Distinct properties of telmisartan on agonistic activities for peroxisome proliferator-activated receptor γ among clinically used angiotensin II receptor blockers: drug-target interaction analyses.

A proportion of angiotensin II type 1 receptor blockers (ARBs) improves glucose dyshomeostasis and insulin resistance in a clinical setting. Of these ARBs, telmisartan has the unique property of being a partial agonist for peroxisome proliferator-activated receptor γ (PPARγ). However, the detailed mechanism of how telmisartan acts on PPARγ and exerts its insulin-sensitizing effect is poorly understood. In this context, we investigated the agonistic activity of a variety of clinically available ARBs on PPARγ using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) system. Based on physicochemical data, we then reevaluated the metabolically beneficial effects of telmisartan in cultured murine adipocytes. ITC and SPR assays demonstrated that telmisartan exhibited the highest affinity of the ARBs tested. Distribution coefficient and parallel artificial membrane permeability assays were used to assess lipophilicity and cell permeability, for which telmisartan exhibited the highest levels of both. We next examined the effect of each ARB on insulin-mediated glucose metabolism in 3T3-L1 preadipocytes. To investigate the impact on adipogenesis, 3T3-L1 preadipocytes were differentiated with each ARB in addition to standard inducers of differentiation for adipogenesis. Telmisartan dose-dependently facilitated adipogenesis and markedly augmented the mRNA expression of adipocyte fatty acid-binding protein (aP2), accompanied by an increase in the uptake of 2-deoxyglucose and protein expression of glucose transporter 4 (GLUT4). In contrast, other ARBs showed only marginal effects in these experiments. In accordance with its highest affinity of binding for PPARγ as well as the highest cell permeability, telmisartan superbly activates PPARγ among the ARBs tested, thereby providing a fresh avenue for treating hypertensive patients with metabolic derangement.

Unique "delta lock" structure of telmisartan is involved in its strongest binding affinity to angiotensin II type 1 receptor.

Angiotensin II type 1 receptor (AT1 receptor) blockers (ARBs) are one of the most popular anti-hypertensive agents. Control of blood pressure (BP) by ARBs is now a therapeutic target for the organ protection in patients with hypertension. Recent meta-analysis demonstrated the possibility that telmisartan was the strongest ARB for the reduction of BP in patients with essential hypertension. However, which molecular interactions of telmisartan with the AT1 receptor could explain its strongest BP lowering activity remains unclear. To address the issue, we constructed models for the interaction between commonly used ARBs and AT1 receptor and compared the docking model of telmisartan with that of other ARBs. Telmisartan has a unique binding mode to the AT1 receptor due to its distal benzimidazole portion. This unique portion could explain the highest molecular lipophilicity, the greatest volume distribution and the strongest binding affinity of telmisartan to AT1 receptor. Furthermore, telmisartan was found to firmly bind to the AT1 receptor through the unique "delta lock" structure. Our present study suggests that due to its "delta lock" structure, telmisartan may be superior to other ARBs in halting cardiovascular disease in patients with hypertension.

Reduction in hepatic non-esterified fatty acid concentration after long-term treatment with atorvastatin lowers hepatic triglyceride synthesis and its secretion in sucrose-fed rats.

The mechanism by which atorvastatin lowers plasma triglyceride (TG) levels is mainly through a decrease in hepatic TG secretion. However, it is not clear why atorvastatin, which does not inhibit TG synthesis in vitro, decreases hepatic TG secretion without a prospective increase in hepatic TG concentration. For the investigation of the mechanisms that underlie the hypotriglyceridemic effects of atorvastatin, we characterized the effect of either a single or an 11 day administration of atorvastatin in sucrose-induced hypertriglyceridemic rats. Atorvastatin (30 mg/kg p.o.) strongly decreased the rate of both very-low-density lipoprotein (VLDL)-TG and VLDL-apolipoprotein B secretion. The inhibitor also decreased hepatic TG concentration. Hepatic TG synthesis activity was also decreased by atorvastatin, and its activity was correlated with both hepatic and plasma TG concentration. There was also a strong correlation between the hepatic TG synthesis and hepatic non-esterified fatty acid (NEFA) concentration (r(2)=0.815). These effects required chronic administration of the inhibitor and were not observed by acute treatment. Repeated administration of atorvastatin also strongly reduced hepatic acyl-coenzyme A synthase mRNA levels. These results suggest that the reduced hepatic NEFA most likely lowers hepatic TG synthesis and TG secretion in sucrose-fed hypertriglyceridemic rats.

Effects of atorvastatin on glucose metabolism and insulin resistance in KK/Ay mice.

Insulin resistance plays an important role not only in the development and progression of diabetes mellitus but also in the establishment of metabolic syndrome. Improvement of insulin resistance is thus of great importance both in improving glucose metabolism and preventing atherosclerosis. Although HMG-CoA reductase inhibitors appear to favorably affect glucose metabolism, as indicated by the results of a subanalysis in the West of Scotland Coronary Prevention Study (WOSCOPS), their effects on glucose metabolism and insulin resistance have not been thoroughly investigated in animal models. In this study, the effects of atorvastatin on the glucose metabolism and insulin resistance of KK/Ay mice, an animal model of type II diabetes, were investigated. Atorvastatin significantly decreased the non-HDL-cholesterol level in the oral glucose tolerance test, inhibited increase in the 30-min glucose level, decreased plasma insulin levels before and 30 and 60 minutes after glucose loading, and decreased the insulin resistance index, compared with corresponding values in controls, indicating that atorvastatin appeared to improve glucose metabolism by improving insulin resistance. Northern blot analysis revealed decreases in levels of mRNA of sterol regulatory element binding protein-1 (SREBP-1) and glucose-6-phosphatase (G6Pase), and it may play a role in the improvement of glucose metabolism and insulin resistance.

Bixalomer, a novel phosphate binder with a small swelling index, improves hyperphosphatemia in chronic kidney disease rat.

In the present study, we evaluated the in vitro characteristics of bixalomer for phosphate binding and swelling and assessed the urinary phosphorus excretion and plasma phosphorus level-lowering effect of bixalomer. The maximum phosphate binding capacity was 6.49 mmol/g and was maximized at pH 6.09. In rats, consuming a high-phosphorus diet resulted in elevated urinary phosphorus excretion, while consuming a diet of bixalomer (0.3-9%) or sevelamer hydrochloride (sevelamer HCl; 3-9%) mixed with a high-phosphorus diet resulted in a dose-dependent reduction in urinary phosphorus excretion. Rats with adenine sulfate-induced chronic kidney disease (CKD) had plasma phosphorus levels of 14.9-18.8 mg/dl, while CKD rats administered a 3% bixalomer or 3% sevelamer HCl diet for 4 weeks had relatively decreased plasma phosphorus levels (6.86 ± 1.42 or 5.32 ± 0.27 mg/dl, respectively). Bixalomer elevated the lowered blood pH in acidemic CKD rats, while sevelamer HCl administration only exacerbated the acidemia. The swelling index, which represents water adsorption capacity, of bixalomer was measured by subtracting the dry weight from the hydrated wet weight of the polymer. The swelling index of bixalomer was four times lower than that of sevelamer HCl. Bixalomer was found to reduce the plasma phosphorus level in CKD rats by binding phosphate in the small intestine and reducing phosphate absorption. Bixalomer showed favorable characteristics of a smaller swelling index than sevelamer HCl and amelioration of metabolic acidosis. These findings suggest that bixalomer may be useful in treating hyperphosphatemia, with fewer gastrointestinal side effects and amelioration of metabolic acidosis than sevelamer HCl.

Experimental model of escape phenomenon in hamsters and the effectiveness of YM-53601 in the model.

1. The aim of this study was to establish an experimental model of the escape phenomenon, in which plasma cholesterol, initially reduced by a 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor such as pravastatin, increases again on long-term administration. We also evaluated the efficacy of YM-53601 ((E)-2-[2-fluoro-2- (quinuclidin-3-ylidene) ethoxy]-9H-carbazole monohydrochloride), a squalene synthase inhibitor, in this model. 2. Pravastatin inhibited cholesterol biosynthesis in hamster primary hepatocytes (IC(50), 14 nM). After pre-treatment with pravastatin, in contrast, almost no effect on cholesterol biosynthesis was seen. 3. In hamsters fed a high fat diet, 3 mg kg(-1) pravastatin for 9 days decreased plasma non-HDL cholesterol (total cholesterol - high density lipoprotein cholesterol) (P<0.01), but this effect was lost between 17 and 27 days of treatment, accompanied by an increase in HMG-CoA reductase activity. No such increase in plasma non-HDL cholesterol was seen with YM-53601 at 30 mg kg(-1) after 9 (P<0.001), 17 (P<0.01) or 27 (P<0.001) days of treatment. Replacement of pravastatin with YM-53601 caused a decrease in plasma non-HDL cholesterol by 53% (P<0.001) and in HMG-CoA reductase activity. 4. This animal model thus satisfactorily replicates the escape phenomenon observed in humans and may therefore be useful in evaluation of lipid-lowering agents, specifically comparison of HMG-CoA reductase inhibitors. Further, YM-53601 may be useful in the treatment of hypercholesterolemia without induction of the escape phenomenon.

Effect of YM-53601, a novel squalene synthase inhibitor, on the clearance rate of plasma LDL and VLDL in hamsters.

1. To better understand how it decreases plasma cholesterol and triglyceride, we evaluated the effect of YM-53601 ((E-2-[2-fluoro-2-(quinuclidin-3-ylidene) ethoxy]-9H-carbozole monohydrochloride) on the clearance rate of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) in hamsters. 2. Treatment with YM-53601 at 50 mg kg(-1) for 5 days in hamsters fed a normal diet enhanced the disappearance of 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-VLDL and DiI-LDL. This effect on DiI-LDL was lost in the early phase after DiI-methyl(met)-LDL, chemically modified to block LDL receptor binding, was injected in hamsters, but was retained in the late phase. Pre-treatment with protamine sulphate, which inhibits the activity of LPL, also failed to enhance DiI-VLDL clearance rate by YM-53601. 3. Even on single oral administration at 30 mg kg(-1), YM-53601 enhanced the disappearance of the high concentration of plasma triglyceride after injection of intrafat, an emulsion of fat. Plasma triglyceride was significantly decreased as soon as 1 h after single administration of YM-53601 in hamsters fed a normal diet. 4. These results indicate that the decrease in plasma total cholesterol and triglyceride after the treatment with YM-53601 is due to its enhancement of the clearance rate of LDL and VLDL, respectively. Moreover, YM-53601 may be effective in decreasing plasma triglyceride levels early in the course of treatment of hypertriglyceridaemia in humans.

YM-53601, a novel squalene synthase inhibitor, suppresses lipogenic biosynthesis and lipid secretion in rodents.

1. To better understand how it decreases plasma cholesterol and triglyceride levels, we evaluated the effect of (E)-2-[2-fluoro-2-(quinuclidin-3-ylidene)ethoxy]-9H-carbazole monohydrochloride(YM-53601) on lipogenic biosynthesis in the liver and lipid secretion from the liver in rats and hamsters. 2. Single administration of YM-53601 in cholestyramine-treated rats inhibited triglyceride and free fatty acid (FFA) biosynthesis at a similar dose range to that at which it inhibited cholesterol biosynthesis. YM-53601 inhibited both triglyceride and FFA biosynthesis in hamsters treated with cholestyramine. 3. YM-53601 by single oral administration decreased the enhanced plasma triglyceride levels in hamsters induced by an injection of protamine sulfate, which inhibits lipoprotein lipase (LPL) and consequently increases plasma very low-density lipoprotein (VLDL) triglyceride levels. YM-53601 also decreased the enhanced plasma triglyceride and cholesterol levels in hamsters treated with Triton WR1339, which also inhibits the degradation of VLDL. Plasma cholesterol was significantly decreased as soon as 1 h after single administration of YM-53601 in hamsters fed a normal diet. 4. This is the first report that a squalene synthase inhibitor suppresses lipogenic biosynthesis in the liver and cholesterol and triglyceride secretion from the liver in vivo. We therefore suggest that the mechanism by which YM-53601 decreases plasma triglyceride might include these effects. The finding that YM-53601 rapidly decreased plasma cholesterol suggests that this compound may be effective in decreasing plasma cholesterol levels early in the course of treatment of hypercholesterolemia in humans.

Experimental model of postprandial hypertriglyceridemia in sucrose-fed rats and the effectiveness of atorvastatin in the model.

Although postprandial hypertriglyceridemia has drawn attention as an independent risk factor of cardiovascular disease, there is no established animal model that shows a physiological transitory change in lipoprotein metabolism after ingestion of a fatty meal. We developed an animal model of postprandial hypertriglyceridemia using sucrose-fed rats, and used this model to evaluate the effect of atorvastatin on this condition. Compared with normal rats, sucrose-fed rats orally loaded with olive oil showed a high and prolonged increase in plasma triglyceride (TG) concentration accompanied by both an increase in TG secretion and decrease in TG clearance. Atorvastatin (30 mg/kg orally) for 2 weeks reduced not only fasting plasma TG concentration, but also the postprandial TG concentration. Atorvastatin also suppressed rates of TG secretion in both chylomicron (CM)-rich (d < 0.96 g/mL) and very-low-density lipoprotein (VLDL) (d = 0.96 to 1.006 g/mL) fractions after oral fat loading. Further, atorvastatin improved the elimination time of exogenous TG emulsion only in the nonfasted, namely, high plasma TG condition. These results indicate that this animal model satisfactorily replicates the postprandial hypertriglyceridemia observed in humans and may therefore be useful in evaluation of lipid-lowering agents. Furthermore, atorvastatin not only improves fasting but also postprandial lipoprotein metabolism, presumably by reducing TG secretion from the liver or intestine or both, and by secondarily increasing TG-rich lipoprotein clearance by eliminating saturation.

Atorvastatin increases hepatic fatty acid beta-oxidation in sucrose-fed rats: comparison with an MTP inhibitor.

We investigated the effects of atorvastatin, a widely used 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and BMS-201038, a microsomal triglyceride transfer protein (MTP) inhibitor, in sucrose-fed hypertriglyceridemic rats to determine whether the activation of beta-oxidation by these compounds plays a role in their hypotriglyceridemic effect. The decrease in plasma triglyceride concentration and post-Triton very low-density lipoprotein (VLDL) triglyceride concentration, a measure of hepatic triglyceride secretion, by atorvastatin (30 mg/kg p.o.) for 2 weeks was to approximately the same degree as those by BMS-201038 (0.3 mg/kg). Atorvastatin (30 mg/kg) increased hepatic beta-oxidation activity by 54% (P < 0.01), while BMS-201038 (0.3 mg/kg) had no significant effect. Atorvastatin decreased hepatic triglyceride, fatty acid and cholesteryl ester concentrations by 21% to 39%, whereas BMS-201038 increased these variables by 28% to 307%. In the atorvastatin-treated groups, a significant relationship was seen not only between hepatic beta-oxidation activity and hepatic triglyceride concentration (R(2) = 0.535, P < 0.01), but also between hepatic and plasma triglyceride concentrations (R(2) = 0.586, P < 0.01). No effect of atorvastatin on hepatic fatty acid synthesis was observed. These results indicate that the activation of hepatic beta-oxidation by atorvastatin may contribute to the decrease in hepatic triglyceride concentration, leading to its hypotriglyceridemic effect.

Brain penetration of telmisartan, a unique centrally acting angiotensin II type 1 receptor blocker, studied by PET in conscious rhesus macaques.

INTRODUCTION: Telmisartan is a widely used, long-acting antihypertensive agent. Known to be a selective angiotensin II type 1 (AT(1)) receptor (AT(1)R) blocker (ARB), telmisartan acts as a partial agonist of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and inhibits centrally mediated effects of angiotensin II in rats following peripheral administration, although the brain penetration of telmisartan remains unclear. We investigated the brain concentration and localization of telmisartan using (11)C-labeled telmisartan and positron emission tomography (PET) in conscious rhesus macaques. METHODS: Three male rhesus macaques were bolus intravenously administered [(11)C]telmisartan either alone or as a mixture with unlabeled telmisartan (1mg/kg). Dynamic PET images were acquired for 95min following administration. Blood samples were collected for the analysis of plasma concentration and metabolites, and brain and plasma concentrations were calculated from detected radioactivity using the specific activity of the administered drug preparation, in which whole blood radioactivity was used for the correction of intravascular blood radioactivity in brain. RESULTS: Telmisartan penetrated into the brain little but enough to block AT(1)R and showed a consistently increasing brain/plasma ratio within the PET scanning period, suggesting slow clearance of the compound from the brain compared to the plasma clearance. Brain/plasma ratios at 30, 60, and 90min were 0.06, 0.13, and 0.18, respectively. No marked localization according to the AT(1)R distribution was noted over the entire brain, even on tracer alone dosing. CONCLUSIONS: Telmisartan penetrated into the brain enough to block AT(1)R and showed a slow clearance from the brain in conscious rhesus macaques, supporting the long-acting and central responses of telmisartan as a unique property among ARBs.

An Angiotensin II (Ang II) Type 1 Receptor Blocker, Telmisartan, Improves Insulin Resistance in KK-A(y) Diabetic Mice.

Metabolic syndrome is strongly associated with insulin resistance and consists of a constellation of factors such as hypertension and hyperlipidemia that raise the risk for cardiovascular diseases and diabetes mellitus. The renin-angiotensin system (RAS) plays a pivotal role in the pathogenesis of diabetes and cardiovascular disease (CVD) in hypertensive patients. Further, recently, the interruption of the RAS has been shown to prevent the onset of diabetes in hypertensive patients. However, whether telmisartan, an angiotensin II type 1 receptor blocker (ARB) with selective peroxisome proliferator-activated receptor-γ (PPAR-γ) agonistic property could improve insulin sensitivity is not fully understood. In this study, we studied the effects of telmisartan on insulin sensitivity in KK-A(y) mice, an obese type 2 diabetic animal. Although there was no significant difference in body weight, food consumption, and glucose levels between the two groups, plasma insulin, triglycerides and non-esterified fatty acid levels were significantly decreased in telmisartan-treated KK-A(y) mice, compared with control KK-A(y) mice. The present findings suggest that telmisartan could exert a beneficial effect on insulin sensitivity in diabetic animals. Inhibition of the RAS by telmisartan, a selective agonist of PPAR-γ, may become a promising strategy for the treatment of hypertensive patients with metabolic syndrome and/or insulin resistance.

Synthesis and biological evaluation of novel propylamine derivatives as orally active squalene synthase inhibitors.

Squalene synthase inhibitors are potentially superior hypolipidemic agents. We synthesized novel propylamine derivatives, as well as evaluated their ability to inhibit squalene synthase and their lipid-lowering effects in rats. 1-Allyl-2-[3-(benzylamino)propoxy]-9H-carbazole (YM-75440) demonstrated potent inhibition of the enzyme derived from HepG2 cells with an IC(50) value of 63 nM. It significantly reduced both plasma total cholesterol and plasma triglyceride levels following oral dosing to rats with a reduced tendency to elevate plasma transaminase levels.

Synthesis and biological evaluation of quinuclidine derivatives incorporating phenothiazine moieties as squalene synthase inhibitors.

Squalene synthase inhibitors have the potential to be superior hypocholesterolemic agents. A series of quinuclidine derivatives incorporating phenothiazine systems was synthesized in order to investigate the effects of their structure on the inhibition of hamster liver microsomal enzyme. (+/-)-3-(10-Methyl-10H-phenothiazin-3-ylmethoxy)quinuclidine hydrochloride (19) was the most potent inhibitor in this series with an IC(50) value of 0.12 microM. Oral dosing of compound 19 to hamsters demonstrated effective reduction of both plasma total cholesterol levels and plasma triglyceride levels. Compound 19 showed a reduced tendency to elevate plasma transaminase levels, an indicator of hepatotoxicity. Enantiomerically pure (-)-19, YM-53546, was found to be more potent than the corresponding (+)-enantiomer.

Syntheses and biological evaluation of novel quinuclidine derivatives as squalene synthase inhibitors.

Squalene synthase (E.C. 2.5.1.21) catalyses the reductive dimerization of two molecules of farnesyl diphosphate to form squalene and is involved in the first committed step in cholesterol biosynthesis. Inhibition of this enzyme is therefore an attractive target for hypocholesterolemic strategies. A series of quinuclidine derivatives incorporating a tricyclic system was synthesized and evaluated for their ability to inhibit squalene synthase in vitro. A 9H-fluorene moiety was found to be optimal as the tricyclic system for potent inhibitory activity. Improved activity can be achieved with a conformationally constrained three-atom linkage connecting the tricyclic system with the quinuclidine nucleus. Among these compounds, (Z)-3-[2-(9H-fluoren-2-yloxy)ethylidene]-quinuclidine hydrochloride 31 was found to be a potent inhibitor of squalene synthase derived from hamster liver and human hepatoma cells with IC(50) values of 76 and 48 nM, respectively. Oral dosing of compound 31 demonstrated effective reduction of plasma non-HDL cholesterol levels in hamsters.

Syntheses of 3-ethylidenequinuclidine derivatives as squalene synthase inhibitors. Part 2: enzyme inhibition and effects on plasma lipid levels.

Squalene synthase (E.C. 2.5.1.21) is a microsomal enzyme which catalyzes the reductive dimerization of two molecules of farnesyl diphosphate to form squalene, and is involved in the first committed step in cholesterol biosynthesis. It is an attractive target for hypocholesterolemic and hypotriglyceridemic strategies. We synthesized a series of 3-ethylidenequinuclidine derivatives, and evaluated their ability to inhibit squalene synthase in vitro and to lower non-HDL cholesterol levels in hamsters. 3-Ethylidenequinuclidine derivatives incorporating an unsubstituted 9H-carbazole moiety reduced plasma non-HDL cholesterol levels and did not affect plasma transaminase levels, indicating a lack of hepatotoxicity. Among the novel compounds, (Z)-2-[2-(quinuclidin-3-ylidene)ethoxy]-9H-carbazole hydrochloride 8 (YM-53579) and (E)-2-[2-fluoro-2-(quinuclidin-3-ylidene)ethoxy]-9H-carbazole hydrochloride 28 (YM-53601) were potent inhibitors of squalene synthase derived from human hepatoma cells, with IC(50) values of 160 and 79 nM, respectively. They also reduced plasma non-HDL cholesterol levels in hamsters by approximately 50 and 70%, respectively, at an oral dose of 50 mg/kg/day for 5 days.