ABSTRACT
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.
Subject(s)
Phosphates , Renal Insufficiency, Chronic , Rats , Mice , Animals , Phosphates/metabolism , Sodium-Hydrogen Exchanger 3 , Rodentia , Intestinal Absorption , Renal Insufficiency, Chronic/metabolism , Sodium-Hydrogen Exchangers/metabolismABSTRACT
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.
Subject(s)
Coronary Artery Disease , Myocardial Ischemia , Sodium-Glucose Transporter 2 Inhibitors , Animals , Canagliflozin/pharmacology , Canagliflozin/therapeutic use , Glucose , Myocardial Ischemia/drug therapy , Myocytes, Cardiac , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Sodium-Glucose Transporter 2 Inhibitors/therapeutic use , Sodium-Hydrogen Exchangers/pharmacology , SwineABSTRACT
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.
Subject(s)
Intestinal Absorption , Phosphates/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIb/antagonists & inhibitors , Animals , CHO Cells , Chelating Agents/administration & dosage , Chelating Agents/pharmacology , Cricetulus , Male , Mice , Mice, Inbred C57BL , Rats , Rats, Sprague-Dawley , Sevelamer/administration & dosage , Sevelamer/pharmacology , Sodium-Phosphate Cotransporter Proteins, Type IIb/metabolism , Species SpecificityABSTRACT
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.
Subject(s)
Coronary Circulation/drug effects , Intercellular Signaling Peptides and Proteins/pharmacology , Myocardial Contraction/drug effects , Animals , Blood Pressure , Coronary Vessels/drug effects , Dogs , Heart Rate , Male , Stroke Volume , Swine , VasodilationABSTRACT
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.
ABSTRACT
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.
Subject(s)
Adenine/analogs & derivatives , Hypolipidemic Agents/pharmacology , Obesity/blood , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Adenine/pharmacology , Animals , Cholesterol, LDL/blood , Cholesterol, LDL/metabolism , Cholesterol, VLDL/biosynthesis , Cholesterol, VLDL/genetics , Gene Expression Regulation/drug effects , Hep G2 Cells , Humans , Lipid Metabolism/drug effects , Lipoproteins, LDL/metabolism , Liver/drug effects , Liver/metabolism , Male , Rats , Rats, Long-Evans , Triglycerides/metabolismABSTRACT
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.
Subject(s)
Aldosterone/metabolism , Blood Pressure/genetics , Cytochrome P-450 CYP11B2/genetics , Sodium Chloride, Dietary/metabolism , Animals , Biomarkers , Cytochrome P-450 CYP11B2/antagonists & inhibitors , Cytochrome P-450 CYP11B2/metabolism , Electrolytes/blood , Fadrozole/pharmacology , Gene Expression , Genotype , Hematocrit , Humans , Hypertension/etiology , Hypertension/metabolism , Hypertension/physiopathology , Kidney/metabolism , Mice , Mice, Transgenic , Phenotype , Renin/blood , Renin/metabolism , Sodium Chloride, Dietary/blood , Steroid 11-beta-Hydroxylase/genetics , Steroid 11-beta-Hydroxylase/metabolismABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/pharmacology , Anticholesteremic Agents/pharmacology , Proprotein Convertases/antagonists & inhibitors , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacokinetics , Anticholesteremic Agents/chemistry , Anticholesteremic Agents/pharmacokinetics , Cholesterol, LDL/blood , Drug Evaluation, Preclinical , Drug Stability , Furin/chemistry , Half-Life , Humans , Hypercholesterolemia/drug therapy , Macaca fascicularis , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Proprotein Convertase 9 , Proprotein Convertases/immunology , Protein Binding , Proteolysis , Serine Endopeptidases/immunology , Treatment OutcomeABSTRACT
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 (nâ=â58) or symptomatic (nâ=â317) 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 (nâ=â189), symptomatic (nâ=â29) or ruptured (nâ=â23). 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 (pâ=â0.025) or AAA (pâ=â0.017). 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.
Subject(s)
Aortic Aneurysm, Abdominal/metabolism , Leukotriene B4/metabolism , Plaque, Atherosclerotic/metabolism , Analysis of Variance , Case-Control Studies , Humans , Immunohistochemistry , Leukotriene B4/bloodABSTRACT
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.
Subject(s)
Apolipoprotein A-I/metabolism , Hepatocytes/metabolism , Liver/metabolism , Animals , Apolipoprotein A-I/genetics , Cholesterol, HDL/genetics , Cholesterol, HDL/metabolism , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase/antagonists & inhibitors , Farnesyltranstransferase/genetics , Farnesyltranstransferase/metabolism , Genome-Wide Association Study , Hep G2 Cells , Humans , Liver/cytology , Mice , Mice, Transgenic , Polyenes/pharmacology , Polyunsaturated Alkamides/pharmacology , RNA, Small Interfering/geneticsABSTRACT
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.
Subject(s)
Metabolic Syndrome/blood , Animals , Hemostasis , Humans , Metabolic Syndrome/epidemiology , Metabolic Syndrome/therapy , Thrombosis/physiopathologyABSTRACT
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.
Subject(s)
Anticholesteremic Agents/pharmacology , Anticholesteremic Agents/therapeutic use , Cholesterol, LDL/blood , Coronary Artery Disease/blood , Coronary Artery Disease/drug therapy , Serine Endopeptidases/drug effects , Serine Endopeptidases/genetics , Animals , Genetic Therapy , Homeostasis/drug effects , Homeostasis/physiology , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Oligonucleotides, Antisense/therapeutic use , Proprotein Convertase 9 , Proprotein Convertases , RNA, Small Interfering/therapeutic use , Receptors, LDL/drug effectsABSTRACT
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.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Angiotensinogen/genetics , Cardiotonic Agents/therapeutic use , Kidney Diseases/drug therapy , Piperidines/therapeutic use , Quinolines/therapeutic use , Renin/antagonists & inhibitors , Administration, Oral , Albuminuria/diagnosis , Albuminuria/drug therapy , Albuminuria/etiology , Aldosterone/blood , Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin II Type 1 Receptor Blockers/therapeutic use , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Angiotensinogen/metabolism , Animals , Benzimidazoles/pharmacology , Biphenyl Compounds/pharmacology , Blood Pressure/drug effects , Blood Pressure/physiology , Cardiotonic Agents/pharmacology , Drug Administration Schedule , Enalapril/pharmacology , Female , Glomerular Filtration Rate/drug effects , Glomerular Filtration Rate/physiology , Humans , Hypertrophy, Left Ventricular/diagnostic imaging , Hypertrophy, Left Ventricular/drug therapy , Hypertrophy, Left Ventricular/physiopathology , Kidney Diseases/prevention & control , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Structure , Piperidines/chemistry , Quinolines/chemistry , Renin/blood , Renin/genetics , Renin-Angiotensin System/drug effects , Renin-Angiotensin System/genetics , Tetrazoles/pharmacology , Time Factors , UltrasonographyABSTRACT
Myriocin, a potent inhibitor of serine palmitoyltransferase (SPT), has been shown to reduce plasma sphingolipids, cholesterol and triglycerides in hyperlipidemic apolipoprotein E knockout (apoE KO) mice. We hypothesized that the inhibition of sphingolipid biosynthesis modulates the composition of atherosclerotic plaque via its lipid-lowering effects. To test this hypothesis, the effect of myriocin on plasma lipids, sphingolipids and atherosclerosis progression, regression and lesion composition was investigated in apoE KO mice. Myriocin was administered to 24-week-old male apoE KO mice for 12 weeks. Myriocin-treated apoE KO mice had significant reductions in plasma total cholesterol, triglycerides, VLDL-cholesterol, ceramide, sphinganine and sphingomyelin (SM) compared to 24- and 36-week-old control mice. The ratio of SM to phosphatidylcholine (SM/PC), an independent risk factor for coronary artery disease, was also reduced by myriocin. Compared to 24- and 36-week controls, atherosclerotic lesion area and macrophage content in the aortic root and brachiocephalic arteries of myriocin-treated ApoE KO mice were reduced but there was only a slight increase in smooth muscle content. However, the content of collagen within aortic root lesions was increased in myriocin-treated apoE KO mice. In summary, the inhibition of SPT lowers plasma sphingolipids and atherogenic plasma lipids leading to the regression of pre-existing atherosclerotic lesions and to the formation of a stable plaque phenotype.
Subject(s)
Apolipoproteins E/genetics , Arteriosclerosis/prevention & control , Fatty Acids, Monounsaturated/therapeutic use , Hyperlipidemias/drug therapy , Hypolipidemic Agents/therapeutic use , Serine C-Palmitoyltransferase/antagonists & inhibitors , Animals , Arteriosclerosis/metabolism , Arteriosclerosis/pathology , Cholesterol/blood , Disease Progression , Fatty Acids, Monounsaturated/administration & dosage , Fatty Acids, Monounsaturated/pharmacology , Hyperlipidemias/metabolism , Hypolipidemic Agents/administration & dosage , Hypolipidemic Agents/pharmacology , Male , Mice , Mice, Knockout , Serine C-Palmitoyltransferase/metabolism , Sphingolipids/blood , Triglycerides/bloodABSTRACT
Among the L-type calcium channel blockers (CCBs), particularly dihydropyridines like nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester], a common adverse effect is vasodilatory edema. Newer CCBs, such as the T- and L-type CCB, mibefradil [(1S,2S)-2-[2[[3-(2-benzimidazolylpropyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl methoxyacetate dihydrochloride hydrate], demonstrate antihypertensive efficacy similar to that of their predecessors but seem to have a reduced propensity to cause edema. Using a magnetic resonance imaging (MRI) T(2) mapping technique, we investigated the ability of mibefradil to reduce extracellular water accumulation caused by the L-type CCB, nifedipine, in the hindleg skeletal muscle of the spontaneously hypertensive rat. Mibefradil (10 mg/kg i.v.) and nifedipine (1 mg/kg i.v.) lowered mean arterial blood pressure by 97 +/- 5 and 77 +/- 4 mm Hg, respectively. MRI edema index (expressed as percentage increase of integral T(2) over predrug control) was significantly higher with nifedipine (2606 +/- 86%; p < 0.05) than with mibefradil (981 +/- 171%) measured 30 to 60 min after the start of drug infusion. The hindleg edema caused by nifedipine was dose dependently decreased by coadministration of mibefradil (0, 0.3, or 3 mg/kg). The hindleg edema formation was not due to albumin leakage into the interstitial space based on immunostaining. However, a 4.2-fold increase in the arterial L-/T-type CC mRNA expression ratio was observed compared with the venous L/T ratio as shown by quantitative reverse transcription polymerase chain reaction. These results demonstrate the novel utility of MRI to measure extravascular water after acute exposure to CCBs and indicate that T-type CCB activity may reduce L-type CCB-induced vasodilatory edema in the skeletal muscle vasculature, possibly by a differential effect on arteriole and venule dilatation.
Subject(s)
Antihypertensive Agents/therapeutic use , Calcium Channel Blockers/therapeutic use , Calcium Channels, L-Type/physiology , Calcium Channels, T-Type/physiology , Edema/chemically induced , Edema/drug therapy , Hypertension/drug therapy , Mibefradil/therapeutic use , Animals , Calcium Channels, L-Type/genetics , Calcium Channels, T-Type/genetics , Edema/pathology , Edema/physiopathology , Femoral Artery/metabolism , Hindlimb , Hypertension/pathology , Hypertension/physiopathology , Magnetic Resonance Imaging , Muscle, Skeletal/drug effects , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Nifedipine/therapeutic use , RNA, Messenger/metabolism , Rats , Rats, Inbred SHR , Rats, Sprague-DawleyABSTRACT
Statins, because of their excellent efficacy and manageable safety profile, represent a key component in the current armamentarium for the treatment of hypercholesterolemia. Nonetheless, myopathy remains a safety concern for this important drug class. Cerivastatin was withdrawn from the market for myotoxicity safety concerns. BMS-423526 [{(3R,5S)-7-[4-(4-fluorophenyl)-6,7-dihydro-2-(1-methylethyl)-5H-benzo[6,7]cyclohepta[1,2-b]pyridin-3-yl]-3,5-dihydroxy-heptenoic acid} sodium salt], similar to cerivastatin in potency and lipophilicity, was terminated in early clinical development due to an unacceptable myotoxicity profile. In this report, we describe the guinea pig as a model of statin-induced cholesterol lowering and myotoxicity and show that this model can distinguish statins with unacceptable myotoxicity profiles from statins with acceptable safety profiles. In our guinea pig model, both cerivastatin and BMS-423526 induced myotoxicity at doses near the ED(50) for total cholesterol (TC) lowering in plasma. In contrast, wide differences between myotoxic and TC-lowering doses were established for the currently marketed, more hydrophilic statins, pravastatin, rosuvastatin, and atorvastatin. This in vivo model compared favorably to an in vitro model, which used statin inhibition of cholesterol synthesis in rat hepatocytes and L6 myoblasts as surrogates of potential efficacy and toxicity, respectively. Our conclusion is that the guinea pig is a useful preclinical in vivo model for demonstrating whether a statin is likely to have an acceptable therapeutic safety margin.
Subject(s)
Guinea Pigs/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/adverse effects , Models, Animal , Animals , Cells, Cultured , Drug Evaluation, Preclinical/methods , Guinea Pigs/blood , Male , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
4-Sulfamoyl pyrroles were designed as novel hepatoselective HMG-CoA reductase inhibitors (statins) to reduce myalgia, a statin-induced adverse effect. The compounds were prepared via a [3+2] cycloaddition of a Münchnone with a sulfonamide-substituted alkyne. We identified compounds with greater selectivity for hepatocytes compared to L6-myocytes than rosuvastatin and atorvastatin. There was an inverse correlation of myocyte potencies and ClogP values. A number of analogs were effective at reducing cholesterol in acute and chronic in vivo models but they lacked sufficient chronic in vivo activity to warrant further development.
Subject(s)
Hydroxymethylglutaryl-CoA Reductase Inhibitors/chemical synthesis , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Muscle Cells/drug effects , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology , Animals , Atorvastatin , Combinatorial Chemistry Techniques , Disease Models, Animal , Fluorobenzenes/pharmacology , Hepatocytes/drug effects , Heptanoic Acids/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/chemistry , Mice , Molecular Structure , Pyrimidines/pharmacology , Pyrroles/chemistry , Rosuvastatin CalciumABSTRACT
An extraordinarily potent and hepatoselective class of HMG-CoA reductase inhibitors containing a pyrazole core was recently reported; however, its development was hampered by a long and difficult synthetic route. We attempted to circumvent this obstacle by preparing closely related analogs wherein the key dihydroxyheptanoic acid sidechain was tethered to the pyrazole core via an oxygen linker ('oxypyrazoles'). This minor change reduced the total number of synthetic steps from 14 to 7. Although the resulting analogs maintained much of the in vitro and cell activity of the pyrazoles, inferior in vivo activity precluded further development. Caco-2 cell permeability data suggest that enhanced cellular efflux of the oxypyrazoles relative to the pyrazoles may be responsible for the poor in vivo activity.
Subject(s)
Drug Design , Hydroxymethylglutaryl CoA Reductases/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/chemistry , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacology , Animals , Cell Line , Cricetinae , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/chemical synthesis , Liver/drug effects , Liver/enzymology , Molecular Structure , Muscle Cells/drug effects , Muscle Cells/enzymology , Pyrazoles/chemical synthesis , Rats , Structure-Activity RelationshipABSTRACT
In a previous report we demonstrated that merging together key structural elements present in an AT(1) receptor antagonist (1, irbesartan) with key structural elements in a biphenylsulfonamide ET(A) receptor antagonist (2) followed by additional optimization provided compound 3 as a dual-action receptor antagonist (DARA), which potently blocked both AT(1) and ET(A) receptors. Described herein are our efforts directed toward improving both the pharmacokinetic profile as well as the AT(1) and ET(A) receptor potency of 3. Our efforts centered on modifying the 2'-side chain of 3 and examining the isoxazolylsulfonamide moiety in 3. This effort resulted in the discovery of 7 as a highly potent second-generation DARA. Compound 7 also showed substantially improved pharmacokinetic properties compared to 3. In rats, DARA 7 reduced blood pressure elevations caused by intravenous infusion of Ang II or big ET-1 to a greater extent and with longer duration than DARA 3 or AT(1) or ET(A) receptor antagonists alone. Compound 7 clearly demonstrated superiority over irbesartan (an AT(1) receptor antagonist) in the normal SHR model of hypertension in a dose-dependent manner, demonstrating the synergy of AT(1) and ET(A) receptor blockade in a single molecule.
Subject(s)
Angiotensin II Type 1 Receptor Blockers/chemistry , Angiotensin II Type 1 Receptor Blockers/pharmacology , Endothelin A Receptor Antagonists , Isoxazoles/chemistry , Isoxazoles/pharmacology , Receptor, Angiotensin, Type 1/drug effects , Sulfonamides/chemistry , Sulfonamides/pharmacology , Administration, Oral , Angiotensin II/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacokinetics , Animals , Antihypertensive Agents/chemistry , Antihypertensive Agents/pharmacology , Biological Availability , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Dogs , Humans , Hypertension/drug therapy , Irbesartan , Isoxazoles/pharmacokinetics , Macaca fascicularis , Male , Rats , Rats, Inbred SHR , Structure-Activity Relationship , Sulfonamides/pharmacokinetics , Tetrazoles/chemistry , Tetrazoles/pharmacologyABSTRACT
Angiotensin II and endothelin-1 activate their respective AT(1) and ET(A) receptors on vascular smooth muscle cells, producing vasoconstriction, and both peptides are implicated in the pathogenesis of essential hypertension. Angiotensin II potentiates the production of endothelin, and conversely endothelin augments the synthesis of angiotensin II. Both AT(1) and ET(A) receptor antagonists lower blood pressure in hypertensive patients; thus, a combination AT(1)/ET(A) receptor antagonist may have greater efficacy and broader utility compared with each drug alone. By rational drug design a biphenyl ET(A) receptor blocker was modified to acquire AT(1) receptor antagonism. These compounds (C and D) decreased Sar-Ile-Angiotensin II binding to AT(1) receptors and endothelin-1 binding to ET(A) receptors, and compound C inhibited angiotensin II- and endothelin-1-mediated Ca(2+) transients. In rats compounds C and D reduced blood pressure elevations caused by intravenous infusion of angiotensin II or big endothelin-1. Compound C decreased blood pressure in Na(+)-depleted spontaneously hypertensive rats and in rats with mineralocorticoid hypertension. Compound D was more efficacious than AT(1) receptor antagonists at reducing blood pressure in spontaneously hypertensive rats, and its superiority was likely due to its partial blockade of ET(A) receptors. Therefore compounds C and D are novel agents for treating a broad spectrum of patients with essential hypertension and other cardiovascular diseases.