| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 2, 2002 |
| PATENT TITLE |
Fatty analogues for the treatment of diabetes |
| PATENT ABSTRACT | The present invention relates to novel fatty acid analogues of the general formula (I): CH.sub.3 --[CH.sub.2 ].sub.m --[x.sub.i --CH.sub.2 ].sub.n --COOR, as defined in the specification, which can be used for the treatment and/or prevention of diabetes. Further, the invention relates to a nutritional composition comprising such fatty acid analogues |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | January 8, 2001 |
| PATENT CT FILE DATE | April 23, 1999 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | November 18, 1999 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED | This data is not available for free |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method for the treatment or prevention of a diabetic condition comprising administering to an animal in need thereof an effective amount of at least one fatty acid analogue of the general formula (I) CH.sub.3 --[CH.sub.2 ].sub.m --[X.sub.i --CH.sub.2 ].sub.n --COOR wherein n is an integer from 1 to 12, and m is an integer from 0 to 23, and i is an odd number which indicates the position relative to COOR, and each X.sub.i is independently selected from the group consisting of O, S, SO, SO.sub.2, Se, and CH.sub.2, and R represents hydrogen or C.sub.1 -C.sub.4 alkyl, with the proviso that at least one of the X.sub.i is not CH.sub.2, or a salt, prodrug, or complex thereof. 2. The method of claim 1, wherein the animal is a human. 3. The method of claim 1, wherein the animal is an agricultural animal. 4. The method of claim 3, wherein the agricultural animal is selected from the group consisting of gallinaceous birds, bovine mammals, ovine mammals, caprine mammals, and porcine mammals. 5. The method of claim 1, wherein said animal is a domestic animal. 6. The method of claim 5, wherein the domestic animal is selected from the group consisting of dogs and cats. 7. The method of claim 1, wherein the diabetic condition is type I diabetes. 8. The method of claim 1, wherein the diabetic condition is type II diabetes. 9. The method of claim 1, wherein the diabetic condition is a form of secondary diabetes selected from the group consisting of pancreatic diabetes, extrapancreatic/endocrine diabetes, drug-induced diabetes, lipoatropic diabetes, myatonic diabetes and diabetes induced by disturbance of insulin receptors. 10. The method of claim 1, wherein m is greater than or equal to 13. 11. The method of claim 1, wherein X.sub.i=3 is selected from the group consisting of O, S, SO, SO.sub.2, and Se, and wherein X.sub.i=5-25 is CH.sub.2. 12. The method of claim 11, wherein X.sub.i=3 is S. 13. The method of claim 11, wherein X.sub.i=3 is Se. 14. The method of claim 11, wherein the at least one fatty acid analogue is administered such that its concentration is maintained substantially continuously in the blood of the animal for the duration of the period of administration. 15. The method of claim 1, wherein the composition is in unit dosage form. 16. The method of claim 1, wherein the at least one fatty acid analogue is administered orally or parenterally. 17. A method for the treatment or prevention of hyperglycemia, comprising administering to an animal in need thereof an effective amount of at least one fatty acid analogue of the general formula (I) CH.sub.3 --[CH.sub.2 ].sub.m --[X.sub.i --CH.sub.2 ].sub.n --COOR wherein n is an integer from 1 to 12, and m is an integer from 0 to 23, and i is an odd number which indicates the position relative to COOR, and each X.sub.i is independently selected from the group consisting of O, S, SO, SO.sub.2, Se, and CH.sub.2, and R represents hydrogen or C.sub.1 -C.sub.4 alkyl, with the proviso that at least one of the X.sub.i is not CH.sub.2, or a salt, prodrug, or complex thereof. 18. The method of claim 17, wherein the animal is a human. 19. The method of claim 17, wherein the animal is an agricultural animal. 20. The method of claim 19, wherein the agricultural animal is selected from the group consisting of gallinaceous birds, bovine mammals, ovine mammals, caprine mammals, and porcine mammals. 21. The method of claim 17, wherein said animal is a domestic animal. 22. The method of claim 21, wherein the domestic animal is selected from the group consisting of dogs and cats. 23. The method of claim 17, wherein m is greater than or equal to 13. 24. The method of claim 17, wherein X.sub.i=3 is selected from the group consisting of O, S, SO, SO.sub.2, and Se, and wherein X.sub.i=5-25 is CH.sub.2. 25. The method of claim 24, wherein X.sub.i=3 is S. 26. The method of claim 24, wherein X.sub.i=3 is Se. 27. The method of claim 17, wherein the at least one fatty acid analogue is administered such that its concentration is maintained substantially continuously in the blood of the animal for the duration of the period of administration. 28. The method of claim 17, wherein the composition is in unit dosage form. 29. The method of claim 17, wherein the at least one fatty acid analogue is administered orally or parenterally. 30. A method for the treatment or prevention of hyperinsulinemia, comprising administering to an animal in need thereof an effective amount of at least one fatty acid analogue of the general formula (I) CH.sub.3 --[CH.sub.2 ].sub.m --[X.sub.i --CH.sub.2 ].sub.n --COOR wherein n is an integer from 1 to 12, and m is an integer from 0 to 23, and i is an odd number which indicates the position relative to COOR, and each X.sub.i is independently selected from the group consisting of O, S, SO, SO.sub.2, Se, and CH.sub.2, and R represents hydrogen or C.sub.1 -C.sub.4 alkyl, with the proviso that at least one of the X.sub.i is not CH.sub.2, or a salt, prodrug, or complex thereof. 31. The method of claim 30, wherein the animal is a human. 32. The method of claim 30, wherein the animal is an agricultural animal. 33. The method of claim 32, wherein the agricultural animal is selected from the group consisting of gallinaceous birds, bovine mammals, ovine mammals, caprine mammals, and porcine mammals. 34. The method of claim 30, wherein said animal is a domestic animal. 35. The method of claim 34, wherein the domestic animal is selected from the group consisting of dogs and cats. 36. The method of claim 30, wherein m is greater than or equal to 13. 37. The method of claim 30, wherein X.sub.i=3 is selected from the group consisting of O, S, SO, SO.sub.2, and Se, and wherein X.sub.i=5-25 is CH.sub.2. 38. The method of claim 37, wherein X.sub.i=3 is S. 39. The method of claim 37, wherein X.sub.i=3 is Se. 40. The method of claim 30, wherein the at least one fatty acid analogue is administered such that its concentration is maintained substantially continuously in the blood of the animal for the duration of the period of administration. 41. The method of claim 30, wherein the composition is in unit dosage form. 42. The method of claim 30, wherein the at least one fatty acid analogue is administered orally or parenterally. 43. A method for the treatment or prevention of reduced sensitivity to insulin comprising administering to an animal in need thereof an effective amount of at least one fatty acid analogue of the general formula (I) CH.sub.3 --[CH.sub.2 ].sub.m --[X.sub.i --CH.sub.2 ].sub.n --COOR wherein n is an integer from 1 to 12, and m is an integer from 0 to 23, and i is an odd number which indicates the position relative to COOR, and each X.sub.i is independently selected from the group consisting of O, S, SO, SO.sub.2, Se, and CH.sub.2, and R represents hydrogen or C.sub.1 -C.sub.4 alkyl, with the proviso that at least one of the X.sub.i is not CH.sub.2, or a salt, prodrug, or complex thereof. 44. The method of claim 43, wherein the animal is a human. 45. The method of claim 43, wherein the animal is an agricultural animal. 46. The method of claim 45, wherein the agricultural animal is selected from the group consisting of gallinaceous birds, bovine mammals, ovine mammals, caprine mammals, and porcine mammals. 47. The method of claim 43, wherein said animal is a domestic animal. 48. The method of claim 47, wherein the domestic animal is selected from the group consisting of dogs and cats. 49. The method of claim 43, wherein m is greater than or equal to 13. 50. The method of claim 43, wherein X.sub.i=3 is selected from the group consisting of O, S, SO, SO.sub.2, and Se, and wherein X.sub.i=5-25 is CH.sub.2. 51. The method of claim 50, wherein X.sub.i=3 is S. 52. The method of claim 50, wherein X.sub.i=3 is Se. 53. The method of claim 43, wherein the at least one fatty acid analogue is administered such that its concentration is maintained substantially continuously in the blood of the animal for the duration of the period of administration. 54. The method of claim 43, wherein the composition is in unit dosage form. 55. The method of claim 43, wherein the at least one fatty acid analogue is administered orally or parenterally. 56. A method for reducing the concentration of glucose in the blood of a human or non-human animal in need thereof, comprising administering to the animal an effective amount of a composition comprising fatty acid analogues of the general formula (I) CH.sub.3 --[CH.sub.2 ].sub.m --[X.sub.i --CH.sub.2 ].sub.n --COOR wherein n is an integer from 1 to 12, and m is an integer from 0 to 23, and i is an odd number which indicates the position relative to COOR, and each X.sub.i is independently selected from the group consisting of O, S, SO, SO.sub.2, Se, and CH.sub.2, and R represents hydrogen or C.sub.1 -C.sub.4 alkyl, with the proviso that at least one of the X.sub.i is not CH.sub.2, or a salt, prodrug or complex thereof. 57. The method of claim 56, wherein the animal is a human. 58. The method of claim 56, wherein the animal is an agricultural animal. 59. The method of claim 58, wherein the agricultural animal is selected from the group consisting of gallinaceous birds, bovine mammals, ovine mammals, caprine mammals, and porcine mammals. 60. The method of claim 56, wherein said animal is a domestic animal. 61. The method of claim 60, wherein the domestic animal is selected from the group consisting of dogs and cats. 62. The method of claim 56, wherein m is greater than or equal to 13. 63. The method of claim 56, wherein X.sub.i=3 is selected from the group consisting of O, S, SO, SO.sub.2, and Se, and wherein X.sub.i=5-25 is CH.sub.2. 64. The method of claim 63, wherein X.sub.i=3 is S. 65. The method of claim 63, wherein X.sub.i=3 is Se. 66. The method of claim 56, wherein the at least one fatty acid analogue is administered such that its concentration is maintained substantially continuously in the blood of the animal for the duration of the period of administration. 67. The method of claim 56, wherein the composition is in unit dosage form. 68. The method of claim 56, wherein the at least one fatty acid analogue is administered orally or parenterally. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION Diabetes mellitus and its complications are now considered to be the third leading cause of death in Canada and the United States, trailing only cancer and cardiovascular disease. Treatment with modified fatty acids represent a new way to treat these diseases. EP 345.038 and PCT/NO95/00195 describes the use of non-.beta.-oxidizable fatty acid analogues. It has now been found that these have broader area of applications. Further, we have now synthesized and characterized novel fatty acid analogous which impose an effect on diabetes. In feeding experiments with the fatty acid the results show that these compounds lower the adipose tissue mass and body weight, and are thus potent drugs for the treatment of obesity and overweight. Further, we have shown that the fatty acid analogues are potent antidiabetic compounds, with a profound effect on the levels of glucose and insulin. Further, the compounds have been proved to have an favourable effect on restenosis, and exhibit good anti-oxidative properties. DIABETES Diabetes mellitus and its complications are now considered to be the third leading cause of death in Canada and the United States, trailing only cancer and cardiovascular disease. Although the acute and often lethal symptoms of diabetes can be controlled by insulin therapy, the long-term complications reduce life expectancy by as much as one third. Compared with rates of incidence in nondiabetic normal persons, diabetic patients show rates which are increased 25-fold for blindness, 17-fold for kidney disease, 5-fold for gangrene, and 2-fold for heart disease. There are 2 major forms of diabetes mellitus. One is type I diabetes, which is also known as insulin-dependent diabetes mellitus (IDDM), and the other is type II diabetes, which is also known as noninsulin-dependent diabetes mellitus (NIDDM). Most patients with IDDM have a common pathological picture: the nearly total disappearance of insulin-producing pancreatic beta cells which results in hyperglycemia. Considerable evidence has been accumulated showing that most IDDM is the consequence of progressive beta-cell destruction during an asymptomatic period often extending over many years. The prediabetic period can be recognized by the detection of circulating islet-cell autoantibodies and insulin autoantibodies. There is a need for a compound which would be nontoxic and have no side effects but which would prevent clinical IDDM and NIDDM. Type I diabetes: severe diabetes mellitus, usually of abrupt onset prior to maturity, characterized by low plasma insulin levels, polydipsia, polyuria, increased appetite, weight loss and episodic ketoacidosis; also referred to as IDDM. Type II diabetes: an often mild form of diabetes mellitus, often of gradual onset, usually in adults, characterized by normal to high absolute plasma insulin levels which are relatively low in relation to plasma glucose levels; also referred to as NIDDM. Type I and II diabetes are in accordance with an etiologic classification considered as < Secondary diabetes comprises pancreatic, extrapancreatic/endocrine or drug-induced diabetes. Further, some types of diabetes are classified as exceptional forms. These include lipoatrophic, myatonic diabetes, and a type of diabetes caused by disturbance of insulin receptors. Considering the high prevalence of diabetes in our society and the serious consequences associated therewith as discussed above, any therapeutic drug potentially useful for the treatment and prevention of this disease could have a profound beneficial effect on their health. There is a need in the art for a drug that will reduce the concentration of glucose in the blood of diabetic subjects without significant adverse side effects. It is therefore an object of the present invention to provide a treatment regimen that is useful in lowering the blood glucose and to treat a diabetic condition. It is yet another object of the invention to provide a treatment regimen that is useful in lowering the concentration of insulin in the blood, and to increase the effect of the remaining insulin. MECHANISMS OF ACTION Minor modifications of natural fatty acids, sulphur, selenium or oxygen replacing one or more of carbons in the fatty acid backbone. The compounds defined by the formula I have properties which give them a unique combination of biological effects. Tetradecylthioacetic acid (TTA) is most thoroughly studied and we have shown several beneficial effects in various test animals. The studies have shown that TTA has properties very similar to natural fatty acids, the main difference being that TTA is not oxidised by the mitochondrial .beta.-oxidation system. However, the presence of compounds of the present invention have been shown to increase the .beta.-oxidation of other (non-substituted fatty acids). Administration of TTA to rats for 12 weeks nearly doubled the hepatic and plasma content of monounsaturated fatty acids (mainly oleic acid), while polyunsaturated fatty acids (mainly linoleic acid and DHA) decreased. Thus the compound of the present invention modifies the composition of the lipids in various tissues. It is also shown that the present compounds modifies the fat content, and it is anticipated that the present compounds also will modify the fat distribution. Feeding moderate doses of TTA to animals like rats, mice, rabbits and dogs decreased both plasma cholesterol and triacylglycerol levels within days of treatment. We have also shown the same effect for TSA, and compounds of the present invention with Sulphur substituted in positions 5 or 7 have been shown to increase the .beta.-oxidation and it is thus anticipated that also these fatty acid analogous will lower the plasma levels of triglycerides and cholesterol. TTA and TSA are far more potent in this respect than polyunsaturated fatty acids like EPA. As mentioned above, an important mechanism of action of 3-thia fatty acids is a significant increased mitochondrial fatty acid oxidation reducing the availability of fatty acids for esterification. The synthesis of triacylglycerol and cholesterol is reduced and the secretion of VLDL from the liver is decreased (10). This has the effect of reducing the production of LDL. All these effects seem to be at least partly mediated by peroxisome proliferator activated receptors (PPAR), ubiquitous transcription factors involved in the regulation of lipid metabolism. We have shown that TTA is a potent ligand of PPAR.alpha., a transcription factor regulating the catabolism of fatty acids and eicosanoids, and a less potent ligand of PPAR.gamma., which is involved in the regulation of adipocyte differentiation. Obesity is a common feature of non insulin dependent diabetes mellitus (NIDDM) and a risk factor for its development. NIDDM is often linked to hypertension, dyslipidemia, elevated levels of plasma free fatty acids and an increased risk of cardiovascular disease. NIDDM patients are characterised by resistance to insulin action on glucose uptake in peripheral tissues and dysregulated insulin secretion. We have shown that TTA decrease hyperinsulinemia and markedly improved insulin action on glucose utilisation. TTA did also prevent diet-induced insulin resistance. In contrast to the prior known antidiabetic glitazones TTA did not increase body weight gain. These effects may at least partly be explained by increased influx of fatty acids and enhanced fatty acid oxidation in the liver. The data thus suggest a role for TTA in both lipid and glucose homeostasis in vivo. As clearly shown in the experimental section the compounds of the present invention inhibit an increase in the body weight and adipose tissue mass of animals given either a high fat or a high sucrose diet. This make the compounds of the present invention very suitable as pharmaceutical and/or nutritional agents for the treatment of obesity, i.e. the compounds can be used as a slimming agent to provide a body weight or adipose tissue weigh reduction. Further the compounds of the present invention can be used as an anti-diabetic drug by reducing the concentration of glucose in the blood. We have also shown that the compounds of the present invention reduce the plasma concentration of insulin in hyperinsulineamic animals. For animals which possesses a reduces sensitivity to insulin, the compounds of the present invention have been shown to strengthen the effect of endogenous insulin. The term < As indicated above the compounds of the present invention have been shown to provide a positive effect on all the conditions mentioned above, i.e. by regulating both the glucose and lipid homeostasis, and thus it is anticipated that the compounds of the present invention will be suitable agents for the regulation of the above defined metabolic disease (sometimes called syndrome X). DETAILED DESCRIPTION OF THE INVENTION The present invention discloses that modified fatty a The present invention discloses that modified fatty acid analogous at non-cytotoxic concentrations can be used for the treatment and/or prevention of obesity, hypertension and fatty liver. The present invention relates to the use of fatty acid analogues of the general formula (I): CH.sub.3 --[CH.sub.2 ].sub.m --[x.sub.i --CH.sub.2 ].sub.n --COOR wherein n is an integer from 1 to 12, and wherein m is an integer from 0 to 23, and wherein i is an odd number which indicates the position relative to COOR, and wherein X.sub.i independent of each other are selected from the group comprising O, S, SO, SO.sub.2, Se and CH.sub.2, and wherein R represents hydrogen or C.sub.1 -C.sub.4 alkyl, with the proviso that at least one of the X.sub.i is not CH.sub.2, or a salt, prodrug and complex thereof, for the preparation of a pharmaceutical composition for the treatment and/or prevention of diabetes. In particular, the invention relates to the use of a compound of the general formula I, wherein the diabetes is type I diabetes. A preferred embodiment of the invention relates to the use of a compound of the general formula I, wherein the diabetes is type II diabetes. Still further embodiments relates types of diabetes selected from the group comprising secondary diabetes such as pancreatic, extrapancreatic/endocrine or drug-induced diabetes, or exceptional forms of diabetes such as lipoatrophic, myatonic or a diabetes caused by disturbance of insulin receptors. One embodiment of the invention is the use of a compound of formula I wherein m.gtoreq.13. A presently preferred embodiment of the invention comprises the formula I, wherein X.sub.i=3 is selected from the group consisting of O, S, SO, SO.sub.2 and Se, and wherein X.sub.i=5-25 is CH.sub.2. Tetradecylthioacetic acid (TTA) and Tetradecylselenoacetic acid (TSA), i. e. X.sub.i=3 is Sulphur and Selenium, respectively is presently preferred compounds. Still a further aspect of the invention relates to the use a compound of the formula I for the preparation of a pharmaceutical composition for the treatment and/or prevention of the multi metabolic syndrome termed < A further aspect of the invention relates to a method for the treatment or prevention of a diabetic condition, said method comprising the step of administering to an animal in need thereof an effective amount of fatty acid analogues of the general formula (I): CH.sub.3 --[CH.sub.2 ].sub.m --[x.sub.i --CH.sub.2 ].sub.n --COOR wherein n is an integer from 1 to 12, and wherein m is an integer from 0 to 23, and wherein i is an odd number which indicates the position relative to COOR, and wherein X.sub.i independent of each other are selected from the group comprising O, S, SO, SO.sub.2, Se and CH.sub.2, and wherein R represents hydrogen or C.sub.1 -C.sub.4 alkyl, with the proviso that at least one of the X.sub.i is not CH.sub.2, or a salt, prodrug or complex thereof. In accordance with the method indicated above, preferred embodiments are as follows: said animal is a human. said animal is an agricultural animal, such as gallinaceous birds, bovine, ovine, caprine or porcine mammals. said animal is a domestic or pet animal, such as dog or cat. The treatment involves administering to a patient in need of such treatment a therapeutically effective concentration which is maintained substantially continuously in the blood of the animal for the duration of the period of its administration. Further, the invention relates to a pharmaceutical composition for the prevention and/or treatment of a diabetic condition. Preferably, the pharmaceutical composition comprises in admixture with the fatty acid analogues a pharmaceutically acceptable carrier or excipient. Further the invention relates to methods for treatment and/or prevention of hyperglycaemia, hyperinsulinemia or reduced sensitivity to insulin, said method comprising the step of administering to an animal in need thereof an effective amount of fatty acid analogues of the general formula (I). The invention also relates to a nutritional composition comprising an amount of fatty acid analogues of the general formula (I): effective to reduce, or to prevent an increase in the concentration of glucose in the blood of a human or non-human animal. The invention also relates to novel fatty acid analogous of the formula I CH.sub.3 --[CH.sub.2 ].sub.m --[x.sub.i --CH.sub.2 ].sub.n --COOR wherein n is an integer from 1 to 12, and wherein m is an integer from 0 to 23, and wherein i is an odd number which indicates the position relative to COOR, and wherein X.sub.i independent of each other are selected from the group comprising O, S, SO, SO.sub.2, Se and CH.sub.2, and wherein R represents hydrogen or C.sub.1 -C.sub.4 alkyl, with the proviso that at least one of the X.sub.i is not CH.sub.2, or a salt, prodrug or complex thereof. FIGURE LEGENDS FIG. 1 shows the effect of TTA on weight gain for rats given a high fat diet. FIG. 2 shows the effect of TTA on weight gain for rats given a high sucrose diet. FIG. 3 shows that TTA treatment prevents high fat diet induced hyperinsulinemia. FIG. 4 shows that TTA treatment prevents high fat diet induced insulin resistance. FIG. 5 shows that TTA treatment reduces blood insulin and glucose concentrations in 5 week old Zucker (fa/fa) rats. FIG. 6 shows that TTA treatment reduces blood insulin and glucose concentrations in 4 month old Zucker (fa/fa) rats (FIG. 5B. FIG. 7 shows that TTA treatment decreases the plasma insulin response to glucose. FIG. 8 shows that TTA increases the mitochondrial .beta.-oxidation. ADMINISTRATION OF THE COMPOUNDS OF THE PRESENT INVENTION As a pharmaceutical medicament the compounds of the present invention may be administered directly to the animal by any suitable technique, including parenterally, intranasally, orally, or by absorption through the skin. They can be administered locally or systemically. The specific route of administration of each agent will depend, e.g., on the medical history of the animal. Examples of parenteral administration include subcutaneous, intramuscular, intravenous, intraarterial, and intraperitoneal administration As a general proposition, the total pharmaceutically effective amount of each of the compounds administered parenterally per dose will preferably be in the range of about 5 mg/kg/day to 1000 mg/kg/day of patient body weight, although, as noted above, this will be subject to a great deal of therapeutic discretion. For TTA it is expected that a dose of 100-500 mg/kg/day is preferable, and for TSA the dosage could probably in the range of from 10 to 100 mg/kg/day. If given continuously, the compounds of the present invention are each typically administered by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The key factor in selecting an appropriate dose is the result obtained, as measured by decreases in total body weight or ratio of fat to lean mass, or by other criteria for measuring control or prevention of obesity or prevention of obesity-related conditions, as are deemed appropriate by the practitioner. For parenteral administration, in one embodiment, the compounds of the present invention are formulated generally by mixing each at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. Generally, the formulations are prepared by contacting the compounds of the present invention each uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier may suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or non-ionic surfactants such as polysorbates, poloxamers, or PEG. 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, ampoules and the like, in conventional dosage forms, such as dry ampulles, and as suppositories and the like. The treatment with the present compounds may occur without, or may be imposed with, a dietary restriction such as a limit in daily food or calorie intake, as is desired for the individual patient. In addition, the compounds of the present invention are appropriately administered in combination with other treatments for combatting or preventing obesity. The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. EXPERIMENTAL SECTION METHODS Obese Zucker (fa/fa) rats. The obese Zucker (fa/fa) rats used in this study were bred at the U 465 INSERM animal facility from pairs originally provided by the Harriet G. Bird Laboratory (Stow, Mass., USA). Unless otherwise stated, the animals were maintained under a constant light-dark cycle (light from 7:00 a.m. to 7:00 p.m.) at 21.+-.1.degree. C. and were given free access to food and water. Three rats were housed per cage. Weight gains were recorded daily. Wistar Rats Male Wistar Charles River rats weighing 280-358 grams were purchased from AnLab Ltd. (Prague, Czech Republic) and housed in wire-mesh cages in a temperature (22.+-.1.degree. C.) and light-controlled (light from 7:00 a.m. to 7:00 p.m.) room. They were given free access to chow and water. Three rats were housed per cage. Weight gain and food intake were recorded daily. Diets (given in weight %) Used in the Feeding Experiments Standard Chow Diet: Rats were fed a Standard Laboratory Rat Chow ST1 from Velaz, Prague, Czech Republic. High Sucrose Diet (HS) 50.3% sucrose, 4.8% gelatin, 3.2% hay, 2.3% vitamins and minerals, 8.7% yeast, 8.7% dried milk, 12.3% casein, 9% beef tallow, 1% sunflower oil. HS+TTA: Same as HS+0.3% TTA dissolved in the beef tallow. HS+Fish Oil (FO): Beef tallow and sunflower oil is replaced by 10% Triomar. Triomar is from Pronova Biocare, Norway and contains 33.4% EPA, 3.1% DPA and 20.2% DHA. High Fat (HF): 1.9% gelatin, 5.7% wheat bran, 7.7% vitamins and minerals, 25.4% corn starch, 25.7% casein, 26.8% beef tallow and 7.1% sunflower oil. HF+TTA: Same+0.4% TTA dissolved in the beef tallow. HF+FO: 10% beef tallow is replaced by 10% Triomar. Intravenous Glucose Tolerance Tests Male Zucker (fa/fa) rats (5 weeks old) were anaesthetised after a 5-hours fast, by intraperitoneal injection of sodium pentobarbital (50 mg/kg). The rats were injected with glucose (0.55 g/kg) in the saphenous vein and blood samples were collected from the tail vein in heparinized tubes at time 0, 5, 10, 15, 20 and 30 minutes after the glucose load. Samples were kept on ice, centrifuged and plasma was stored at -20.degree. C. until analysis. Hyperinsulinemic Euglycemic Clamp. After 21 days on their respective diets (see above), the rats were anaesthetised by injection of xylazine hydrochloride (Rometar SPOFA, Prague, Czech Republic; 10 mg/ml) and ketamine hydrochloride (Narkamon SPOFA, Prague, Czech republic; 75 mg/ml), and fitted with chronic carotid artery and jugular vein cannulas as described by Koopmans et al. (Koopmans, S. J., et al., Biochim Biophys Acta, 1115, 2130-2138 1992.). The cannulated rats were allowed to recover for two days after surgery before the clamping studies which were carried out according to Kraegen et al. (Kraegen, E. W., et al., Am J Physiol, 248, E353-E362 1983.). Thus, on the third day after surgery, unrestrained conscious rats were given a continuous infusion of porcine insulin (Actrapid, Novo Nordisk, Denmark) at a dose of 6.4 mU per kg per min to achieve plasma insulin levels in the upper physiological range. The arterial blood glucose concentration was clamped at the basal fasting level, by variable infusion of a 30% w/v glucose solution (Leciva, Prague, Czech Republic). Blood samples for determination of plasma glucose and insulin concentrations were obtained every 15 minutes from the start of the glucose infusion. After 90 minutes, the rats were disconnected from the infusions and immediately decapitated, blood was collected for plasma separation, liver and epididymal adipose tissue pads were dissected out and weighed. Measurement of Plasma Parameters Glucose (GLU, Boehringer Mannheim, Germany), free fatty acids (NEFA, C ACS-ACOD kit; Wako Chemicals, Dalton, USA) and b-hydroxybutyrate (310-A kit; Sigma Diagnostics Inc., St. Louis, USA) concentrations were measured using enzymatic methods. Insulin concentrations were determined with radioimmunoassay by (CIS bio International, Gif sur Yvette, France) using rat insulin as standard in the Zucker rats. In the Wistar Charles River rats, plasma glucose concentrations were measured with the aid of Beckman Glucose Analyzer (Fullerton, Calif., USA). Plasma insulin levels were measured using a RIA kit from Linco Research Inc. (St. Charles, Mo., USA). Phospholipids were measured by the enzymatic method of bioMerieux, Marcy-l Etoile, France, Triacylglycerol by the Technicon Method no. SA4-0324L90, USA and Cholesterol by the Technicon Method no. SA4-0305L90, USA. Preparation of Post-nuclear and Mitochondrial Fractions and Measurement of Enzyme Activities Freshly isolated livers from individual old Zucker rats, were homogenised in ice-cold sucrose buffer (0.25 M sucrose, 10 mM HEPES (pH 7.4) and 2 mM EDTA). Post-nuclear and mitochondrial fractions were prepared using preparative differential centrifugation according to DeDuve et al. (De Duve, C., et al., Biochem. J., 60, 604-617 1955.) Modifications, purity and yield were as described earlier (Garras, A., et al., Biochim. Biophys. Acta, 1255, 154-160 1995.). Acid soluble products were measured in post-nuclear and mitochondrial enriched fractions, using [1-.sup.14 C]-palmitoyl-CoA and [1-.sup.14 C]-palmitoyl-L-carnitine (Radiochemical Centre, Amersham, England) as substrates as described earlier (Willumsen, N., et al., J. Lipid Res., 34, 13-22 1993. Carnitine palmitoyltransferase-I and -II activities were measured in the post-nuclear and mitochondrial fractions essentially as described by Bremer (Bremer, J., Biochim. Biophys. Acta, 665, 628-631 1981.) and 3-hydroxy-3-methylglutharyl-CoA synthase was measured according to Clinkenbeard et al. (Clinkenbeard, K. D., et al., J. Biol. Chem, 250, 3108-3116 1975.) in the mitochondrial fractions. RNA Analysis RNA extraction (Chomczynski, P., et al., Anal. Biochem., 162, 156-159 1987.), Northern blot analysis and slot blotting of RNA onto nylon filters, and hybridisation to immobilised RNA were performed as earlier described (Vaagenes, H., et al., Biochem. Pharmacol., 56, 1571-1582 1998.). The following cDNA fragments were used as probes: CPT-I, (Esser, V. et al., J. Biol. Chem., 268,5817-5822 1993), CPT-II (Woeltje, K. F., et al., J. Biol. Chem., 265, 10720-10725 1990.), 3-hydroxy-3-methylglutharyl-CoA synthase (Ayte, J., et al., Proc. Natl. Acad. Sci. USA., 87, 3874-3878 1990.), and hormone sensitive lipase (Holm, C., et al., Biochim. Biophys. Acta, 1006, 193-197 1989.). The relative levels of RNA expression were estimated as the amounts of radioactive probe hybridised to the respective levels of 28S rRNA. |
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