The invention relates to 3-substituted fatty acids acting on PPAR receptors, and a new way to prevent and treat the syndrome-X disease, a term to express the link between NIDDM, obesity, atherosclerosis, hypertension and premature cardiovascular disease, CD.

Hyperlipidemia and obesity afflict an increasing proportion of the population in Western societies and are associated with the development of serious conditions such as atherosclerosis, hypertension and insulin resistance.

These conditions may eventually lead to the clinical mani- festations of coronary heart diseases (CD) and non-insulin dependent diabetes mellitus (NIDDM). The link between NIDDM, atherosclerosis, hypertension and CD has been termed Syndrome-X, and elevated levels of plasma fatty acids appear to play a central role in the development of this class of diseases. More closely the term Syndrome X is inter alial related to conditions of low levels of high density lipoprotein-cholesterol (HDL-C), high levels of low density lipoprotein-cholesterol (LDL-C), raised triglyce- rides, glucose intolerance, increased blood pressure and abdominal obesity and restinose.

Treatment with modified fatty acids represent a new way to treat these diseases.

Modified fatty acids reduce the degree of obesity and reduce the levels of plasma fatty acids. Secondly, they are

potent antidiabetic compounds that lower the hyperglycaemia and hyperinsulinemia observed in animal models of non- insulin dependent diabetes (NIDDM) and in human NIDDM.

Thirdly, they promote adipocyte differentiation. Finally, they have an anti-atheriosclerotic potential by a) lowering blood, lipids, b) protect LDL against oxidation, c) and lowering of homocysteine.

Thus the present invention relates to the use of certain non-ß-oxidazable fatty acid analogues (3-substi- tuted) for the manufacture of medicaments for patients with a variety of Syndrome X-conditions, namely low levels of high density lipoprotein-cholesterol (HDL-C), high levels of low density lipoprotein-cholesterol (LDL-C), raised triglycerides, glucose intolerance, increased blood pressure, NIDDM, abdominal obesity, and restinose.

The diseases to be treated may include obesity and non-insulin dependent diabetes mellitus (NIDDM), coronary heart diseases (CD), atherosclerosis, hypertension, and represents an improvement of existing therapy (omega 3- fatty acids, fibrates, statines and thiazolidinediones).

Syndrome-X is also sometimes considered as a link between these individual diseases.

Fibrates, fatty acids and antidiabetic compounds such as thiazolidinediones exert their mode of actions via peroxisome proliferator-activated receptors (PPAR).

Fibrates and fatty acids via the a receptors (PPAR-a) and thiazolidinediones are ligands for PPAR-y. The non-ß- oxidizable fatty acid analogues, however, have dual functions: 1) regulate intracellular metabolism including fatty acid catabolism and extracellular lipoprotein metabolism, and also by afflicting gene expression mediated via PPAR-a.

2) improve insulin sensitivity in insulin resistant subjects which is mediated via PPAR-y and further emphasising the intricate network combining the regularly

circuits involved in adipocyte differentiation and fatty acid metabolism disorders as obesity and insulin resi- stance.

Thus, both liver and adipose tissue are important targets for the effect of non-ß-oxidizable fatty acids on fatty acid catabolism (PPAR-a) and insulin sensitivity (PPAR-y), respectively.

In this connection reference is made to European Patent Specification No. 345.038 (NORSK HYDRO A. S., priority of GB-8813012 of June 1988) (patent I) and International Patent Application No. WO 97/03663, (patent II), which disclose the use of non-b-oxidizable fatty acid analogues of the general formula (I): Alkyl-X-CH2COOR wherein alkyl represents a saturated or unsaturated hydro- carbon group of from 8-22 carbon atoms, X represents O, S, SO, and S02, Se and R represents hydrogen or Cl-C4 alkyl, for the manufacture of a medicament for the treatment of hyperlipidaemic conditions and for reducing the concen- tration of cholesterol and triglycerides in the blood of mammals. The EP-specification also discloses the pre- paration of compounds of the actual non-ß-oxidizable fatty acid analogues wherein the substitute X represents O, S, SO, S02. The EP-specification reports that the compounds in question exhibit favourable lipid lowering effects in blood of mammals, such as rats, and possess low toxicity measured as increase in liver weight and increased paroxysmal S-oxidation.

Further it has been found that analogues with the general formula alkyl-S-CH2COOR and alkyl-Se-CH2COOR functions as antioxidants and inhibit the LDL oxidative modifications. Thus, the antiatherosclerotic properties of these compounds are supposed to be related to their anti- oxidantic and hypolipidemic effects in blood of animals, i. e. by reducing the concentration of cholesterol and tri- glycerides.

It has now been found that the analogues of the above mentioned non-ß-oxidizable fatty acids, i. e. the 3- substituted fatty acid analogues have broader area of applications.

New strategies have been developed to search for compounds that both are ligands for PPAR-a (lipid-lowering effects and anti-obesity) and a ligand for PPARy (insulin- sensitivity effect).

In feeding experiments with such fatty acid analogues, the results show that they are potent antidiabetic compounds that lower the hyperinsulinemia, hypertri- glyceridemia and also reduce the body fat content in animal models of non-insulin dependent diabetes (NIDDM).

The compounds of the present invention probably act by enhancing the peripheral sensitivity to insulin. The fatty acids analogues upregulate the lipoprotein lipase LPL expression (LPL), which hydrolyses lipoprotein triglycerid- es. This upregulation is probably linked to activation of PPARy. On the other hand, PPARy is a key factor for adipo- cyte differentiation and these fatty acid analogues are efficient promoters of adipocyte differentiation in vitro.

The thiazolidinediones also promote adipocyte differentiation in vitro. Thus, it could be questioned whether a thiazolidinedione therapy aimed at improving insulin sensitivity would promote the recruitment of new adipocytes in vivo, an effect which could be deleterious since most of the NIDDM patients are already obese. In contrast to thiazolidinediones, the fatty acid analogues reduce adipose tissue accumulation in vivo.

The link between NIDDM, atherosclerosis, hypertension and coronary heart diseases has been termed syndrome X, and elevated levels of plasma fatty acids appears to play a central role in the development of this class of diseases.

As 3-substituted fatty acids are ligands for PPAR-a and thereby involved in fatty acids catabolism by increasing the fatty acid oxidation, a 3-substituted fatty acid therapy will increase the diversion of fatty acids to

mitochondrial 9-oxidation and increase the rate of transfer of fatty acids from the serum compartment into hepatocytes giving a net reduction of the non-esterified fatty acid (NEFA) levels in plasma.

Accordingly the present invention relates to the use of non-ß-oxidizable fatty acid analogues of the general formula (I): Alkyl-X-CH2COOR wherein alkyl represents a saturated or unsaturated hydro- carbon group of from 8-22 carbon atoms, X represents O, S, SO, S02, and Se, and R represents hydrogen or Cl-C4 alkyl, for the preparation of a pharmaceutical composition for the treatment and/or for prevention of Syndrome-X conditions, with the exception of the Syndrome-X conditions claimed in European patent application 345.038 and International patent application WO 97/003663.

According to a preferred embodiment, a pharmaceutical composition is prepared for the treatment and/or for pre- vention of conditions of low levels of high density lipo- protein-cholesterol (HDL-C), raised triglycerides, glucose intolerance, increased blood pressure (hypertension) and abdominal obesity, NIDDM and restinose.

According to a preferred embodiment, use is made of a pharmaceutical composition wherein the compound of formula (I) is tetradecylthioacetic acid, or tetradecylseleoacetic acid.

According to another aspect, the invention relates to a method for the treatment and/or for prevention of Syndrome-X conditions, with the exception of the Syndrome-X conditions claimed in European patent application 345.038 and International patent application WO 97/003663, said method comprising administering to a mammal in need there- of, an effective amount of a non-ß-oxidizable fatty acid analogues of the general formula (I): Alkyl-X-CH2COOR

wherein alkyl represents a saturated or unsaturated hydro- carbon group of from 8-22 carbon atoms, X represents O, S, SO, S02, and Se, and R represents hydrogen or Cl-C4 alkyl.

More specific there is disclosed a method for the pre- paration of a pharmaceutical composition for the treatment and/or for prevention of: -metabolic conditions of low levels of high density lipoprotein-cholesterol (HDL-C), and/or high levels of low density lipoprotein-cholesterol (LDL-C).

-raised triglycerides, -glucose intolerance, -increased blood pressure, -NIDDM, -restinose, and/or -abdominal obesity.

Preferably, according to the method, the compound of formula (I) is tetradecylthioacetic acid, or the compound of formula (I) is tetradecylselenocetic acid, or in the compound of formula I, X is Oxygen (O), Sulfur-I-oxide (SO) or Sulfurdioxide (SO2).

RESTENOSIS.

THE RESTENOSIS AFTER PERCUTANEOUS TRANSLUMINAL CORONARY ANGIOPLASTY (PTCA).

The compounds specified according to the present invention are also suitable for the treatment of resteno- sis. Restenosis remains a major problem limiting the long- term success of catheter based balloon interventions in the coronary arteries and in the main arteries of the head, kidneys and lower limbs. Restenosis occurs between 1-6 months after the intervention. The mechanism involves proliferation of smooth muscle and hyperplasia of the endothelium which results in narrowing of the arteries in the same site as the original balloon dilatation. There are two mechanisms by which restenosis may be reduced, either via reduced thickening of the vessel wall or by remodell-

ing. Remodelling is when the vessel changes shape, the lumen diameter increases and the net effect is a dilatation of the vessel.

It has now been found that the tetradecylthioacetic acid (i. e. the fatty acid, 3-THIA-) probably via its anti- oxidant properties influences favourably the remodelling of the vessel wall, while the arterial wall thickening is un- affected. We assume that the quite similar selen containing fatty acid will exert the same properties, and also at the same time is more potent in its action.

OBESITY Obesity is an important medical problem leading to high blood pressure, heart failure, diabetes, and myo- cardial infarction. Weight reduction with various diets have a high recurrence rate, and drug therapy has been limited mainly due to severe side effects. Compound I has weight reducing properties by its actions on adipose tissue. Thus, the non-ß-oxidizable fatty acid analogues may also be used for the manufacture of a weight reducing slimming-product for persons who wish to go through a slimming program.

The present invention provides fatty acid analogues to be potent antidiabetic compounds that can lower hyper- glycaemia, hyperinsulinemia, hypertriglyceridmia, hyper- cholesterolemia and to reduce obesity, reduce susceptibili- ty to LDL oxidation in human NIDDM.

The compounds used according to the present invention wherein the substituent X is a sulphur atom or selenium atom may be prepared according to the following general procedure: X is a sulphur atom: The thio-substituted compound used according to the present invention may be prepared by the general procedure indicated below:

Base Alkyl-Hal + HS-CH2COOR ===> Alkyl-S-CH2-COOR The sulphur-compound, namely, etradecylthioaceticacid, (CH3- (CH2) 13-S-CH2-COOH was prepared as shown in EP- 345.038, page 3, last paragraph.

X is a selenium atom: the seleno-substituted compound used according to the present invention may be prepared by the following general procedure 1. Alkyl-Hal + KSeCN > Alkyl-SeCN...

2. Alkyl-SeCN + BH4-Alkyl-Se- 3. Alkyl-Se-+ 02 Alkyl-Se-Se-Alkyl This compound is purified by carefully crystallisation from ethanol or methanol.

BH4- 4. Alkyl-Se-Se-Alkyl 2 Alkyl-Se- 5. Alkyl-Se~ + Hal-CH2_COOH => Alkyl-Se-CH2-COOH The final compound, e. g. when alkyl is tetradecyl, (CH3- (CH2) 13-Se-CH2-COOH can be purified by crystallisation from diethyl ether and hexane. This product may be fully characterized by NMR, IR and molecular weight deter- mination.

The methods for the synthesis and isolation of these Sulphur and Selenium compounds, and the compound wherein X of formula I is Oxygen (0), Sulphur-I-oxide (SO) and Sulphurdioxide (SO2) are disclosed in the abovementioned

European Patent Specification No. 345.038 (patent I) and International Patent Application No. WO 97/03663, (patent II).

EXPERIMENTS EXPERIMENT 1 Hvrolicidemic effect Male obese Zucker fa/fa rats, weighing 100 g at the start of the experiment, were housed in pairs in metal wire cages in a room maintained at 12 h light-dark cycles and a con- stant temperature of 203 OC. The animals were acclimatised for at least one week under these conditions before the start of the experiment.

Compound I (tetradecylthioacetic acid) prepared in accord- ance with procedure described previously, and palmitic acid (control), was suspended in 0.5% (w/v) carboxymethyl cellu- lose (CMC). Six animals were used in both groups. Compound I (tetradecylthioacetic acid) and palmitic acid were administered at a dose of 300 mg/day/kg body weight, by gastric intubation (gavage) once daily for 10 days. The rats were fasted for 2 hours before termination of the experiment. Blood and organs were collected. Very low density lipoproteins (VLDL) were prepared by sequential ultracentrifugation. Lipid concentrations in plasma and very low density lipoproteins (VLDL) were determined using an autoanalyzer. Results obtained are reported in Table 1.

TABLE 1.

Effect of Compound I (tetradecylthioacetic acid) on lipid levels in obese Zucker fa/fa rats. Decreased lipid level in plasma (% of control) TriglyceridesCholesterol Phospholipides Compound I727371 Decreased lipid level in VLDL (% of control) Triglycerides Cholesterol Phospholipides Compound I 63 115 88

Table 1 shows that Compound I (tetradecylthioacetic acid), exhibits a good hypolipidemic effect in blood of obese Zucker fa/fa rats. A sub-chronic toxicity study has been performed in dog by Corning Hazleton, Europe. The compound possesses low toxicity. Compound I (tetradecylthioacetic acid) are therefore potentially useful as a medical compound in this respect.

Hypolvcaemia and increased insulin sensitivity The effect of tetradecylthioacetic acid (Compound I) on the plasma levels of insulin and glucose.

TABLE 2 Effect of Compound I (tetradecylthioacetic acid) on insulin n and glucose levels in obese Zucker fa/fa rats.

Decreased levels of insulin in plasma after 10 days of treatment (% of control) Insulin Glucose Compound I 60 90 These results show that the tetradecylthioacetic acid affects the insulin and glucose levels in obese Zucker fa/fa rats.

The results also suggest that Compound I (tetradecyl- thioacetic acid) increases the insulin sensitivity and glucose tolerance in obese Zucker fa/fa rats. The body weight and weight gain was not altered in the treated rats, compared to controls.

TABLE 3 Effect of Compound I (tetradecylthioacetic acid) on g epidydimal fat/g body weight in Zucker fa/-fa rats.

Decreased levels of epidydimal fat after 10 days of treatment (g epidydimal fat/g body weight Control Compound I 0,0044 +0,0001 0, 0039 +0,0001 1 The amount (or ratio of) epidydimal fat/body weight however decreased slightly but significant during the treatment (table 3). When obese Zucker fa/fa rats are treated with the thiazolidinedione or pioglitazone, which lower hyper- glycaemia and hyperinsulinemia, there is a marked increase in weight gain during the treatment. Table 4 shows that Compound I (tetradecylthioacetic acid) significantly increases the activity of LPL. Also mRNA levels of LPL increased (data not shown).

Experiment.

A. Male Wistar rats were treated with compound 1 for 7 days. Total RNA was isolated from epidydimal adipose tissue and electrophoresis followed by northern blotting was performed. The blot was hybridised with LPL cDNA. The results are illustrated in the accompanying Figure 1. This figure shows that the mRNA of LPL in epidymal adipose tissue is increased in normal male Wistar rats after 1 week of treatment with compound 1.

B. The mouse preadipocyte cell line, 3T3-L1 (ATCC), was maintained in Dulbecco's modified Eagle's minimal essential medium and supplemented with 10% dilapidated and charcoal- treated fetal calf serum, L-glutamine and antibiotics.

Compound 1 was solved in ethanol and added to the medium at a concentration of 100 mM. Control cells received ethanol

only. Northern-blotting with total RNA was performed and the filters were hybridised using human LPL cDNA. The results are illustrated in the accomnanying Figure 2. This figure shows that compound 1 induce adipocyte differenti- ation measured by the appearance of LPL mRNA levels in 3T3- L1 cells.

TABLE 4.

Effect of Compound I (tetradecylthioacetic acid) on LPL activity in inguinal adipose tissue in Zucker ta/ta rats.

LPC activity (g epidydimal fat/g body weight) Control (compound I 91 13 1137 + 6 Figure 1 panel A, shows that the mRNA levels of LPL in epidydimal adipose tissue is increased after 1 week of treatment with compound 1 in normal male Wistar rats. Panel B shows that compound 1 induce adipocyte differentiation measured by the appearance of LPL mRNA levels in 3T3-L1 cells.

Antioxidant effect Male obese Zucker. fa/fa rats, weighing 100 g at the start of the experiment, were housed in pairs in metal wire cages in a room maintained at 12 h light-dark cycles and a con- stant temperature of 20h3 °C. The animals were acclimatised for at least one week under these conditions before the start of the experiment.

Compound I (tetradecylthioacetic acid) prepared in accord- ance with the procedures disclosed above, and palmitic acid (control), was suspende in 0.5% (w/v) carboxymethyl cellu- lose (CMC). Six animals were used in both groups. Compound

I (tetradecylthioacetic acid) and palmitic acid were administered at a dose of 300 mg/day/kg body weight, by gastric intubation (gavage) once daily for 10 days. The rats were fasted for 2 hours before termination of the experiment. Blood and organs were collected. Homocystein levels were determined using an autoanalyzer. The homo- cystein level was reduced 45% compared to control. Total lipids were extracted from liver and plasma. The lipids were evaporated, saponified and esterified prior to separation using a Carlo Erba 2900 gas-chromatograph.

Table 5 Effect of Compound I (tetradecylthioacetic acid) on fatty acid composition in obese Zucker fa/fa rats.

Fatty acid composition in liver (% of total) Oleic acid Monounsaturated tetradecylthioacetic acid Control 9.9 _ Compound I 14.9 0.2 1 Fatty acid composition in plasma (% of total) Oleic acid Monounsaturated tetradecylthioacetic acid Control 18.3 0.9 0.0 Compound I 22.1 0.5 0.2 0.1 1 Table 5 shows that oral administration of compound I (tetradecylthioacetic acid) increases the level of oleic acid in both liver and plasma. Also a delta-9-desaturated product of tetradecylthioacetic acid accumulated in both plasma and liver.

RESTINOSIS Experimental methods: 3-THIA was administered to pig coronary arteries by local drug delivery via a special angioplasty balloon catheter with side holes in the balloon. Coronary balloon angio- plasty injury to the vessel wall was performed to the LAD or Cx using an oversized balloon, thereafter the substance 3-THIA (tetradecylthioacetic acid) was infused via the balloon with side holes. Twenty minipigs were randomised to this treatment or infusion of placebo using the same technique. Radiolabelled 3-THIA was also infused into 2 extra pigs in which presence of the radioactive substance was confirmed after 4-6 weeks. The luminal diameter of coronary arteries was measured in control pigs (placebo group) and pigs treated with compound I.

TABLE 6 The luminal diameter of coronary arteries in the placebo and compound I pigs. Diameter (mm) Placebo Treated with Compound I Before injury2, 72,6 NS Follow up (after 6 2,2 1,3 (p<O, 001) weeks) *NS = not significant Results: The luminal diameter at angiographic and ultra sound follow-up at 4 weeks was significantly smaller in the placebo group than in the active treatment group with 3- THIA (tetradecylthioacetic acid). Histology showed no difference in wall thickening between the two groups. We conclude that local application of the antioxidant agent tetradecylthioacetic acid alters vessel remodelling rather

than intimal hyperplasia after balloon angioplasty.

In another experiment rabbits were treated orally with a medicament containing the 3-THIA-compound (tetradecyl- thioacetic acid) before and after balloon angioplasty of the iliac arteries. The same results were found showing that remodelling was favourably influenced with open arteries in the group of rabbits treated with active compound, and in stenosed arteries in the placebo group (by angiography) at follow-up.

We conclude from these studies that a medicament containing the 3-THIA-compound reduces restenosis. This was judged by angiography after balloon angioplasty, admini- stered either orally or locally. The effect is the same in coronary and peripheral arteries.

OBESITY. Experiments.

2 groups of 6 male Wistar Rats were randomly selected, and studied for weight development over a period of 12 week.

The body weight of each wistar rat was measured at the start of the experiment. All animals in both groups received individually the same amount of food (nutrition) during the experimental period of 12 weeks. All animals in one of the groups were orally administrated with the medicament comprising tetradecylthioacetic acid (compound I). The other group was the control group. After the 12 week period the body weight of rats were measured again.

The results of the experiment are shown in the following table.

Table 7.

Effect of compound I (tetradecylthioacetic acid) on body weight of male Wistar rats after 12 weeks of treatment. Body weight gain < control (rats not treated 293 27 with compound I) Compound I 234 + 20 (p<0,05) The results show that oral administration of tetra- decylthioacetic acid leads to significant weight loss in obese individuals with concomitant medical conditions (examples: heart disease and hypertension), and induces weight loss in obese individuals which are otherwise healthy. Said effects are assumed to be similar when the sulphur-compound is exchanged with the selenium compound, i. e. tetradecyl-seleno-acetic acid, and with oxygen 0, SO and S02 as mentioned above.

The compounds used according to the present invention may be administered to patients suffering from non-insulin dependent diabetes mellitus, coronary heart diseases and obesity. Alternatively by dietary they may prevent the disease.

The dosage range for the compounds according to the present application is contemplated to be from 5 to 100 mg/ day for the average adult patient. Of course, the actual dose necessary will depend on the patient's condition and will have to be determined by the attending physical from case-to-case.

For oral pharmacological compositions such carrier material as, for example, water, gelatine, gums, lactose, starches, magnesium-stearate, talc, oils, polyalkene glycol, petroleum jelly and the like may be used. Such pharmaceutical preparation may be in unit dosage form and may additionally contain other therapeutically valuable

substances or conventional pharmaceutical adjuvants such as preservatives, stabilising agents, emulsifiers, buffers and the like. The pharmaceutical preparations may be in conventional liquid forms such as tablets, capsules, dragees and the like, in conventional dosage forms, such as dry ampulles, and as suppositories and the like.

For parenteral administration the compounds according to the present invention may be administered as solutions, suspensions or emulsions using conventional pharmaceutical carrier materials such for example water for injection, oils, polyalkylene glycols and the like. These pharma- ceutical preparations may further include conventional pharmaceutical adjuvants, such as preservatives, stabilis- ing agents, emulsiers, salts for the adjustment of the osmotic pressure, buffers and the like. The preparations may also contain other therapeutically active materials.