Effect of an NHE3 inhibitor in combination with an NPT2b inhibitor on gastrointestinal phosphate absorption in Rodent models.

Many of the pathological consequences of chronic kidney disease can be attributed to an elevation in serum phosphate levels. Current therapies focused on decreasing intestinal phosphate absorption to treat hyperphosphatemia are inadequate. The most effective therapeutic strategy may be to target multiple absorptive pathways. In this study, the ability of a novel inhibitor of the intestinal sodium hydrogen exchanger 3 (NHE3), LY3304000, which inhibits paracellular, diffusional uptake of phosphate, to work in combination with an inhibitor of the active transporter, sodium dependent phosphate cotransporter 2b (NPT2b), LY3358966, was explored. LY3304000 modestly inhibited the acute uptake of phosphate into plasma of rats, while surprisingly, it doubled the rate of phosphate uptake in mice, an animal model dominated by NPT2b mediated acute phosphate uptake. In rats, LY3004000 and LY3358966 work in concert to inhibit acute phosphate uptake. On top of LY3358966, LY3304000 further decreased the acute uptake of phosphate into plasma. Studies measuring the recovery of radiolabeled phosphate in the intestine demonstrated LY3304000 and LY3358966 synergistically inhibited the absorption of phosphate in rats. We hypothesize the synergism is because the NHE3 inhibitor, LY3304000, has two opposing effects on intestinal phosphate absorption in rats, first it decreases diffusion mediated paracellular phosphate absorption, while second, it simultaneously increases phosphate absorption through the NPT2b pathway. NHE3 inhibition decreases proton export from enterocytes and raises the cell surface pH. In vitro, NPT2b mediated phosphate transport is increased at higher pHs. The increased NPT2b mediated transport induced by NHE3 inhibition is masked in rats which have relatively low levels of NPT2b mediated phosphate transport, by the more robust inhibition of diffusion mediated phosphate absorption. Thus, the inhibition of NPT2b mediated phosphate transport in rats in the presence of NHE3 inhibition has an effect that exceeds its effect in the absence of NHE3 inhibition, leading to the observed synergism on phosphate absorption between NPT2b and NHE3 inhibition.

Diabetic cardiomyopathy: the need for adjusting experimental models to meet clinical reality.

Diabetic cardiomyopathy (CM), occurring in the absence of hypertension, coronary artery disease, and valvular or congenital heart disease, is now recognized as a distinct, multifactorial disease leading to ventricular hypertrophy and abnormal myocardial contractility that correlates with an array of complex molecular and cellular changes. Animal models provide the unique opportunity to investigate mechanistic aspects of diabetic CM, but important caveats exist when extrapolating findings obtained from preclinical models of diabetes to humans. Indeed, animal models do not recapitulate the complexity of environmental factors, most notably the duration of the exposure to insulin resistance that may play a crucial role in the development of diabetic CM. Moreover, most preclinical studies are performed in animals with uncontrolled or poorly controlled diabetes, whereas patients tend to undergo therapeutic intervention. Finally, whilst type 2 diabetes mellitus prevalence trajectory mainly increases at 40- < 75 years (with a currently alarming increase at younger ages, however), it is a legitimate concern how closely rodent models employing young animals recapitulate the disease developing in old people. The aim of this review is to identify the current limitations of rodent models and to discuss how future mechanistic and preclinical studies should integrate key confounding factors to better mimic the diabetic CM phenotype.

Acute SGLT-2i treatment improves cardiac efficiency during myocardial ischemia independent of Na+/H+ exchanger-1.

AIMS: Sodium glucose co-transporter 2 inhibitors (SGLT2i) demonstrate cardioprotective benefits independent of a glucose lowering effect including preservation of cardiac function during a myocardial ischemia. Sodium­hydrogen exchanger-1 (NHE-1), has been hypothesized to contribute to the cardiac effects of SGLT2i. We characterized the beneficial effects of acute pre-ischemia exposure to SGLT2i and explored the possibility that these effects are explained by NHE-1 inhibition. METHODS AND RESULTS: Swine were anesthetized and instrumented for invasive hemodynamic measurements. After baseline data collection, swine received a 15-30 min intravenous infusion of vehicle (DMSO), the SGLT2i canagliflozin (~1 mg/kg), or the NHE-1 inhibitor cariporide (~0.03 mg/kg) ending immediately prior to occlusion of the left circumflex artery. Measurements were obtained at baseline, during a 60-min complete occlusion of the circumflex coronary artery, and during a 2-h reperfusion period. Blood pressure, heart rate, left anterior descending artery flow, and associated myocardial oxygen consumption were unaffected by acute pre-treatment with canagliflozin or cariporide during ischemia and reperfusion. Acute pre-ischemic treatment with canagliflozin significantly increased diastolic filling and stroke work, producing a rightward shift in the Frank-Starling relationship, and also improved cardiac work efficiency relative to untreated control hearts during ischemia. Effects of NHE-1 inhibition with cariporide were modest and dissimilar. Examination of AP-1 cells transfected with wild-type NHE-1 and iPSC-derived cardiomyocytes confirmed dose-dependent-inhibition of NHE-1 activity by cariporide, while canagliflozin had no significant effect on NHE-1 activity. CONCLUSION: Acute pre-treatment with SGLT2i produces cardioprotective effects during ischemia, including improved work efficiency. These effects are not explained by NHE-1 inhibition. TRANSLATIONAL PERSPECTIVE: SGLT2 inhibitors have been shown to improve cardiac outcomes in patient including reducing myocardial infarction incidence and mortality. The mechanism(s) explaining this effect are not clear. This manuscript demonstrates a protective effect from acute SGLT2i exposure, as short as 15 min, prior to experimental infarction in swine. These effects were independent of NHE1 inhibition. These observations suggest that SGLT2 inhibitors can confer cardioprotective effects on a very short time scale. It is possible that such effects provide an ongoing contribution to ischemic protection even in the setting of chronic treatment.

Effects of pharmacological inhibition of the sodium-dependent phosphate cotransporter 2b (NPT2b) on intestinal phosphate absorption in mouse and rat models.

An excess phosphate burden in renal disease has pathological consequences for bone, kidney, and heart. Therapies to decrease intestinal phosphate absorption have been used to address the problem, but with limited success. Here, we describe the in vivo effects of a novel potent inhibitor of the intestinal sodium-dependent phosphate cotransporter NPT2b, LY3358966. Following treatment with LY3358966, phosphate uptake into plasma 15 min following an oral dose of radiolabeled phosphate was decreased 74% and 22% in mice and rats, respectively, indicating NPT2b plays a much more dominant role in mice than rats. Following the treatment with LY3358966 and radiolabeled phosphate, mouse feces were collected for 48 h to determine the ability of LY3358966 to inhibit phosphate absorption. Compared to vehicle-treated animals, there was a significant increase in radiolabeled phosphate recovered in feces (8.6% of the dose, p < .0001). Similar studies performed in rats also increased phosphate recovered in feces (5.3% of the dose, p < .05). When used in combination with the phosphate binder sevelamer in rats, there was a further small, but not significant, increase in fecal phosphate. In conclusion, LY3358966 revealed a more prominent role for NPT2b on acute intestinal phosphate uptake into plasma in mice than rats. However, the modest effects on total intestinal phosphate absorption observed in mice and rats with LY3359866 when used alone or in combination with sevelamer highlights the challenge to identify new more effective therapeutic targets and/or drug combinations to treat the phosphate burden in patients with renal disease.

Selective Phosphodiesterase 1 Inhibitor BTTQ Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats: Role of Peripheral Vasodilation.

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1-11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and, thus, peripheral resistance. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (BTTQ) on the cardiovascular system. Normal Sprague Dawley, spontaneously hypertensive (SHR), and Dahl salt-sensitive rats were used to test in vivo the efficacy of the compound. Phosphodiesterase radiometric enzyme assay revealed that BTTQ inhibited all three isoforms of PDE1 in nanomolar concentration, while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study conducted on mesenteric arteries revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriols reflected by the rise in the temperature. Furthermore, BTTQ proved a high efficacy in lowering the blood pressure about 9, 36, and 24 mmHg in normal Sprague Dawley, SHR and, Dahl salt-sensitive rats, respectively, compared to the vehicle-treated group. Moreover, additional blood pressure lowering of about 22 mmHg could be achieved when BTTQ was administered on top of ACE inhibitor lisinopril, a current standard of care in the treatment of hypertension. Therefore, PDE1 inhibition induced efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Administration of BTTQ was also associated with increased heart rate in both models of hypertension as well as in the normotensive rats. Thus, PDE1 appears to be an attractive therapeutic target for the treatment of resistant hypertension, while tachycardia needs to be addressed by further compound structural optimization.

Distinct hemodynamic responses to (pyr)apelin-13 in large animal models.

This study tested the hypothesis that (pyr)apelin-13 dose-dependently augments myocardial contractility and coronary blood flow, irrespective of changes in systemic hemodynamics. Acute effects of intravenous (pyr)apelin-13 administration (10 to 1,000 nM) on blood pressure, heart rate, left ventricular pressure and volume, and coronary parameters were measured in dogs and pigs. Administration of (pyr)apelin-13 did not influence blood pressure (P = 0.59), dP/dtmax (P = 0.26), or dP/dtmin (P = 0.85) in dogs. However, heart rate dose-dependently increased > 70% (P < 0.01), which was accompanied by a significant increase in coronary blood flow (P < 0.05) and reductions in left ventricular end-diastolic volume and stroke volume (P < 0.001). In contrast, (pyr)apelin-13 did not significantly affect hemodynamics, coronary blood flow, or indexes of contractile function in pigs. Furthermore, swine studies found no effect of intracoronary (pyr)apelin-13 administration on coronary blood flow (P = 0.83) or vasorelaxation in isolated, endothelium-intact (P = 0.89) or denuded (P = 0.38) coronary artery rings. Examination of all data across (pyr)apelin-13 concentrations revealed an exponential increase in cardiac output as peripheral resistance decreased across pigs and dogs (P < 0.001; R2 = 0.78). Assessment of the Frank-Starling relationship demonstrated a significant linear relationship between left ventricular end-diastolic volume and stroke volume across species (P < 0.001; R2 = 0.70). Taken together, these findings demonstrate that (pyr)apelin-13 does not directly influence myocardial contractility or coronary blood flow in either dogs or pigs.NEW & NOTEWORTHY Our findings provide much needed insight regarding the pharmacological cardiac and coronary effects of (pyr)apelin-13 in larger animal preparations. In particular, data highlight distinct hemodynamic responses of apelin across species, which are independent of any direct effect on myocardial contractility or perfusion.

Rare DEGS1 variant significantly alters de novo ceramide synthesis pathway.

The de novo ceramide synthesis pathway is essential to human biology and health, but genetic influences remain unexplored. The core function of this pathway is the generation of biologically active ceramide from its precursor, dihydroceramide. Dihydroceramides have diverse, often protective, biological roles; conversely, increased ceramide levels are biomarkers of complex disease. To explore the genetics of the ceramide synthesis pathway, we searched for deleterious nonsynonymous variants in the genomes of 1,020 Mexican Americans from extended pedigrees. We identified a Hispanic ancestry-specific rare functional variant, L175Q, in delta 4-desaturase, sphingolipid 1 (DEGS1), a key enzyme in the pathway that converts dihydroceramide to ceramide. This amino acid change was significantly associated with large increases in plasma dihydroceramides. Indexes of DEGS1 enzymatic activity were dramatically reduced in heterozygotes. CRISPR/Cas9 genome editing of HepG2 cells confirmed that the L175Q variant results in a partial loss of function for the DEGS1 enzyme. Understanding the biological role of DEGS1 variants, such as L175Q, in ceramide synthesis may improve the understanding of metabolic-related disorders and spur ongoing research of drug targets along this pathway.

Author Correction: Salt-dependent Blood Pressure in Human Aldosterone Synthase-Transgenic Mice.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Identification of 4-Aminopyrazolopyrimidine Metabolite That May Contribute to the Hypolipidemic Effects of LY2584702 in Long Evans Diet-Induced Obese Rats.

LY2584702 is an inhibitor of p70 S6 kinase-1 previously developed for the treatment of cancer. In two phase 1 trials in oncology patients, significant reductions of total cholesterol, low-density lipoprotein cholesterol (LDL-C), and triglyceride were observed. In the current study, we sought to understand the potential mechanism of action of this compound in regulating lipid metabolism. In Long Evans diet-induced obese (DIO) rats, oral administration of LY2584702 for 3-4 weeks led to robust reduction of LDL-C up to 60%. An unexpected finding of liver triglyceride (TG) increase implicated a metabolite of LY2584702, 4-aminopyrazolo[3,4-day]pyrimidine (4-APP), in modulation of lipid metabolism in these rats. We showed that low-dose 4-APP, when administered orally for 3-4 weeks to Long Evans DIO rats, produced lipoprotein profile changes that were strikingly similar to LY2584702. Kinetic studies suggested that both LY2584702 and 4-APP had no effect on chylomicron-TG secretion and only exerted a modest effect on hepatic very low-density lipoprotein (VLDL)-TG secretion. In human hepatoma HepG2 cells, 4-APP, but not LY2584702, increased LDL uptake. We hypothesize that generation of the 4-APP metabolite may contribute to the efficacy of LY2584702 in lowering LDL-C in rats and potentially in humans as well. This mechanism of LDL-C lowering may include inhibition of VLDL production and increase in LDL clearance.

Salt-dependent Blood Pressure in Human Aldosterone Synthase-Transgenic Mice.

Hypertension is one of the most important, preventable causes of premature morbidity and mortality in the developed world. Aldosterone is a major mineralocorticoid hormone that plays a key role in the regulation of blood pressure and is implicated in the pathogenesis of hypertension and heart failure. Aldosterone synthase (AS, cytochrome P450 11B2, cyp11B2) is the sole enzyme responsible for the production of aldosterone in humans. To determine the effects of increased expression of human aldosterone synthase (hAS) on blood pressure (BP), we established transgenic mice carrying the hAS gene (cyp11B2). We showed that hAS overexpression increased levels of aldosterone in hAS+/- mice. On high salt diet (HS), BPs of hAS+/- mice were significantly increased compared with WT mice. Fadrozole (an inhibitor of aldosterone synthase) treatment significantly reduced BPs of hAS+/- mice on HS. This is the first time overexpression of AS in a transgenic mouse line has shown an ability to induce HP. Specifically inhibiting AS activity in these mice is a promising therapy for reducing hypertension. This hAS transgenic mouse model is therefore an ideal animal model for hypertension therapy studies.

Proteolytic cleavage of antigen extends the durability of an anti-PCSK9 monoclonal antibody.

Lilly PCSK9 antibody LY3015014 (LY) is a monoclonal antibody (mAb) that neutralizes proprotein convertase subtilisin-kexin type 9 (PCSK9). LY decreases LDL cholesterol in monkeys and, unlike other PCSK9 mAbs, does not cause an accumulation of intact PCSK9 in serum. Comparing the epitope of LY with other clinically tested PCSK9 mAbs, it was noted that the LY epitope excludes the furin cleavage site in PCSK9, whereas other mAbs span this site. In vitro exposure of PCSK9 to furin resulted in degradation of PCSK9 bound to LY, whereas cleavage was blocked by other mAbs. These other mAbs caused a significant accumulation of serum PCSK9 and displayed a shorter duration of LDL-cholesterol lowering than LY when administered to mice expressing the WT human PCSK9. In mice expressing a noncleavable variant of human PCSK9, LY behaved like a cleavage-blocking mAb, in that it caused significant PCSK9 accumulation, its duration of LDL lowering was reduced, and its clearance (CL) from serum was accelerated. Thus, LY neutralizes PCSK9 and allows its proteolytic degradation to proceed, which limits PCSK9 accumulation, reduces the CL rate of LY, and extends its duration of action. PCSK9 mAbs with this property are likely to achieve longer durability and require lower doses than mAbs that cause antigen to accumulate.

Development of a high-throughput assay for aldosterone synthase inhibitors using high-performance liquid chromatography-tandem mass spectrometry.

Aldosterone plays a key role in the pathogenesis of hypertension, congestive heart failure, and chronic kidney disease. Aldosterone biosynthesis involves three membrane-bound enzymes: aldosterone synthase, adrenodoxin, and adrenodoxin reductase. Here, we report the development of a mass spectrometry-based high-throughput whole cell-based assay for aldosterone synthesis. A human adrenal carcinoma cell line (H295R) overexpressing human aldosterone synthase cDNA was established. The production of aldosterone in these cells was initiated with the addition of 11-deoxycorticosterone, the immediate substrate of aldosterone synthase. An automatic liquid handler was used to gently distribute cells uniformly to well plates. The adaption of a second automated liquid handling system to extract aldosterone from the cell culture medium into organic solvent enabled the development of 96- and 384-well plate formats for this cellular assay. A high-performance liquid chromatography-tandem mass spectrometry method was established for the detection of aldosterone. Production of aldosterone was linear with time and saturable with increasing substrate concentration. The assay was highly reproducible with an overall average Z' value=0.49. This high-throughput assay would enable high-throughput screening for inhibitors of aldosterone biosynthesis.

Leukotriene B4 levels in human atherosclerotic plaques and abdominal aortic aneurysms.

BACKGROUND: Leukotriene B4 (LTB4) has been associated with the initiation and progression of atherosclerosis and abdominal aortic aneurysm (AAA) formation. However, associations of LTB4 levels with tissue characteristics and adverse clinical outcome of advanced atherosclerosis and AAA are scarcely studied. We hypothesized that LTB4 levels are associated with a vulnerable plaque phenotype and adverse clinical outcome. Furthermore, that LTB4 levels are associated with inflammatory AAA and adverse clinical outcome. METHODS: Atherosclerotic plaques and AAA specimens were selected from two independent databases for LTB4 measurements. Plaques were isolated during carotid endarterectomy from asymptomatic (n = 58) or symptomatic (n = 317) patients, classified prior to surgery. LTB4 levels were measured without prior lipid extraction and levels were corrected for protein content. LTB4 levels were related to plaque phenotype, baseline patient characteristics and clinical outcome within three years following surgery. Seven non-diseased mammary artery specimens served as controls. AAA specimens were isolated during open repair, classified as elective (n = 189), symptomatic (n = 29) or ruptured (n = 23). LTB4 levels were measured similar to the plaque measurements and were related to tissue characteristics, baseline patient characteristics and clinical outcome. Twenty-six non-diseased aortic specimens served as controls. RESULTS: LTB4 levels corrected for protein content were not significantly associated with histological characteristics specific for vulnerable plaques or inflammatory AAA as well as clinical presentation. Moreover, it could not predict secondary manifestations independently investigated in both databases. However, LTB4 levels were significantly lower in controls compared to plaque (p = 0.025) or AAA (p = 0.017). CONCLUSIONS: LTB4 levels were not associated with a vulnerable plaque phenotype or inflammatory AAA or clinical presentation. This study does not provide supportive evidence for a role of LTB4 in atherosclerotic plaque destabilization or AAA expansion. However, these data should be interpreted with care, since LTB4 measurements were performed without prior lipid extractions.

Genome-wide screen for modulation of hepatic apolipoprotein A-I (ApoA-I) secretion.

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25-40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.

Bile-acid-mediated decrease in endoplasmic reticulum stress: a potential contributor to the metabolic benefits of ileal interposition surgery in UCD-T2DM rats.

Post-operative increases in circulating bile acids have been suggested to contribute to the metabolic benefits of bariatric surgery; however, their mechanistic contributions remain undefined. We have previously reported that ileal interposition (IT) surgery delays the onset of type 2 diabetes in UCD-T2DM rats and increases circulating bile acids, independently of effects on energy intake or body weight. Therefore, we investigated potential mechanisms by which post-operative increases in circulating bile acids improve glucose homeostasis after IT surgery. IT, sham or no surgery was performed on 2-month-old weight-matched male UCD-T2DM rats. Animals underwent an oral fat tolerance test (OFTT) and serial oral glucose tolerance tests (OGTT). Tissues were collected at 1.5 and 4.5 months after surgery. Cell culture models were used to investigate interactions between bile acids and ER stress. IT-operated animals exhibited marked improvements in glucose and lipid metabolism, with concurrent increases in postprandial glucagon-like peptide-1 (GLP-1) secretion during the OFTT and OGTTs, independently of food intake and body weight. Measurement of circulating bile acid profiles revealed increases in circulating total bile acids in IT-operated animals, with a preferential increase in circulating cholic acid concentrations. Gut microbial populations were assessed as potential contributors to the increases in circulating bile acid concentrations, which revealed proportional increases in Gammaproteobacteria in IT-operated animals. Furthermore, IT surgery decreased all three sub-arms of ER stress signaling in liver, adipose and pancreas tissues. Amelioration of ER stress coincided with improved insulin signaling and preservation of ß-cell mass in IT-operated animals. Incubation of hepatocyte, adipocyte and ß-cell lines with cholic acid decreased ER stress. These results suggest that postoperative increases in circulating cholic acid concentration contribute to improvements in glucose homeostasis after IT surgery by ameliorating ER stress.

Vertical sleeve gastrectomy improves glucose and lipid metabolism and delays diabetes onset in UCD-T2DM rats.

Vertical sleeve gastrectomy (VSG) has gained interest as a low morbidity bariatric surgery, which is effective in producing weight loss and causing type 2 diabetes resolution. However, the efficacy of VSG to prevent the onset of type 2 diabetes has not been previously investigated. VSG or sham surgery was performed on 2-month-old prediabetic male University of California Davis-type 2 diabetes mellitus rats. Sham-operated animals were either sham-operated ad libitum fed (S-AL) or were weight-matched to VSG-operated animals (S-WM). Diabetes onset was determined by weekly nonfasting blood glucose measurements. Animals underwent oral glucose tolerance tests at 1 and 4 months after surgery and indirect calorimetry at 1.5 months after surgery. VSG surgery significantly delayed diabetes onset compared with both S-AL and S-WM animals. VSG-operated animals ate 23% less and weighed 20% less than S-AL. Energy expenditure did not differ between VSG-operated animals and controls. Results from the oral glucose tolerance tests demonstrate improved glucose tolerance and islet function in VSG-operated animals compared with S-AL and S-WM. Nutrient-stimulated glucagon-like peptide (GLP)-1, GLP-2, and peptide YY excursions were greater in VSG-operated animals. VSG surgery resulted in decreased fasting plasma insulin, ghrelin and lipid concentrations, and markedly higher fasting plasma adiponectin and bile acid concentrations, independent of body weight. Increases of circulating bile acid concentrations were due to selective increases of taurine-conjugated bile acids. Thus, VSG delays type 2 diabetes onset in the University of California Davis-type 2 diabetes mellitus rat, independent of body weight. This is potentially mediated by increases of circulating bile acids, adiponectin, and nutrient-stimulated GLP-1 secretion and decreased circulating ghrelin concentrations.

Opportunities for pharmacotherapy at the intersection of metabolic syndrome and hemostasis.

Hemostatic balance is regulated by many factors that may become perturbed by cardio-metabolic abnormalities. Indeed, patients with multiple components of the metabolic syndrome have increased risk of atherosclerosis, hemostatic disorders and thrombotic events. This review focuses on the interrelationship between the metabolic syndrome components and thrombotic and thromboembolic events, the potential underlying mechanisms that lead to metabolic and hemostatic disorders in metabolic syndrome patients, the existing therapeutics aimed at reducing major cardiovascular events, and new therapeutic approaches to address pro-coagulant states.

Discovery of novel hepatoselective HMG-CoA reductase inhibitors for treating hypercholesterolemia: a bench-to-bedside case study on tissue selective drug distribution.

The design of drugs with selective tissue distribution can be an effective strategy for enhancing efficacy and safety, but understanding the translation of preclinical tissue distribution data to the clinic remains an important challenge. As part of a discovery program to identify next generation liver selective HMG-CoA reductase inhibitors we report the identification of (3R,5R)-7-(4-((3-fluorobenzyl)carbamoyl)-5-cyclopropyl-2-(4-fluorophenyl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoic acid (26) as a candidate for treating hypercholesterlemia. Clinical evaluation of 26 (PF-03491165), as well as the previously reported 2 (PF-03052334), provided an opportunity for a case study comparison of the preclinical and clinical pharmacokinetics as well as pharmacodynamics of tissue targeted HMG-CoA reductase inhibitors.

Further LDL cholesterol lowering through targeting PCSK9 for coronary artery disease.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that belongs to the proprotein convertase family. PCSK9 is synthesized as a zymogen and its prodomain is cleaved by its own catalytic activity. The cleaved prodomain forms a protein complex with the rest of the PCSK9 carboxyl terminus within the endoplasmic reticulum and is secreted. Secreted PCSK9 has been shown to be able to reduce low-density lipoprotein receptor (LDLR) levels in vitro and in vivo. Thus PCSK9 has emerged as an important player modulating LDLR levels and plasma LDL cholesterol. Furthermore, PCSK9 deficiency leads to significantly lowered LDL cholesterol levels in humans and provides dramatic protection against coronary heart disease. We review here the current understanding of PCSK9 and its potential as a therapeutic target through which to reduce LDL cholesterol for prevention and treatment of coronary heart disease.

A nonpeptide, piperidine renin inhibitor provides renal and cardiac protection in double-transgenic mice expressing human renin and angiotensinogen genes.

INTRODUCTION: Controlling hypertension by angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB), mechanisms that inhibit later pathway steps in the renin-angiotensin system (RAS), have clinically afforded protection against cardiac and renal disease. MATERIALS AND METHODS: In order to determine if blocking the RAS rate-limiting step of angiotensin II generation via renin inhibition could afford similar end organ protection in a human-relevant preclinical model, this study investigated the cardiac and renal effects of a nonpeptide, piperidine renin inhibitor (RI; 100 mg/kg/day PO) in double transgenic mice (dTGM) which express both human renin and angiotensinogen genes. RI was compared to the ARB, candesartan (3 mg/kg/day PO), and to the ACEI, enalapril (60 mg/kg/day PO) in a 4-week dosing paradigm. These doses of RI, ACEI and ARB were previously found to normalize mean blood pressure (MBP) to 110 + 3, 109 + 7 and 107 + 6 mmHg, respectively, after 1 day of treatment. RESULTS AND DISCUSSION: In the dTGM, PRA, plasma aldosterone, GFR, microalbuminuria and left ventricular free wall thickness (LVH) were higher than in the wild type C57BL/6 mice. Microalbuminuria and LVH were significantly reduced by 93% and 9% for the RI, 83% and 13% for enalapril and 73% and 6% for candesartan, respectively. PRA and aldosterone were reduced by the RI 56% and 23%, respectively. These results suggest that the RI provides protection against cardiac and renal disease, similar to ARB and ACEI.