleen. In addition, dark or pale areas in the liver lobes were reported in two male rats. These liver abnormalities could have occurred as a sequel of circulatory alterations caused by a mechanical trauma. However, these changes were considered as an expression of inflammatory reactions, due to the route of administration. Microscopic observations

Microscopic evaluation was carried out as described in Example 4.

Unscheduled deaths

One dead rat receiving the test compound showed multifocal, mild centrilobular/midzonal hepatocytic necrosis, moderate congestion and minimal steatosis. The other two dead animals only showed slight to mild steatosis in the liver.

Final sacrifice

Slight microvesicular steatosis, mainly localised in the periportal/midzonal area,

histochemically confirmed with positive intensity to Oil red O stain, was seen in animals dosed with the compound of Example 3 (Group 3).

Negative or weakly positive PAS stains, indicative of glycogen depletion, were recorded in the fat diet plus ethanol fed rats, either receiving the vehicle alone or test items, in comparison to the controls receiving the standard diet.

However, decreased severity and distribution in the hepatic lobule of steatosis were noted in rats receiving the test compound (Group 3), when compared with controls, fed with the fat diet plus ethanol and receiving the vehicle (Group 2).

Diffused and moderate hepatocytic steatosis was observed at histopathological examination of the liver in animals fed with the fat diet plus ethanol for 8 consecutive weeks. An evident reduction of hepatic steatosis with a decreased severity and distribution in the hepatic lobule, was noted in animals receiving the test compound, when compared with controls, fed with the fat diet plus ethanol and receiving the vehicle (Groups 1 and 2). These results indicate that four weeks of continuous intravenous infusion of the test compound (100 mg/kg/day) showed a therapeutic activity in rats with hepatic steatosis.

Example 6 - Protective effect of the compound of Example 3 in a D-galactosamine- induced model of hepatotoxicity.

This study investigated the potential effect of the test compound of Example 3 above following five days intravenous administration to Wistar Hannover rats in which liver damage had been induced by D-galactosamine prior to treatment with the test compound.

Animal management, diet, and preparation including surgery were carried out essentially as described in Example 4 above.

Each group comprised ten female rats. The treatment schedule is summarised below.

Pre-Dose: After acclimatisation, and before dosing commenced, samples of blood were withdrawn under isofluorane anaesthesia from the retro-orbital sinus of all allocated animals.

Days 1, 3 and 4: Animals of the control group (group 1) were dosed with isotonic saline by i.p. injection at a dose volume of lOml/kg body weight. Animals of both toxicant control and test groups were dosed with D-galactosamine hydrochloride by i.p. injection at a dose volume of 1 Oml/kg body weight (giving a dose level of 800mg/kg D-galactosamine hydrochloride). Days 5 and 7: Prior to dosing, samples of blood were withdrawn under isofluorane anaesthesia from the retro-orbital sinus of all surviving animals. Days 5,6,7,8 and 9: Animals of control and toxicant groups were dosed with the vehicle selected for use by i.v. injection into the tail vein. Animals of the test group (Group 3) were dosed daily with the test compound at a dose of 1 OOmg/kg body weight (body weight as determined on Day 5 of the study). Day 10: Animals were terminated according to standard procedures.

Results

Two animals of Group 2 and four animals of Group 3 were found dead from day 3 to 5.

These deaths were related to toxicity induced by D-galactosamine administration. A statistically significant increase in serum liver enzymes and a marked increase in bilirubin content were observed at day 5 in Groups 2 and 3 compared to control, Group 1. A statistically significant decrease in serum triglyceride serum levels was also observed for Groups 2 and 3. From Day 7 to 10, animals of group 2 and 5 showed progressive recovery in serum liver enzymes to normal values with a statistically significant increase of AST (aspartate transaminase) levels only in animals of Group 3.

Microscopic Observations: All animals treated with D-galactosamine showed Kupffer cell PAS positive reaction. A slight decrease in the incidence of levels of hepatocytic vacuolation, increased lipid accumulation and single cell apoptosis/necrosis were observed in Group 3. Group 3 showed an overall improvement in liver condition compared to Group 2 as no case of mixed inflammatory cell infiltration, lipid accumulation, single cell apoptosis/necrosis and hepatocyte pigmentation was recorded for surviving animals.