Tetrahydroisoquinolines as MCH-R1 antagonists.

A series of potent and selective inhibitors of h-MCH-R1 has been developed based on the piperidine glycineamide compounds I and II. These structurally more rigid tetrahydroisoquinolines (III and IV) showed better pharmacokinetics. The highly potent compounds 12d and 12g displayed excellent rat pk.

Ezetimibe selectively inhibits intestinal cholesterol absorption in rodents in the presence and absence of exocrine pancreatic function.

1. Ezetimibe potently inhibits the transport of cholesterol across the intestinal wall, thereby reducing plasma cholesterol in preclinical animal models of hypercholesterolemia. The effect of ezetimibe on known absorptive processes was determined in the present studies. 2. Experiments were conducted in the hamster and/or rat to determine whether ezetimibe would affect the absorption of molecules other than free cholesterol, namely cholesteryl ester, triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid. In addition, to determine whether exocrine pancreatic function is involved in the mechanism of action of ezetimibe, a biliary anastomosis model, which eliminates exocrine pancreatic function from the intestine while maintaining bile flow, was established in the rat. 3. Ezetimibe reduced plasma cholesterol and hepatic cholesterol accumulation in cholesterol-fed hamsters with an ED(50) of 0.04 mg kg(-1). Utilizing cholesteryl esters labelled on either the cholesterol or the fatty acid moiety, we demonstrated that ezetimibe did not affect cholesteryl ester hydrolysis and the absorption of fatty acid thus generated in both hamsters and rats. The free cholesterol from this hydrolysis, however, was not absorbed (92 - 96% inhibition) in the presence of ezetimibe. Eliminating pancreatic function in rats abolished hydrolysis of cholesteryl esters, but did not affect the ability of ezetimibe to block absorption of free cholesterol (-94%). Ezetimibe did not affect the absorption of triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid in rats. 4. Ezetimibe is a potent inhibitor of intestinal free cholesterol absorption that does not require exocrine pancreatic function for activity. Ezetimibe does not affect the absorption of triglyceride as a pancreatic lipase inhibitor (Orlistat) would, nor does it affect the absorption of vitamin A, D or taurocholate, as a bile acid sequestrant (cholestyramine) would.

Ezetimibe, a potent cholesterol absorption inhibitor, normalizes combined dyslipidemia in obese hyperinsulinemic hamsters.

Ezetimibe potently and selectively inhibits cholesterol absorption in the intestine, thereby reducing plasma cholesterol in preclinical models of hypercholesterolemia. Clinical trials have demonstrated that ezetimibe lowers LDL cholesterol and raises HDL cholesterol in humans. The effect of ezetimibe on other dyslipidemias, particularly hypertriglyceridemia, is not yet known. In the present studies, we assessed the effect of ezetimibe on combined hypercholesterolemia and hypertriglyceridemia in obese hyperinsulinemic hamsters. Hamsters were fed chow, chow with cholesterol (0.12%), or the same cholesterol diet containing different dietary triglycerides (15%) in the absence or presence of 1 mg/kg ezetimibe (in diet) for up to 84 days. Body weight, serum insulin, leptin, glucose, cholesterol, and triglyceride levels were analyzed. Cholesterol and triglyceride levels were also determined in VLDL+IDL, LDL, and HDL. Hamsters maintained on high-fat diets became obese, hyperinsulinemic, hyperleptinemic, hypercholesterolemic, and hypertriglyceridemic. Ezetimibe did not affect body weight, insulin, or leptin, but ablated the combined hypercholesterolemia and hypertriglyceridemia induced by high-fat diets. Ezetimibe normalized VLDL+IDL cholesterol and triglyceride and significantly decreased LDL cholesterol to below chow-fed levels. The ratio of HDL to LDL cholesterol increased significantly with the addition of ezetimibe. Ezetimibe completely eliminated the accumulation of cholesteryl ester and free cholesterol in liver that was induced under the various dietary conditions in the absence of drug. In conclusion, ezetimibe is very effective in correcting the combined dyslipidemia in diet-induced obese hyperinsulinemic hamsters and may be an effective therapy for ameliorating combined dyslipidemia in obese insulin-resistant and/or type 2 diabetic humans.

The cholesterol absorption inhibitor, ezetimibe, decreases diet-induced hypercholesterolemia in monkeys.

Ezetimibe (1-(4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-hydroxypropyl]-(4S)-(4-hydroxyphenyl)-2-azetidinone) potently and selectively inhibits the intestinal absorption of cholesterol, thereby reducing plasma cholesterol in preclinical models of hypercholesterolemia. In rhesus monkeys fed a diet containing 375 mg/day of cholesterol, 0.1 mg/kg of ezetimibe completely prevented the doubling of plasma cholesterol normally induced under these dietary conditions (ED(50)=0.0005 mg/kg). Low-density lipoprotein cholesterol (LDL) was dose-dependently reduced, while high-density lipoprotein cholesterol (HDL) and plasma triglyceride were unchanged. A single dose of an ezetimibe analog administered to cynomolgus monkeys fed a single cholesterol-containing meal caused a significant reduction (-69%) of cholesterol in chylomicrons during the postprandial phase without affecting triglyceride content. In rhesus monkeys, apolipoprotein (apo) B(48) concentrations in chylomicrons did not differ between control and the ezetimibe analog, but apo B(100) was significantly reduced in LDL (-41%). These data indicate that these cholesterol absorption inhibitors reduce cholesterol content in chylomicrons, which indirectly leads to a decrease in LDL cholesterol and particle number.

Comparison of the activity and disposition of the novel cholesterol absorption inhibitor, SCH58235, and its glucuronide, SCH60663.

Previous studies described the metabolism-based discovery of a potent, selective inhibitor of intestinal absorption of cholesterol, SCH58235 (Ezetimibe). Here we demonstrate that the phenolic glucuronide (SCH60663) of SCH58235, was more potent at inhibiting cholesterol absorption in rats than SCH58235, when administered by the intraduodenal route. To understand the increased potency of the glucuronide, the metabolism and distribution of SCH58235 and SCH60663 were studied in bile duct-cannulated rats. One minute after intraduodenal delivery of SCH58235, significant levels of compound were detected in portal plasma; >95% was glucuronidated, indicating that the intestine was metabolizing SCH58235 to its glucuronide. When intraduodenally delivered as SCH58235, the compound was glucuronidated, moved through the intestinal wall, into portal plasma, through the liver, and into bile. However, when delivered as SCH60663, >95% of the compound remained in the intestinal lumen and wall, which may explain its increased potency. Significant inhibition of cholesterol absorption and glucuronidation of SCH58235 occurred when SCH58235 was intravenously injected into bile duct-cannulated rats. Autoradiographic analysis demonstrated that drug related material was located throughout the intestinal villi, but concentrated in the villus tip. These data indicate that (a) SCH58235 is rapidly metabolized in the intestine to its glucuronide; (b) once glucuronidated, the dose is excreted in the bile, thereby delivering drug related material back to the site of action and (c) the glucuronide is more potent than the parent possibly because it localizes to the intestine. Taken together, these data may explain the potency of SCH58235 in the rat (ID(50) = 0.0015 mg kg(-1)) and rhesus monkey (ID(50) = 0.0005 mg kg(-1)).

Cholesteryl hydroperoxyoctadecadienoate from oxidized low density lipoprotein inactivates platelet-derived growth factor.

Both oxidized low density lipoprotein (ox-LDL) and platelet-derived growth factor (PDGF) have been implicated in the genesis of various inflammatory responses, including atherosclerosis. We demonstrate here a novel interaction between specific oxidized lipids derived from ox-LDL and PDGF. The lipid moieties of ox-LDL caused concentration-dependent inactivation of PDGF as measured by loss of its mitogenic activity and its binding to high affinity receptors. Reverse-phase and normal-phase HPLC were used to purify the inactivating component in the lipid mixture. By fast atom bombardment mass spectrometry and infrared spectroscopy, we identified the inactivating lipids as the 9- and 13-hydroperoxy derivatives of cholesteryl linoleate, cholesteryl hydroperoxyoctadecadienoate. When a series of cholesteryl esters were subjected to oxidizing conditions, only those containing two or more double bonds caused inactivation of PDGF; the extent of inactivation increased with increased levels of oxidation. Exposing PDGF to cumene hydroperoxide, t-butyl hydroperoxide, or hydrogen peroxide did not affect the activity of the mitogen. The oxidized lipid had no effect on the mitogenic activity of epidermal growth factor but did abolish the mitogenic activity of basic fibroblast growth factor and the antiproliferative activity of transforming growth factor beta1. The inactivation of PDGF and other cytokines by lipid hydroperoxides may occur in such processes as vascular disease, inflammation, and wound healing.

Effect of chronic central administration of glucagon-like peptide-1 (7-36) amide on food consumption and body weight in normal and obese rats.

Glucagon-like peptide (7-36) amide (GLP-1) acutely inhibits food and water consumption in rats after intracerebroventricular (icv) administration. To assess the potential for desensitization of these effects, we investigated the effects of chronic icv administration of GLP-1 on food consumption and body weight in Sprague-Dawley (SD) rats and Zucker (fa/fa) obese rats. In vitro functional densensitization of the GLP-1 receptor was not observed after overnight exposure of Rin m5F insulinoma cells to GLP-1 at concentrations up to 10 nM. Administration of GLP-1 to SD rats (30 microg icv twice a day for 6 days) resulted in significant reductions in 24-hour food consumption each day (25 +/- 1%). Continuous icv infusion of GLP-1 for 7 and 14 days significantly inhibited cumulative food consumption and reduced body weight in SD rats. In the genetically obese Zucker rat, chronic dosing with GLP-1 (30 microg icv) once a day for 6 days caused significant reductions in food consumption each day and a reduction in body weight. These results indicate that the GLP-1 pathways in the central nervous system controlling food consumption do not desensitize after chronic exposure to GLP-1 and suggest that agonists of the central GLP-1 receptor may be effective agents for the treatment of obesity.

In vivo metabolism-based discovery of a potent cholesterol absorption inhibitor, SCH58235, in the rat and rhesus monkey through the identification of the active metabolites of SCH48461.

SCH48461 is a selective and highly potent inhibitor of cholesterol absorption. In rats, SCH48461 is rapidly and completely metabolized in the first pass through the body. To compare the activity of the metabolites of SCH48461 with SCH48461 itself, an intestinally cannulated, bile duct-cannulated rat model for cholesterol absorption was developed. SCH48461 inhibited the absorption of cholesterol by 70%, whereas bile containing the metabolites of SCH48461 (henceforth, "metabolite bile") inhibited the absorption by greater than 95%. Very little of the recovered radioactive dose of SCH48461 was located in the intestinal lumen (7%) or wall (4%), whereas 85% appeared in bile. However, in rats treated with metabolite bile, 62% of the dose remained in the lumen, 13% was associated with the wall and only 24% reappeared in bile, which suggests that the activity of the metabolite bile may be related to its higher retention in the intestinal wall. Rats treated with metabolite bile had 64% and 84% less drug-related radioactivity in their plasma and livers, respectively, compared with animals treated with SCH48461, which indicates that the metabolites are systemically less available than SCH48461. The metabolites in bile were separated by high-performance liquid chromatography; the most active fraction in the bile duct-cannulated rat model was identified by mass spectrometry as the glucuronide of the C4-phenol of SCH48461. The other fractions had moderate or no activity. Through the identification of the most active biliary metabolites of SCH48461 in the rat, we have discovered SCH58235, a novel cholesterol absorption inhibitor which is 400 times more potent than SCH48461 in the cholesterol-fed rhesus monkey.

Leptin increases energy expenditure and selectively promotes fat metabolism in ob/ob mice.

Obesity occurs whenever energy intake exceeds energy expenditure. The ob gene product leptin is a potent anorectic agent when administered to ob/ob mice, but its effects on energy expenditure have not been investigated in detail. The present study was designed to analyze the acute metabolic effects of leptin in vivo. Analysis of oxygen consumption in ob/ob mice demonstrated a reduction in energy expenditure compared with lean controls; this reduction showed a diurnal fluctuation and was most evident during the light cycle. A single intraperitoneal dose of leptin increased oxygen consumption during the light cycle in ob/ob mice, ablating the circadian fluctuation in this parameter. In addition, leptin had a profound effect on fuel selection: the respiratory quotient was markedly reduced, indicating a reduction in carbohydrate oxidation and an increase in fat oxidation. These acute effects of leptin on metabolic parameters are consistent with the selective loss of body fat observed on chronic leptin treatment and suggest that increased energy utilization plays an important role in the anti-obese actions of leptin.

Diet-induced obese mice develop peripheral, but not central, resistance to leptin.

Leptin administration reduces obesity in leptin-deficient ob/ob mice; its effects in obese humans, who have high circulating leptin levels, remain to be determined. This longitudinal study was designed to determine whether diet-induced obesity in mice produces resistance to peripheral and/or central leptin treatment. Obesity was induced in two strains of mice by exposure to a 45% fat diet. Serum leptin increased in proportion to body weight (P < 0.00001). Whereas C57BL/6 mice initially responded to peripherally administered leptin with a marked decrease in food intake, leptin resistance developed after 16 d on high fat diet; mice on 10% fat diet retained leptin sensitivity. In AKR mice, peripheral leptin significantly decreased food intake in both 10 and 45% fat-fed mice after 16 d of dietary treatment. However, after 56 d, both groups became resistant to peripherally administered leptin. Central administration of leptin to peripherally leptin-resistant AKR mice on 45% fat diet resulted in a robust response to leptin, with a dose-dependent decrease in food intake (P < 0.00001) and body weight (P < 0.0001) after a single intracerebroventricular infusion. These data demonstrate that, in a diet-induced obesity model, mice exhibit resistance to peripherally administered leptin, while retaining sensitivity to centrally administered leptin.

The leptin haemopoietic cytokine fold is stabilized by an intrachain disulfide bond.

Structure prediction algorithms have tagged leptin as the newest member of the haemopoietic cytokine family, a diverse class of secreted hormone-like factors with pleiotropic effects in immunity and haemopoietic development. While haemopoietic cytokines typically lack sequence similarity, they conserve a distinctive three-dimensional fold, a four-alpha-helix bundle structure that is recognized by the cognate family of haemopoietic cellular receptors. We have constructed a detailed molecular model of the human leptin helical fold that places the two cysteine residues of the leptin chain, Cys96 and Cys146, in close spatial proximity to each other. In this report, we present evidence that these cysteines are involved in an intrachain disulfide bridge that is critical for the structural integrity and stability of leptin. A leptin variant that is unable to form the disulfide link shows a reduced biological response when administered to leptin-deficient, ob/ob mice.

The relationship of tissue localization, distribution and turnover to feeding after intraperitoneal 125I-leptin administration to ob/ob and db/db mice.

Brain and whole body localization and distribution of 125I-leptin was determined after intraperitoneal administration to ob/ob and db/db mice, and was compared to inhibition of food intake. Food intake was not significantly inhibited at3 hours post-injection, but was decreased significantly at 6 h (p < 0.0007) and 24 h (p < 0.02) in ob/ob mice, times at which > 97 % of the radioactive dose was found in the urine. The highest concentrations of 125I-leptin at all time-points were found in the serum, liver and kidneys. These findings were verified by whole body autoradiography. Virtually no 125I-leptin was found in the CNS at later timepoints in either ob/ob or db/db mice. Coronal sectioning of entire brains from ob/ob and db/db mice revealed 125I radioactivity localized to the choroid plexus and in the ventricular space, but not in other CNS regions. No differences in localization, accumulation, or clearance of 125I-leptin in ob/ob vs. db/db mice were found in any of the tissues studied. The present studies demonstrate that the inhibitory effect of leptin on food intake in the ob/ob mouse persists for up to 24 hours after a single dose, despite the complete degradation and elimination of the labeled leptin during the first several hours after injection.

Purification of milligram quantities of human leptin from recombinant E. coli.

Leptin, the product of the obese (ob) gene, is a 16 kilodalton protein secreted from adipose tissue. Restoration of leptin to obese ob/ob mice leads to normalization of body weight. The effect of leptin in larger animals has not been explored, in part because of limited supplies of leptin. To date, the potency and yield of recombinant leptin purified from a variety of eukaryotic sources or from E. coli has been highly variable. While purification of leptin from E. coli inclusion bodies has afforded the greatest yield of protein, its potency is at least an order of magnitude lower than that of leptin secreted from E. coli or eukaryotic cells. The mechanistic basis of this difference in potency is not clear at present. The ability to purify significant quantities of highly active leptin will be crucial for the evaluation of leptin structure, as well as its function in additional animal models of obesity. We now report a facile protocol for the preparation of recombinant leptin using an E. coli expression system. 75-85 milligrams of leptin with a purity of greater than 97 % was prepared from a liter of recombinant E. coil. The procedure can be performed in less than 48 h and requires no chromatography. Intraperitoneal injection of 0.1 mg/kg renatured leptin into ob/ob mice results in a significant reduction in food consumption. The potency of this material is similar to the most potent recombinant leptin described to date. The ability to rapidly prepare large quantities of high specific activity material will hasten the definition of leptin's role in non-rodent models of obesity.

Mechanisms of hypertriglyceridemia in the coconut oil/cholesterol-fed rabbit. Increased secretion and decreased catabolism of very low density lipoprotein.

Rabbits fed a 14% coconut oil/0.5% cholesterol (CNO/Chol) diet develop mild to severe hypertriglyceridemia compared with rabbits fed a 14% olive oil/0.5% cholesterol (OO/Chol) diet. Lipids and apolipoprotein (apo) B were significantly higher in the very low density lipoprotein (VLDL) and intermediate density lipoprotein fractions from CNO/Chol than from OO/Chol rabbits. Yet, the particle diameters of these lipoproteins were similar in both diet groups, indicating that CNO/Chol rabbits had a much larger number of VLDL and intermediate density lipoprotein particles in plasma. Although the composition of CNO/Chol VLDL differed from that of OO/Chol VLDL, the rates of triglyceride hydrolysis of CNO/Chol VLDL and OO/Chol VLDL by postheparin lipoprotein lipase in vitro were the same, suggesting that VLDLs from the two diet groups were equally good substrates for lipoprotein lipase. To determine the mechanisms of hypertriglyceridemia in the CNO/Chol rabbit, triglyceride and apo B of CNO/Chol VLDL and OO/Chol VLDL were labeled with tritium-containing triolein and iodine-131 and injected intravenously into CNO/Chol and OO/Chol rabbits. The fractional clearance rate for triglyceride in OO/Chol rabbits was twice that of CNO/Chol rabbits, which parallels the previously observed differences in postheparin lipoprotein lipase activity. Although the average fractional removal of apo B did not differ between diet groups, there was a significant inverse relation between plasma cholesterol and apo B fractional clearance rate. We conclude that the hypertriglyceridemia and the enhanced hypercholesterolemia in the CNO/Chol rabbit results primarily from increased hepatic secretion of VLDL and a modest decrease in VLDL triglyceride clearance capacity.

Postprandial lipemia and lipoprotein lipase in the rabbit are modified by olive and coconut oil.

Feeding a 14% coconut oil/0.5% cholesterol (CNO/chol) diet to rabbits resulted in plasma triglycerides that were, on average, 15 times higher than basal levels. Plasma triglycerides in rabbits fed a 14% olive oil/0.5% cholesterol (OO/chol) diet were significantly below baseline levels. Differences in postprandial triglyceride response and postheparin plasma lipoprotein lipase activity (LPL) in various feeding conditions were studied to determine the mechanism of the hypertriglyceridemia. Postprandial triglyceride responses after the first high fat/cholesterol meal were more prolonged in CNO/chol rabbits than in OO/chol rabbits; postprandial triglyceride responses after chronic CNO/chol feeding were significantly greater compared to OO/chol rabbits. When long-term CNO/chol rabbits were given one OO/chol or corn oil/chol meal, postprandial triglyceride peaks were greatly diminished, suggesting that these unsaturated fat meals may alter triglyceride clearance capacity. LPL activity was 400% higher than basal levels in chronically fed OO/chol rabbits but changed very little in chronically fed CNO/chol rabbits. Twenty-four hours after a single OO/chol meal was fed to chow-fed rabbits, LPL doubled; one CNO/chol meal was associated with only a 40% increase. Feeding a single OO/chol or corn oil/chol meal to chronically fed CNO/chol rabbits resulted in a 30% to 50% increase in LPL by 24 hours. Thus, the hypertriglyceridemia in CNO/chol rabbits may result in part from a decreased clearance capacity due to a lack of increase in LPL activity, while increased LPL may be partially responsible for the hypotriglyceridemia observed in OO/chol feeding. Aortic cholesterol was substantially higher in CNO/chol rabbits. Triglyceride was approximately eight times greater in livers from CNO/chol-fed rabbits than in those fed OO/chol, but liver cholesterol was only about one-third as much as that in OO/chol rabbits.

Evidence for an inverse relation between plasma triglyceride and aortic cholesterol in the coconut oil/cholesterol-fed rabbit.

Rabbits fed a commercial chow diet containing 0.5% cholesterol and 14% coconut oil developed more severe hyperlipidemia and atherosclerosis than rabbits fed the same diet containing olive oil in place of coconut oil. Average plasma cholesterol was twice as high in the coconut oil/cholesterol-fed rabbits than in olive oil/cholesterol-fed rabbits. Final plasma triglycerides, although highly variable, were approx. 20-fold higher than basal plasma triglyceride in coconut oil/cholesterol-fed rabbits; plasma triglyceride in olive oil/cholesterol-fed rabbits remained unchanged throughout the study period. In coconut oil/cholesterol-fed rabbits, a direct relationship between plasma triglyceride and aortic cholesterol was not found. Plasma cholesterol and aortic cholesterol were also not correlated at a statistically significant level (r = 0.26, P greater than 0.25). However, when both plasma cholesterol and triglyceride were simultaneously introduced as predictors of aortic cholesterol, the correlation between these plasma lipids and aortic cholesterol became highly significant (r = 0.64, P less than 0.02). Aortic cholesterol increased in proportion to plasma cholesterol concentrations but appeared to be inversely related to plasma triglyceride levels.