The present invention relates to coumarin derivatives, particularly hymecromone, to pharmaceutical compositions containing them and to their use in the treatment of disorders of the liver, kidney, pancreas, bladder and gastro-intestinal tract.

The liver is a complex organ with many diverse functions. It is the central organ of metabolism of carbohydrates, proteins, and fat. It stores glycogen and takes part in regulating blood sugar, and stores other essential substances such as vitamins and factors concerned in haemopoiesis. It synthesises fibrinogen, prothrombin, heparin, and plasma proteins, and is a site of destruction of deteriorated red blood cells. It is also the chief detoxicating organ of the body rendering unwanted substances innocuous.

Viral hepatitis refers to infection of the liver caused by a group of hepatitis viruses. Those so far identified are designated A, B, C, D and E. Other viruses such as Epstein-Barr virus and yellow fever virus may be secondary causes of hepatitis and nonviral infections, drugs, chemicals and alcoholism may also cause hepatitis. One of the main treatments for the various viral hepatitis infections is alpha or beta interferon, but in many cases it is not particularly effective. Other drugs that may produce a therapeutic response include lamivudine, ursodeoxycholic acid and vidarabine.

Primary biliary cirrhosis is a chronic liver disease of unknown aetiology, which develops due to progressive destruction of small and intermediate bile ducts within the liver, subsequently evolving to fibrosis and cirrhosis. Over 90% of patients are female, usually aged between 40 and 60 years. The disease is thought to be

autoimmune in nature, perhaps triggered by a micro- organism in the environment and most patients exhibit autoantibodies to mitochondria. The disease is slowly progressive but no specific treatment is available.

Wilson's disease is due to an inborn error of liver metabolism leading to the accumulation of toxic concentrations of copper. Gilbert's syndrome is an inherited disorder that affects the way bilirubin is handled by the liver. Symptoms include mild jaundice, fatigue and abdominal pain. Again, in both Wilson's disease and Gilbert's syndrome, there is no real effective treatment.

Accordingly there is a constant need for new compounds to treat the various liver disorders.

The inventor has now found that certain organic compounds (including naturally occurring extracts) are effective in the treatment of various liver and other disorders.

Accordingly, in a first aspect of the invention there is provided use of a compound of the Formula 1: Formula 1 wherein R'and R'are independently H, OH, halogen, CF3, a glucopyranosyloxy group or an optionally substituted Cl6 straight or branched chain alkyl group, Cl6 alkoxy group, aryl C26 alkenyl group, C26 alkynyl group, C36 cycloalkyl

group, (C36cycloalkyl) C16alkyl group, a carboxylic acid group, an aryl group, or NR3R4 wherein R3 and R4 are independently H, C,-, alkyl, aryl or halogen, wherein R1 can be substituted at one or more positions; and R3 is H or a carboxylic acid group; and pharmacologically acceptable derivatives thereof in the manufacture of a medicament for the treatment or prophylaxis of liver kidney, pancreatic, bladder, and gastro-intestinal disorders (other than as a choleretic or biliary antispasmodic) or for reducing the concentration or activity of transaminase enzymes.

A preferred sub-group of compounds of Formula 1 is that defined by Formula 2: Formula 2 wherein: W is H or a P-D-glucopyranosyloxy group, X, Y and Z are independently H, OH, Cl6 alkyl C16 alkoxy, CFl, a glucopyranosyloxy group, or NR3R4 wherein R'and R4 are independently H, Cl6alkyl, aryl or halogen.

Preferably at least X or Y in Formula 2 is OH, a. nd/or Z is methyl.

Examples of known compounds covered by Formula 2 are as follows: coumarin, fraxin (7,8-dihydroxy-6- methoxycoumarin-8-p-D-glucoside), esculatin (6,7- dihydroxycoumarin), esculin (6, 7-dihydroxycoumarin-6- glucoside), scorparone (6,7-dimethoxycoumarin),

umbelliferone (7-hydroxycoumarin), skimmin (7- (glucosyloxy) coumarin), and hymecromone (7-hydroxy-4- methylcoumarin).

Many of the compounds of Formula 1 and 2 are novel and as such the compounds per se form a further aspect of the invention. Furthermore, of the known compounds, most do not have a recognised medical use, and therefore pharmaceutical compositions containing such novel and known compounds and their use in medical therapy form yet a further aspect of the invention.

All compounds of Formula 1 (and Formula 2) are hereinafter referred to as"compounds of the invention".

The compound that the inventor has done most work on and which has been found to be particularly effective is hymecromone (CAS Registry no. 90-33-5).

Although hymecromone is primarily used for the fluorometric determination of enzyme activity (Clin.

Chim. Acta., 39,49 (1972); Anal. Biochem., 54,40 (1973)) it is also known as a choleretic (agent which aids the excretion of bile) and a biliary antispasmodic, and has been administered in doses of 300mg-400mg. (The Merck Index, 12th Edition, 4903 and Martindale, The Extra Pharmacopoeia, 31st Edition pl715). A more soluble form of hymecromone is disclosed in EP-B-0240874, and tablets of hymecromone for improving the excretion of bile are known from US-A-3 175 943.

Hymecromone is commercially available from ABCR GmbH & Co KG (Karlsruhe, Germany, Acros Organics (Geel, Belgium), Loba Feinchemie AG (Fischamend, Austria), Sigma-Aldrich Fine Chemicals (Poole, Dorset, UK).

Hymecromone is also available as a natural occurring extract of Manna Ash known as Fraxin.

By pharmacologically acceptable derivatives of the compounds of formulas 1 and 2, it is meant to include prodrugs, salts, solvates, esters, ethers, amides, glycosylated derivatives, and including methylated, aminated and acetylated derivatives.

By prodrug is meant any compound, which is capable of being metabolised in vivo to give a compound of Formula 1.

By salt it is meant to include pharmaceutically acceptable salts derived from an appropriate base such as alkali metal salts (e. g. sodium or potassium), alkaline earth metals (e. g. magnesium or calcium), ammonium and NX4+ (wherein X is C14 alkyl), or salts of a hydrogen atom including salts formed from organic and inorganic acids such as those formed from hydrochloric, hydrobromic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic and isethionic acids.

The compounds of the invention, particularly hymecromone, have hepatoprotective and/or hepatoregenerative properties. Liver disorders which can be treated comprise hepatitis including infective hepatitis (e. g. viral hepatitis of types A, B, C, D and E), chronic active hepatitis, acute infective hepatitis, toxic hepatitis (e. g. as caused by drugs and narcotics, X-rays, solvents, chemotherapy, and alcohol abuse), steatosis hepatitis, acute parenchymatous hepatitis, amoebic hepatitis, cytomegalic hepatitis, enzootic hepatitis, familial hepatitis, homologous serum hepatitis, intestitial hepatitis, suppurative hepatitis, and trophopathic hepatitis. Various cirrhosis of the liver conditions which can be treated include primary and

secondary biliary cirrhosis, alcoholic cirrhosis, annular cirrhosis, atrophic cirrhosis, bacterial cirrhosis, capsular cirrhosis, cardiac cirrhosis, fatty cirrhosis lymphatic cirrhosis and pigmentary cirrhosis. Yet further liver disorders against which the compounds of the invention are effective are Wilson's disease and Gilbert's syndrome.

The compounds of the invention are particularly effective in protecting the liver from the toxic effects of anabolic steroids, alcohol, chemotherapy, solvents, drugs, and environmental pollution.

Early investigations also suggest that compounds of the invention such as hymecromone will also be effective in the treatment or prophylaxis of disorders of the kidney (e. g. cirrhosis of the kidney), pancreas (e. g. pancreatitis), bladder and gastro-intestinal tract (e. g. cirrhosis of the stomach).

The invention can also be used to rejuvenate healthy people, especially those exposed to heavy physical labour, the elderly and otherwise healthy people recovering from illness, and sportsmen (especially those using anabolic steroids).

Without being bound by theory, it is thought that the compounds of the invention are at least partly effective by reducing the concentration or activity in the peripheral blood of transaminase enzymes, such as serum glutamic oxaloacetic transaminase (SGOT) or aspartate aminotransferase (AST), serum glutamic pyruvic transaminase (SGPT) or alanine transaminase (ALT), and gamma glutamyl transpeptidase (GGT).

Accordingly in another aspect of the invention there is provided the use of a compound of the invention in the manufacture of a medicament for the treatment of

conditions which are provided a beneficial therapeutic effect by a reduction in the concentration or activity of transaminase enzymes.

A further aspect provides the use of a compound of the invention as an inhibitor of one or more transaminase enzymes, particularly those disclosed herein.

A yet further aspect of the invention relates to a method for the treatment or prophylaxis of a liver disorder, or a kidney, pancreatic, bowel, or gastro- intestinal disorder or a disorder benefiting from a decrease in the activity or concentration of transaminase enzymes which comprises administering to the patient a beneficial amount of a compound of the invention.

The scope of the invention does not include gastro- intestinal disorders relating to the use of hymecromone as a choleretic or biliary antispasmodic.

Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Such a unit may contain for example 50mg to 2000mg preferably 200mg to 50Omg depending on the condition being treated, the route of administration and the age, weight and condition of the patient. In the case of a compound of the invention suspended in honey, a unit dosage would be relative to one or more spoonfuls of the honey. The patient should preferably receive in the region of 15g to 50g per month, advantageously about 25g to 28g/month of a compound of the invention. Each unit dose may be administered once, twice, three or more times daily.

Pharmaceutical formulations may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal),

vaginal or parenteral (including subcutaneous, intramuscular, intravenous or iniradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier (s) or excipient (s).

Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or enemas, and those adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above

recited, or an appropriate fraction thereof, of an active ingredient.

In a preferred oral composition, a compound of the invention is present together with one or more of the following other components preferably at the given relative quantities: Component Preferred More Range (q) Preferred Cancre(cor) Compound of the invention 15-30 25-28 (e. g. hymecromone) Bee Pollen 2 to 10 About 5 Matricaria Camomile (Camomile) 2 to 10 About 5 pulv. Asperula Oderata (Sweet Woodruff) 2 to 10 About 5 pulv. Royal Jelly 5 to 15 About 10 The composition is preferably suspended in honey and presented as a rejuvenating dietary supplement.

In a particularly preferred oral composition, there is provided a capsule containing from 100 to 300mg (preferably about 210mg) of Compound of the invention (e. g. hymecromone), from 3 to 7mg (preferably about 5mg) of camomile, and from 3 to 7mg (preferably about 5mg) of bee pollen.

The preferred source of a Compound of the invention (hymecromone) is Loba Feinchemie AG (Fischamend, Austria).

The invention will now be illustrated by way of the following examples.

Example 1 A composition of the invention suspended in honey is as follows: Ingredient Weicrht Bee Pollen 0.5g Matricaria Camomile (Camomile) pulv. 0.5g Matricaria Camomile (Camomile) extr. sic. 2.5g Asperula Oderata (Sweet Woodruff) pulv. 0.5g Compound of the invention [Fraxinus Ornus 2. Og (Manna Ash) extr. sic.] Bee Honey 250. Og Example 2 A further composition of the invention is as follows: Ingredient Weictht Bee Pollen 0.5g Matricaria Camomile (Camomile) pulv. 0.5g Matricaria Camomile (Camomile) extr. sic. 0.5g Asperula Oderata (Sweet Woodruff) pulv. 0.5g Compound of the invention [4-Methyl-7-5. Og hydroxycoumarin (hymecromone)] Royal Jelly 1.5g Bee Honey 250. Og Example 3 A composition of the invention according to Example 1 was administered to 30 patients (20 males and 10 females) over a 3-month period.

During the three months of monitoring, the clinical diagnosis remained constant, however, changes were reported in the biochemical activity, evident in a reduction in enzymatic activities, SGOT (AST) and SGPT (ALT) in 70% of cases, as well as an in improvement in symptomatic levels. In 65% of cases, fatigue and overall

exhaustion were eliminated. In patients with chronic pancreatitis, pain in the area of the pancreas disappeared and sexual instinct reappeared. During testing there were no harmful side effects present.

Example 4 Patient A was diagnosed as having hypertrigliceridaemia. His liver was increased in size for 2p. p. of discrete ehogenous and homogenous parenhim (steatiosis hepatitis). He felt constantly tired, had chest and intestinal rumblings (especially after meals), complained of poor digestion (particularly stomach heaviness after any type of food), and experienced repeated heartburn and belching.

Enzyme Levels: GGT: 90.0; SGPT: 41.3; SGOT: 33.8.

The patient was given a course of a composition of the invention according to Example 1 at 2x5ml spoonfuls, 3 times per day for 40 days (hymecromone content equivalent to 140mg/5ml spoonful or 25gms per month).

The patient's transaminase levels were then measured again and were found to have decreased as follows: GGT: 40.3; SGPT: 18.4; SGOT: 21.

Example 5 Patient B was diagnosed as having steatosis hepatitis (an early stage of hepatitis). Liver size within normal, of ehogenous and homogenous parenhim, which indicates liver steatosis. The patient complained of chronic pains below the right rib. After trying to digest food, he experienced a bloated or heavy stomach and constant belching. He also complained of occasional lethargy.

Biochemical Analysis: GGT: 220; SGPT: 110; SGOT: 65.

After a course of a composition of the invention as in Example 1, the steatosis hepatitis was found to be in regression. The patient's transaminase enzyme levels also decreased as follows: GGT: 146; SGPT: 26.2; SGOT: 24.2.

Example 6 Patient C was diagnosed as having laesio hepatitis diffusa chr. and hepatomegalia. The patient had suffered from liver problems for some time, he felt depressed and tired, had a high fever and was jaundiced. He had an enlarged liver of hyperehogenous and inhomogenous parenhim, indicating chronic diffused liver lesion.

Biochemical Analysis: GGT: 685; SGPT: 113; SGOT: 124.

After a course of a composition of the invention as in Example 1, the patient's transaminase enzyme levels decreased as follows: GGT: 57; SGPT: 27.3; SGOT: 23.9.

Example 7 The preparation of Example 2 was used in the treatment of several patients suffering from various kinds of liver disease, some very advanced, but in all cases showing high levels of enzymes, particularly transaminase enzymes.

In all cases, the patients received the preparation of the invention in addition to the standard treatments (e. g. B-vitamins, K-vitamins, Paraaminometilbenzoic acid, fursemid, aldactone, propranolol and others). Pains found under the right ribs, bend, fatigue and general exhaustion were the predominant complaints of the patients.

Liver Profile, Blood Analysis and general health conditions were monitored on a monthly basis.

The following enzymes were measured in the biochemical analysis: Serum Glutamic Oxaloacetic Transaminase (SGOT) or aspartate aminotransferase (AST): (NORMAL VALUE: 10 to 40 international units per decilitre); + Serum Glutamic Pyruvic Transaminase (SGPT) or alanine transaminase (ALT): (NORMAL VALUE: 10 to 30 international units per decilitre); Gamma Glutamyl Transpeptidase (GGT): (NORMAL VALUE: 0-45 international units per decilitre).

On the basis of the biochemical determination, the patients were classified into four groups.

GROUP 1: Patients diagnosed with: cirrhosis and chronic active hepatitis, with high transaminase (AST [SGOT] & ALT [SGPT] over 120); GROUP 2: Patients diagnosed with: viral hepatitis (chronic), both B and C chronic type in active state (AST [SGOT] and ALT [SGPT] over 200); GROUP 3: Patients diagnosed with: toxic alcoholic liver lesion in acute state, with high transaminase (AST [SGOT] and ALT [SGPT] over 350), acute alcoholic hepatitis;

GROUP 4: Patients diagnosed with: syndromes and high transaminase.

In summary, the patients, who were using the composition of the invention for more than two months in conjunction with standard therapy, recovered quicker and felt much better. In addition to an improved general state of health, the patients reported improvements in appetite and physical fitness.

The biochemical analysis showed a rapid reduction of blood transaminase (AST [SGPT] and ALT [SGOT] to 100 units or less.

Patients in the first (1) third (3) and fourth (4) group responded better and quicker when using the invention and the transaminase fell quickly to between 60 and 80.

The patients in the second (2) group, those with chronic B or C hepatitis, whether receiving Interferon or not, responded slower. Initially there was no physical signs of improvement, but after a time a reduction in transaminase concentration in blood (AST [SGOT] and ALT [SGPT]), was noticed concomitant with regression of the disease.

Example 8 The hepatoprotective effect of a composition in accordance with the invention was investigated in mice with acetaminophen (paracetamol) induced hepatotoxicity.

CBA/H Zgr inbred mice were raised in an animal colony unit. Mice of both sexes aged 3 to 4 month were used in the experiments. Mice were maintained under standard laboratory conditions, fed with commercially available murine food pellets and allowed water ad

1 ibi tum.

The procedure of Guarner was followed (see Hepatology 1988; 3: 248-53). To induce hepatic drug-metabolising enzymes, mice were given phenobarbitone-sodium in drinking water for 7 days (0.3g/L). Thereafter, mice were fasted overnight.

Three different doses (10,50 or 250mg/kg) of a Composition of the invention (Hymecromone) were given to mice intraperitoneally in volumes of O. 5ml 30 minutes before intragastric administration of acetaminophen (300mg/kg).

The above ingredients were dissolved in phosphate buffered saline (PBS) to which several drops of Tween 20tam (polyoxyethylene 20 sorbitan monolaurate) were added (50y1/ml). The resulting white milk suspension was administered to mice intraperitoneally.

At the same time, control animals were given 0.5ml of pyrogen-free saline to which several drops of Tween 20TM (50y1/ml) were added.

Acetaminophen, dissolved in heated PBS, was given intragastrically, by stomach tube, in a volume of 0.5ml.

Animals were allowed food 4 hours later. Mortality of mice was followed for 48 hours, as previous results have shown that control mice (given saline rather than the composition before acetaminophen) either die within this period or fully recover and survive indefinitely.

Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured 24 hours after the administration of acetaminophen, because it has been observed that the levels of AST and ALT are at their peak values at this time. Mice were given 250 units of Hymecromone intraperitoneally 15 minutes before bleeding.

Blood was obtained by puncture of medial orbital angle using glass capillary tubes. Plasma was stored at-20°C until aminotransferase determination, which was carried out using standard laboratory techniques (see Expert Panel on Enzymes, Committee on Standards, Clin. Chem.

Biochem. 1977; 15: 39-51 and Clin. Chem. Acta. 1980; 105: 147F-54F).

Differences in survival between groups of mice were compared test. Plasma AST and ALT concentrations are expressed as means SEM and differences between groups were compared by Student s t-test.

Table 1 shows the mortality of mice 48 hours after acetaminophen administration. In comparison with the control group of mice given saline, pretreatment of mice with 250mg/kg of Hymecromone significantly reduced the mortality of mice (P<0.01). The mortality of mice given 10 or 50mg/kg of the composition was similar to that observed in control mice given saline. Tables 2 and 3 show the mortality of mice of two further experiments.

As shown, pretreatment of mice with Hymecromone (250mg/kg) statistically significant reduced the mortality of mice in comparison with control mice given saline (P<0.001).

Table 1. The effect of different doses of Hymecromone on survival of mice with AAP-induced hepatotoxicity Treatmenta Dose of No of dead mice/ Hymecromone Total No of mice (t) b (mg/kg) Saline + AAP-13/13 (100) Hymecromone + 10 12/12 (100) AAP Hymecromone + 50 11/12 (92) AAP Hymecromone + 250 2/7 (29) c AAP a Mice were given 300mg/kg AAP intragastrically in volume of 0.5ml. Saline or different doses of Hymecromone were given intraperitoneally 30 minutes before AAP administration b Mortality was recorded 48 hours after AAP administration c Significantly different in relation to the control group of mice treated with saline (P<0.01) Table 2. The effect of different doses of Hymecromone on survival of mice with AAP-induced hepatotoxicity Treatmenta Dose of No of dead mice/ Hymecromone Total No of mice (o) b (mg/kg) Saline + AAP-10/13 (77) Hymecromone + 50 9/13 (93) AAP Hymecromone + 250 0/13 (O) ° AAP a Mice were given 250mg/kg AAP intragastrically in volume of 0.5ml. Saline or Hymecromone were given intraperitoneally 30 minutes before AAP administration. b Mortality was recorded 48 hours after AAP administration. c Significantly different in relation to the control group of mice treated with saline (P<0.001).

Table 3. The effect of Hymecromone on survival of mice with AAP-induced hepatotoxicity Treatment No of dead mice/ Total No of mice (%) b Saline + AAP 16/16 (100) Hymecromone + AAP 0/15 (O) ° a Mice were given 250mg/kg AAP intragastrically in volume of 0.5ml. Saline or Hymecromone (250mg/kg) were given intraperitoneally 30 minutes before AAP administration. b Mortality was recorded 48 hours after AAP administration.

Significantly different in relation to the control group of mice treated with saline (P<0.001)

Example 9 The effect of a Compound of the invention (Hymecromone) was investigated on plasma aminotransferase levels in mice with AAP-induced hepatotoxicity.

Hymecromone (250mg/kg) or a control of saline was given to mice 30 minutes before administration of acetaminophen (220mg/kg). Plasma AST and ALT were measured 24 hours after acetaminophen administration.

Table 4 shows plasma aminotransferase levels in normal and in mice treated with Hymecromone or saline. As shown, in comparison with normal mice, the administration of acetaminophen increased AST and ALT by approximately 19 and 100 times, respectively. Pretreatment of mice with the composition significantly reduced the increase of AST and ALT in comparison with control mice pretreated with saline (AST five times and ALT three times; P<0.005).

Table 4. Plasma aminotransferase levels in mice with AAP-induced hepatotoxicity pretreated with saline or Hymecromone Treatmenta AST (U/L) b ALT (U/L) b Normal mice 754 231 (n=19) (n=19) Saline + AAP 1417355 2300376 (n=16) (n=16) Hymecromone + 26756° 776+166C AAP (n=16) (n=16) a Saline or Hymecromone (250mg/kg) were given 30 minutes before AAP administration (220mg/kg) <BR> <BR> <BR> <BR> <BR> <BR> b Determined 24 hours after AAP administration; mean SEM c Significantly different in relation to the control group of mice given saline before AAP administration (P<0.005)

The results of the investigation have shown that the composition in accordance with the invention has hepatoprotective effect in mice with acetaminophen-induced acute hepatotoxicity. The composition in a dose of 250mg/kg given intraperitoneally to mice 30 minutes before administration of acetaminophen, significantly reduced the mortality of mice and reduced plasma aminotransferase levels in comparison with control mice given saline before administration of acetaminophen.

The invention is therefore very effective in the treatment of different kinds of liver, kidney, pancreatic and gastro-intestinal disorders. It reduces the transaminase levels in peripheral blood, and reduces the activity and aggressiveness of the illness.