RESUMEN
BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.
Asunto(s)
Anticolesterolemiantes , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Hipercolesterolemia , Adulto , Humanos , Anticolesterolemiantes/efectos adversos , Colesterol , LDL-Colesterol , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Péptidos/uso terapéutico , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
Cholesteryl ester transfer protein (CETP) inhibitors reduce the transfer of cholesteryl esters from the high-density lipoprotein (HDL-C) to apolipoprotein such as VLDL/LDL, with exchange of triglycerides. Thus, this inhibition increases the HDL-C levels, which is believed to lower the risk for heart disease and stroke. We report here a series of CETP inhibitors based on the cyclic, bicyclic urea and sulfamide cores. These CETP inhibitors exemplified by 15, 31, and 45 demonstrated in vitro potency in inhibiting the CETP transfer activity, and 15, 31 showing in vivo efficacy to increase HDL-C levels in cynomolgus-CETP transgenic mice. The synthesis and biological evaluations of these CETP inhibitors are described.
Asunto(s)
Anticolesterolemiantes/síntesis química , Compuestos Bicíclicos con Puentes/química , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Sulfonamidas/química , Urea/análogos & derivados , Animales , Anticolesterolemiantes/metabolismo , Anticolesterolemiantes/uso terapéutico , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , HDL-Colesterol/sangre , Ciclización , Dislipidemias/tratamiento farmacológico , Dislipidemias/patología , Humanos , Ratones , Ratones Transgénicos , Relación Estructura-Actividad , Sulfonamidas/metabolismo , Sulfonamidas/uso terapéutico , Urea/metabolismo , Urea/uso terapéuticoRESUMEN
Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, a zebrafish model of doxorubicin-induced cardiomyopathy was previously established for small-molecule screening. Using this model, several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish and in mouse models have previously been identified. In this study, exploration of doxorubicin cardiotoxicity is expanded by screening 2271 small molecules from a proprietary, target-annotated tool compound collection. It is found that 120 small molecules can prevent doxorubicin-induced cardiotoxicity, including 7 highly effective compounds. Of these, all seven exhibited inhibitory activity towards cytochrome P450 familyâ 1 (CYP1). These results are consistent with previous findings, in which visnagin, a CYP1 inhibitor, also prevents doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection.
Asunto(s)
Cardiomiopatías/prevención & control , Familia 1 del Citocromo P450/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Cardiomiopatías/inducido químicamente , Cardiomiopatías/patología , Familia 1 del Citocromo P450/antagonistas & inhibidores , Familia 1 del Citocromo P450/genética , Modelos Animales de Enfermedad , Doxorrubicina/toxicidad , Insuficiencia Cardíaca , Mutagénesis , Fenotipo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-Actividad , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
Anacetrapib is an inhibitor of cholesteryl ester transfer protein (CETP), associated with reduction in LDL cholesterol and increase in HDL cholesterol in hypercholesterolemic patients. Anacetrapib was not taken forward into filing/registration as a new drug for coronary artery diease, despite the observation of a â¼9% reduction in cardiovascular risk in a large phase III cardiovascular outcomes trial (REVEAL). Anacetrapib displayed no adverse effects throughout extensive preclinical safety evaluation, and no major safety signals were observed in clinical trials studying anacetrapib, including REVEAL. However, anacetrapib demonstrated a long terminal half-life in all species, thought to be due, in part, to distribution into adipose tissue. We sought to understand the dependence of anacetrapib's long half-life on adipose tissue and to explore potential mechanisms that might contribute to the phenomenon. In mice, anacetrapib localized primarily to the lipid droplet of adipocytes in white adipose tissue; in vitro, anacetrapib entry into cultured human adipocytes depended on the presence of a mature adipocyte and lipid droplet but did not require active transport. In vivo, the entry of anacetrapib into adipose tissue did not require lipase activity, as the distribution of anacetrapib into adipose was-not affected by systemic lipase inhibition using poloaxamer-407, a systemic lipase inhibitor. The data from these studies support the notion that the entry of anacetrapib into adipose tissue/lipid droplets does not require active transport, nor does it require mobilization or entry of fat into adipose via lipolysis.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Gotas Lipídicas/metabolismo , Oxazolidinonas/farmacología , Adipocitos/citología , Tejido Adiposo/citología , Animales , Línea Celular , HDL-Colesterol/sangre , HDL-Colesterol/metabolismo , LDL-Colesterol/sangre , LDL-Colesterol/metabolismo , Semivida , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Hipercolesterolemia/sangre , Hipercolesterolemia/tratamiento farmacológico , Lipasa/antagonistas & inhibidores , Lipasa/metabolismo , Lipólisis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Oxazolidinonas/administración & dosificación , Oxazolidinonas/uso terapéutico , Poloxámero/farmacología , Distribución Tisular/efectos de los fármacosRESUMEN
Drug discovery and development efforts are largely based around a common expectation, namely, that direct or indirect action on a cellular process (e.g., statin-mediated enzyme inhibition or insulin-stimulated receptor activation) will have a beneficial impact on physiologic homeostasis. To expand on this, one could argue that virtually all pharmacologic interventions attempt to influence the flow of "traffic" in a biochemical network, irrespective of disease or modality. Since stable isotope tracer kinetic methods provide a measure of traffic flow (i.e., metabolic flux), their inclusion in study designs can yield novel information regarding pathway biology; the application of such methods requires the integration of knowledge in physiology, analytical chemistry, and mathematical modeling. Herein, we review the fundamental concepts that surround the use of tracer kinetics, define basic terms, and outline guiding principles via theoretical and experimental problems. Specifically, one needs to 1) recognize the types of biochemical events that change isotopic enrichments, 2) appreciate the distinction between fractional turnover and flux rate, and 3) be aware of the subtle differences between tracer kinetics and pharmacokinetics. We hope investigators can use the framework presented here to develop applications that address their specific questions surrounding biochemical flux, and thereby gain insight into the pathophysiology of disease states, and examine pharmacodynamic mechanisms.
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Descubrimiento de Drogas/métodos , Análisis de Flujos Metabólicos/métodos , Animales , Humanos , Marcaje Isotópico , Isótopos/química , Agua/química , Agua/metabolismoRESUMEN
Cholesteryl ester transfer protein (CETP) inhibitors are a new class of therapeutics for dyslipidemia that simultaneously improve two major cardiovascular disease (CVD) risk factors: elevated low-density lipoprotein (LDL) cholesterol and decreased high-density lipoprotein (HDL) cholesterol. However, the detailed molecular mechanisms underlying their efficacy are poorly understood, as are any potential mechanistic differences among the drugs in this class. Herein, we used electron microscopy (EM) to investigate the effects of three of these agents (Torcetrapib, Dalcetrapib and Anacetrapib) on CETP structure, CETP-lipoprotein complex formation and CETP-mediated cholesteryl ester (CE) transfer. We found that although none of these inhibitors altered the structure of CETP or the conformation of CETP-lipoprotein binary complexes, all inhibitors, especially Torcetrapib and Anacetrapib, increased the binding ratios of the binary complexes (e.g., HDL-CETP and LDL-CETP) and decreased the binding ratios of the HDL-CETP-LDL ternary complexes. The findings of more binary complexes and fewer ternary complexes reflect a new mechanism of inhibition: one distal end of CETP bound to the first lipoprotein would trigger a conformational change at the other distal end, thus resulting in a decreased binding ratio to the second lipoprotein and a degraded CE transfer rate among lipoproteins. Thus, we suggest a new inhibitor design that should decrease the formation of both binary and ternary complexes. Decreased concentrations of the binary complex may prevent the inhibitor was induced into cell by the tight binding of binary complexes during lipoprotein metabolism in the treatment of CVD.
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Proteínas de Transferencia de Ésteres de Colesterol , Lipoproteínas HDL/química , Lipoproteínas LDL/química , Complejos Multiproteicos , Oxazolidinonas/química , Quinolinas/química , Compuestos de Sulfhidrilo/química , Amidas , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Proteínas de Transferencia de Ésteres de Colesterol/química , Ésteres , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructuraRESUMEN
BACKGROUND: This study determined whether relaxin or matrix metalloproteinase (MMP)-9 influences angiotensin II (AngII)-induced abdominal aortic aneurysms (AAA).MethodsâandâResults:Male C57BL/6 or apolipoprotein E-/-mice were infused with AngII with or without relaxin. Relaxin did not influence AngII-induced AAA in either mouse strain. Infusion of AngII reduced, but relaxin increased, MMP-9 mRNA in macrophages. We then determined the effects of MMP-9 deficiency on AAA in apolipoprotein E-/-mice. MMP-9 deficiency led to AAA formation in the absence of AngII, and augmented AngII-induced aortic rupture and AAA incidence. CONCLUSIONS: MMP-9 deficiency augmented AngII-induced AAA.
Asunto(s)
Angiotensina II/efectos adversos , Aneurisma de la Aorta Abdominal/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Relaxina/biosíntesis , Angiotensina II/farmacología , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/patología , Apolipoproteínas E/deficiencia , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Noqueados , Relaxina/genéticaRESUMEN
Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is â¼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Oxazolidinonas/farmacología , Triglicéridos/metabolismo , Animales , Lipoproteínas HDL/sangre , Macaca mulatta , Masculino , Modelos Biológicos , Triglicéridos/sangreRESUMEN
Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) that affect a broad range of physiological processes, including cell proliferation, inflammation, inflammation resolution, and vascular function. Moreover, oxylipins are readily detectable in plasma, and certain subsets of oxylipins have been detected in human atherosclerotic lesions. Taken together, we set out to produce a detailed quantitative assessment of plasma and plaque oxylipins in a widely used model of atherosclerosis, to identify potential biomarkers of disease progression. We administered regular chow or regular chow supplemented with 0.5% cholesterol (HC) to male New Zealand white rabbits for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our targeted lipidomic analyses of oxylipins on plaques isolated from rabbits fed the HC diet detected 34 oxylipins, 28 of which were in compliance with our previously established quality control acceptance criteria. The arachidonic acid (AA) metabolite derived from the COX pathway, 6-keto-PGF1α was the most abundant plaque oxylipin, followed by the linoleic acid (LA) metabolites 9-HODE, 13-HODE and 9,12,13-TriHOME and the arachidonic acid (AA)-derivatives 11-HETE and 12-HETE. We additionally found that the most abundant oxylipins in plasma were three of the five most abundant oxylipins in plaque, namely 11-HETE, 13-HODE, and 9-HODE. The studies reported here make the first step towards a comprehensive characterization of oxylipins as potentially translatable biomarkers of atherosclerosis.
Asunto(s)
Hipercolesterolemia/sangre , Oxilipinas/sangre , Placa Aterosclerótica/sangre , Animales , Cromatografía Líquida de Alta Presión , Ácidos Grasos Insaturados/metabolismo , Humanos , Hipercolesterolemia/metabolismo , Masculino , Espectrometría de Masas , Oxilipinas/metabolismo , Placa Aterosclerótica/metabolismo , ConejosRESUMEN
Our ability to understand the pathogenesis of problems surrounding lipid accretion requires attention towards quantifying lipid kinetics. In addition, studies of metabolic flux should also help unravel mechanisms that lead to imbalances in inter-organ lipid trafficking which contribute to dyslipidemia and/or peripheral lipid accumulation (e.g. hepatic fat deposits). This review aims to outline the development and use of novel methods for studying lipid kinetics in vivo. Although our focus is directed towards some of the approaches that are currently reported in the literature, we include a discussion of the older literature in order to put "new" methods in better perspective and inform readers of valuable historical research. Presumably, future advances in understanding lipid dynamics will benefit from a careful consideration of the past efforts, where possible we have tried to identify seminal papers or those that provide clear data to emphasize essential points. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.
Asunto(s)
Tejido Adiposo/metabolismo , Metabolismo de los Lípidos , Lípidos/biosíntesis , Triglicéridos/metabolismo , Distribución de la Grasa Corporal , Colesterol/biosíntesis , Colesterol/metabolismo , Metabolismo Energético , Humanos , Cinética , Triglicéridos/químicaRESUMEN
Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2weeks, or from normal human subjects treated either placebo, 20mg, or 150mg anacetrapib daily for 2weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells.
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Antiinflamatorios/farmacología , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Lipoproteínas HDL/farmacología , Oxazolidinonas/farmacología , Células Cultivadas , Selectina E/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
RATIONALE: The ability to quantify rates of formation, regression and/or remodeling of atherosclerotic plaque should facilitate a better understanding of the pathogenesis and management of cardiovascular disease. In the current study, we coupled a stable isotope labeled tracer protocol with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to examine spatial and temporal lipid dynamics in atherosclerotic plaque. METHODS: To promote plaque formation in the aorta region, ApoE KO mice were fed a high cholesterol diet (0.15% cholesterol) and orally dosed with (2,2,3,4,4,6-d(6))-cholesterol over several weeks. Tissue sections of ~10 µm thickness were analyzed by MALDI-MSI using matrix deposition by either chemical sublimation or acoustic droplet ejection. RESULTS: MALDI-MSI yielded distinct spatial distribution information for a variety of lipid classes including specific lysophosphatidylcholines typically associated with atherosclerosis-related tissue damage such as phospholipase 2 (Lp-PLA(2)) that mediate chemotactic responses to inflammation (e.g. LPC 16:0, LPC 18:0 and LPC 18:1) as well as free cholesterol and cholesteryl esters that contribute to atheroma formation. MALDI mass spectra acquired from aorta tissue sections clearly distinguished non-esterified and esterified versions of (2,2,3,4,4,6-d(6))-cholesterol within aortic plaque regions and showed distinct spatial accumulation of the cholesterol tracer. CONCLUSIONS: The ability to couple stable isotope based protocols with MALDI-MSI enables a novel strategy to characterize the effects of therapeutic treatments on atherosclerotic plaque formation, regression and potential remodeling of the complex lipid components with high chemical specificity and spatiotemporal information.
RESUMEN
RATIONALE: The ability to measure low levels of (2)H-labeling is important in studies of metabolic flux, e.g. one can estimate lipid synthesis by administering (2)H2O and then measuring the incorporation of (2)H into fatty acids. Unfortunately, the analyses are complicated by the presence of more abundant naturally occurring stable isotopes, e.g. (13)C. Conventional approaches rely on coupling gas chromatographic separation of lipids with either quadrupole-mass spectrometry (q-MS) and/or pyrolysis-isotope ratio mass spectrometry (IRMS). The former is limited by high background labeling (primarily from (13)C) whereas the latter is not suitable for routine high-throughput analyses. METHODS: We have contrasted the use of continuous flow-pyrolysis-IRMS against high-resolution mass spectrometry (i.e. Qq-FT-ICR MS) for measuring the (2)H-enrichment of fatty acids and peptides. RESULTS: In contrast to IRMS, which requires ~30 min per analysis, it is possible to measure the (2)H-enrichment of palmitate via direct infusion high-resolution mass spectrometry (HRMS) in ~3 min per sample. In addition, Qq-FT-ICR MS enabled measurements of the (2)H-enrichment of peptides (which is not possible using IRMS). CONCLUSIONS: High-resolution mass spectrometry can be used to measure low levels of (2)H-labeling so we expect that this approach will enhance studies of metabolic flux that rely on (2)H-labeled tracers, e.g. (2)H2O. However, since the high-resolution analyses require greater amounts of a given analyte one potential limitation centers on the overall sensitivity. Presumably, future advances can overcome this barrier.
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Deuterio/análisis , Ácidos Grasos/química , Marcaje Isotópico/métodos , Espectrometría de Masas/métodos , Animales , Chlorocebus aethiops , Deuterio/química , Deuterio/metabolismo , Óxido de Deuterio/administración & dosificación , Ácidos Grasos/metabolismo , Femenino , Modelos Lineales , Macaca mulatta , Masculino , Péptidos/química , Péptidos/metabolismoRESUMEN
The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome.
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Hipercolesterolemia/metabolismo , Metabolismo de los Lípidos , Placa Aterosclerótica/metabolismo , Animales , Ceramidas/sangre , Ésteres del Colesterol/sangre , Ácidos Grasos/sangre , Glicerofosfolípidos/sangre , Hipercolesterolemia/complicaciones , Masculino , Placa Aterosclerótica/etiología , Conejos , Esfingomielinas/sangre , Triglicéridos/sangreRESUMEN
Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol and lowers LDL cholesterol in dyslipidemic patients. We previously demonstrated that ANA increases macrophage-to-feces reverse cholesterol transport and fecal cholesterol excretion in hamsters, and increased preß HDL-dependent cholesterol efflux via ABCA1 in vitro. However, the effects of ANA on in vivo preß HDL have not been characterized. In vitro, ANA inhibited the formation of preß, however in ANA-treated dyslipidemic hamsters, preß HDL levels (measured by two-dimensional gel electrophoresis) were increased, in contrast to in vitro findings. Because changes in plasma preß HDL have been proposed to potentially affect markers of cholesterol absorption with other CETP inhibitors, a dual stable isotope method was used to directly measure cholesterol absorption in hamsters. ANA treatment of hamsters (on either dyslipidemic or normal diet) had no effect on cholesterol absorption, while dalcetrapib-treated hamsters displayed an increase in cholesterol absorption. Taken together, these data support the notion that ANA promotes preß HDL functionality in vivo, with no effects on cholesterol absorption.
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Anticolesterolemiantes/farmacología , Colesterol/metabolismo , Dislipidemias/tratamiento farmacológico , Lipoproteínas de Alta Densidad Pre-beta/sangre , Absorción Intestinal/efectos de los fármacos , Oxazolidinonas/farmacología , Amidas , Animales , Anticolesterolemiantes/uso terapéutico , Área Bajo la Curva , Azetidinas/farmacología , Azetidinas/uso terapéutico , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Cricetinae , Dieta Alta en Grasa/efectos adversos , Evaluación Preclínica de Medicamentos , Dislipidemias/sangre , Dislipidemias/etiología , Ésteres , Ezetimiba , Humanos , Masculino , Mesocricetus , Oxazolidinonas/uso terapéutico , Compuestos de Sulfhidrilo/farmacología , Compuestos de Sulfhidrilo/uso terapéuticoRESUMEN
Isotopic tracers have been used to examine lipid trafficking for many years, and data from those studies have typically yielded novel insight regarding the pathophysiology of dyslipidemia. Previous experimental designs were suitable for studies in humans because relatively large volumes of plasma could be regularly sampled. We have expanded on the earlier logic by applying high-throughput analytical methods that require reduced sample volumes. Specifically, we have examined the possibility of coupling gel-based separations of lipoproteins (e.g., lipoprint) with LC-MS/MS analyses of complex lipid mixtures as a way to routinely measure the labeling profiles of distinct lipids in discrete lipoprotein subfractions. We demonstrate the ability to measure the incorporation of [U-(13)C]oleate into triglycerides (TG), PLs (PL), and cholesterol esters (CE) in VLDL, LDL, and HDL particles in mice. Although rodent models of dyslipidemia are inherently different from humans because of alterations in enzyme activities and underlying metabolism, rodent models can be used to screen novel compounds for efficacy in altering a given biochemical pathway and therein enable studies of target engagement in vivo. We expect that it is possible to translate our approach for application in other systems, including studies in humans.
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Fraccionamiento Químico/métodos , Dislipidemias/metabolismo , Ácidos Grasos/metabolismo , Lipoproteínas/aislamiento & purificación , Lipoproteínas/metabolismo , Animales , Transporte Biológico , Modelos Animales de Enfermedad , Humanos , Marcaje Isotópico , Lipoproteínas/química , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
We have previously reported on a liquid chromatography-mass spectrometry method to determine the disposition of [(13)C18]-oleic acid following intravenous and oral administration in vivo. This approach has enabled us to study a variety of aspects of lipid metabolism including a quantitative assessment of triglyceride synthesis. Here we present a more rigorous evaluation of the constraints imposed upon the analytical method in order to generate accurate data using this stable-isotope tracer approach along with more detail on relevant analytical figures of merit including limits of quantitation, precision, and accuracy. The use of mass isotopomer distribution analysis (MIDA) to quantify plasma triglyceride synthesis is specifically highlighted, and a re-evaluation of the underlying mathematics has enabled us to present a simplified series of equations. The derivation of this MIDA model and the significance of all underlying assumptions are explored in detail, and examples are given of how it can successfully be applied to detect differences in plasma triglyceride synthesis in lean and high-fat diet fed mouse models. More work is necessary to evaluate the applicability of this approach to triglyceride stores with slower rates of turnover such as in adipose or muscle tissue; however, the present report provides investigators with the tools necessary to conduct such studies.
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Espectrometría de Masas/métodos , Ácido Oléico/análisis , Triglicéridos/biosíntesis , Triglicéridos/sangre , Animales , Isótopos de Carbono , Ratones , Ratones Endogámicos C57BL , Obesidad/sangre , Obesidad/diagnóstico , Ácido Oléico/administración & dosificaciónRESUMEN
NADPH oxidase isoform-2 (NOX2) generates reactive oxygen species (ROS) that contribute to neurodegenerative and cardiovascular pathologies. However, validation of NOX2 as a pharmacotherapeutic target has been hampered by a lack of mechanistically-defined inhibitors. Using cellular and biochemical assays, we explored previously reported inhibitors of ROS production (perhexiline, suramin, VAS2870 and two Shionogi patent compounds) as direct NOX2 inhibitors. All but suramin, which presumably lacks cell penetrance, inhibit cellular ROS production. However, only perhexiline and suramin inhibit biochemical NOX2 activity. Indeed, our data suggest that NOX2 inhibition by perhexiline may contribute significantly to its demonstrated cardioprotective effects. Inhibition of protein kinase CßII explains the cellular activity of the Shionogi compounds, whereas VAS2870 inhibits by an as-yet unidentified mechanism unrelated to direct NOX2 function or subunit assembly. These data delineate the mechanisms of action of these compounds and highlight their strengths and limitations for use in future target validation studies.
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Inhibidores Enzimáticos/farmacología , Glicoproteínas de Membrana/antagonistas & inhibidores , NADPH Oxidasas/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Secuencia de Bases , Benzoxazoles/farmacología , Fármacos Cardiovasculares/farmacología , Células Cultivadas , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Neutrófilos/metabolismo , Perhexilina/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C beta , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Suramina/farmacología , Triazoles/farmacologíaRESUMEN
Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.
Asunto(s)
Apolipoproteínas/biosíntesis , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Agua/administración & dosificación , Animales , Apolipoproteínas/sangre , Apolipoproteínas/metabolismo , Marcaje Isotópico , Masculino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/metabolismo , ProteolisisRESUMEN
Intracellular proteins are in a state of flux, continually being degraded into amino acids and resynthesized into new proteins. The rate of this biochemical recycling process varies across proteins and is emerging as an important consideration in drug discovery and development. Here, we developed a triple-stage quadrupole mass spectrometry assay based on product ion measurements at unit resolution and H(2)(18)O stable tracer incorporation to measure relative protein synthesis rates. As proof of concept, we selected to measure the relative in vivo synthesis rate of ApoB100, an apolipoprotein where elevated levels are associated with an increased risk of coronary heart disease, in plasma-isolated very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in a mouse in vivo model. In addition, serial time points were acquired to measure the relative in vivo synthesis rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two potential therapeutic targets to reduce plasma ApoB100 levels at 2 and 6 h post-tracer-injection. The combination of H(2)(18)O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment.