Apixaban Use in Obese Patients: A Review of the Pharmacokinetic, Interventional, and Observational Study Data.

Relatively little is known about the influence of extreme body weight on the pharmacokinetics (PK), pharmacodynamics (PD), efficacy, and safety of drugs used in many disease states. While direct oral anticoagulants (DOACs) have an advantage over warfarin in that they do not require routine drug monitoring, some may regard this convenience as less compelling in obese patients. Some consensus guidelines discourage using DOACs in patients weighing > 120 kg or with a body mass index > 35-40 kg/m2, given a sparsity of available data in this population and the concern that fixed dosing in obese patients might lead to decreased drug exposure and lower efficacy. Per the prescribing information, apixaban does not require dose adjustment in patients weighing above a certain threshold (e.g., ≥ 120 kg). Data from healthy volunteers and patients with nonvalvular atrial fibrillation (NVAF) or venous thromboembolism (VTE) have shown that increased body weight has a modest effect on apixaban's PK. However, the paucity of exposure data in individuals > 120 kg and the lack of guideline consensus on DOAC use in obese patients continue to raise concerns about potential decreased drug exposure at extreme weight. This article is the first to comprehensively review the available PK data in obese individuals without NVAF or VTE, and PK, PD, efficacy, effectiveness, and safety data for apixaban in obese patients with either NVAF or VTE, including subgroup analyses across randomized controlled trials and observational (real-world) studies. These data suggest that obesity does not substantially influence the efficacy, effectiveness, or safety of apixaban in these patients. Trial Registration ARISTOTLE: NCT00412984; AVERROES: NCT00496769; AMPLIFY: NCT00643201; AMPLIFY-EXT: NCT00633893; ADVANCE-1: NCT00371683; ADVANCE-2: NCT00452530; ADVANCE-3: NCT00423319 Apixaban Use in Obese Patients: A Review of the Pharmacokinetic, Interventional, and Observational Study Data (MP4 161.22 MB).

Identification of a Hydroxypyrimidinone Compound (21) as a Potent APJ Receptor Agonist for the Potential Treatment of Heart Failure.

This paper describes our continued efforts in the area of small-molecule apelin receptor agonists. Recently disclosed compound 2 showed an acceptable metabolic stability but demonstrated monodemethylation of the dimethoxyphenyl group to generate atropisomer metabolites in vitro. In this article, we extended the structure-activity relationship at the C2 position that led to the identification of potent pyrazole analogues with excellent metabolic stability. Due to the increased polarity at C2, the permeability for these compounds decreased. Further adjustment of the polarity by replacing the N1 2,6-dimethoxyphenyl group with a 2,6-diethylphenyl group and reoptimization for the potency of the C5 pyrroloamides resulted in potent compounds with improved permeability. Compound 21 displayed excellent pharmacokinetic profiles in rat, monkey, and dog models and robust pharmacodynamic efficacy in the rodent heart failure model. Compound 21 also showed an acceptable safety profile in preclinical toxicology studies and was selected as a backup development candidate for the program.

Identification of 6-Hydroxypyrimidin-4(1H)-one-3-carboxamides as Potent and Orally Active APJ Receptor Agonists.

The apelin receptor (APJ) is a significant regulator of cardiovascular function and is involved in heart failure and other cardiovascular diseases. (Pyr1)apelin-13 is one of the endogenous agonists of the APJ receptor. Administration of (Pyr1)apelin-13 increases cardiac output in preclinical models and humans. Recently we disclosed clinical lead BMS-986224 (1), a C3 oxadiazole pyridinone APJ receptor agonist with robust pharmacodynamic effects similar to (Pyr1)apelin-13 in an acute rat pressure-volume loop model. Herein we describe the structure-activity relationship of the carboxamides as oxadiazole bioisosteres at C3 of the pyridinone core and C5 of the respective pyrimidinone core. This study led to the identification of structurally differentiated 6-hydroxypyrimidin-4(1H)-one-3-carboxamide 14a with pharmacodynamic effects comparable to those of compound 1.

Identification of 6-hydroxy-5-phenyl sulfonylpyrimidin-4(1H)-one APJ receptor agonists.

Heart failure (HF) treatment remains a critical unmet medical need. Studies in normal healthy volunteers and HF patients have shown that [Pyr1]apelin-13, the endogenous ligand for the APJ receptor, improves cardiac function. However, the short half-life of [Pyr1]apelin-13 and the need for intravenous administration have limited the therapeutic potential for chronic use. We sought to identify potent, small-molecule APJ agonists with improved pharmaceutical properties to enable oral dosing in clinical studies. In this manuscript, we describe the identification of a series of pyrimidinone sulfones as a structurally differentiated series to the clinical lead (compound 1). Optimization of the sulfone series for potency, metabolic stability and oral bioavailability led to the identification of compound 22, which showed comparable APJ potency to [Pyr1]apelin-13 and exhibited an acceptable pharmacokinetic profile to advance to the acute hemodynamic rat model.

Discovery of a Hydroxypyridinone APJ Receptor Agonist as a Clinical Candidate.

Apelin-13 is an endogenous peptidic agonist of the apelin receptor (APJ) receptor with the potential for improving cardiac function in heart failure patients. However, the low plasma stability of apelin-13 necessitates continuous intravenous infusion for therapeutic use. There are several approaches to increase the stability of apelin-13 including attachment of pharmacokinetic enhancing groups, stabilized peptides, and Fc-fusion approaches. We sought a small-molecule APJ receptor agonist approach to target a compound with a pharmacokinetic profile amenable for chronic oral administration. This manuscript describes sequential optimization of the pyrimidinone series, leading to pyridinone 14, with in vitro potency equivalent to the endogenous ligand apelin-13 and with an excellent oral bioavailability and PK profile in multiple preclinical species. Compound 14 exhibited robust pharmacodynamic effects similar to apelin-13 in an acute rat pressure-volume loop model and was advanced as a clinical candidate.

Discovery and SAR of aryl hydroxy pyrimidinones as potent small molecule agonists of the GPCR APJ.

This article describes the discovery of aryl hydroxy pyrimidinones and the medicinal chemistry efforts to optimize this chemotype for potent APJ agonism. APJ is a G-protein coupled receptor whose natural agonist peptide, apelin, displays hemodynamic improvement in the cardiac function of heart failure patients. A high throughput screen was undertaken to identify small molecule hits that could be optimized to mimic the apelin in vitro response. A potent and low molecular weight aryl hydroxy pyrimidinone analog 30 was identified through optimization of an HTS hit and medicinal chemistry efforts to improve its properties.

Biphenyl Acid Derivatives as APJ Receptor Agonists.

The APJ receptor and its endogenous peptidic ligand apelin have been implicated as important modulators of cardiovascular function, and APJ receptor agonists may be beneficial in the treatment of heart failure. In this article, we describe the discovery of a series of biphenyl acid derivatives as potent APJ receptor agonists. Following the identification of initial high-throughput screen lead 2, successive optimization led to the discovery of lead compound 15a. Compound 15a demonstrated comparable in vitro potency to apelin-13, the endogenous peptidic ligand for the APJ receptor. In vivo, compound 15a demonstrated a dose-dependent improvement in the cardiac output in male Sprague Dawley rats with no significant changes in either mean arterial blood pressure or heart rate, consistent with the hemodynamic profile of apelin-13 in an acute pressure volume loop model.

Genetic Regulation of Adipose Gene Expression and Cardio-Metabolic Traits.

Subcutaneous adipose tissue stores excess lipids and maintains energy balance. We performed expression quantitative trait locus (eQTL) analyses by using abdominal subcutaneous adipose tissue of 770 extensively phenotyped participants of the METSIM study. We identified cis-eQTLs for 12,400 genes at a 1% false-discovery rate. Among an approximately 680 known genome-wide association study (GWAS) loci for cardio-metabolic traits, we identified 140 coincident cis-eQTLs at 109 GWAS loci, including 93 eQTLs not previously described. At 49 of these 140 eQTLs, gene expression was nominally associated (p < 0.05) with levels of the GWAS trait. The size of our dataset enabled identification of five loci associated (p < 5 × 10-8) with at least five genes located >5 Mb away. These trans-eQTL signals confirmed and extended the previously reported KLF14-mediated network to 55 target genes, validated the CIITA regulation of class II MHC genes, and identified ZNF800 as a candidate master regulator. Finally, we observed similar expression-clinical trait correlations of genes associated with GWAS loci in both humans and a panel of genetically diverse mice. These results provide candidate genes for further investigation of their potential roles in adipose biology and in regulating cardio-metabolic traits.

Genetic Architecture of Atherosclerosis in Mice: A Systems Genetics Analysis of Common Inbred Strains.

Common forms of atherosclerosis involve multiple genetic and environmental factors. While human genome-wide association studies have identified numerous loci contributing to coronary artery disease and its risk factors, these studies are unable to control environmental factors or examine detailed molecular traits in relevant tissues. We now report a study of natural variations contributing to atherosclerosis and related traits in over 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). The mice were made hyperlipidemic by transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). The mice were examined for lesion size and morphology as well as plasma lipid, insulin and glucose levels, and blood cell profiles. A subset of mice was studied for plasma levels of metabolites and cytokines. We also measured global transcript levels in aorta and liver. Finally, the uptake of acetylated LDL by macrophages from HMDP mice was quantitatively examined. Loci contributing to the traits were mapped using association analysis, and relationships among traits were examined using correlation and statistical modeling. A number of conclusions emerged. First, relationships among atherosclerosis and the risk factors in mice resemble those found in humans. Second, a number of trait-loci were identified, including some overlapping with previous human and mouse studies. Third, gene expression data enabled enrichment analysis of pathways contributing to atherosclerosis and prioritization of candidate genes at associated loci in both mice and humans. Fourth, the data provided a number of mechanistic inferences; for example, we detected no association between macrophage uptake of acetylated LDL and atherosclerosis. Fifth, broad sense heritability for atherosclerosis was much larger than narrow sense heritability, indicating an important role for gene-by-gene interactions. Sixth, stepwise linear regression showed that the combined variations in plasma metabolites, including LDL/VLDL-cholesterol, trimethylamine N-oxide (TMAO), arginine, glucose and insulin, account for approximately 30 to 40% of the variation in atherosclerotic lesion area. Overall, our data provide a rich resource for studies of complex interactions underlying atherosclerosis.

The genetic architecture of NAFLD among inbred strains of mice.

To identify genetic and environmental factors contributing to the pathogenesis of non-alcoholic fatty liver disease, we examined liver steatosis and related clinical and molecular traits in more than 100 unique inbred mouse strains, which were fed a diet rich in fat and carbohydrates. A >30-fold variation in hepatic TG accumulation was observed among the strains. Genome-wide association studies revealed three loci associated with hepatic TG accumulation. Utilizing transcriptomic data from the liver and adipose tissue, we identified several high-confidence candidate genes for hepatic steatosis, including Gde1, a glycerophosphodiester phosphodiesterase not previously implicated in triglyceride metabolism. We confirmed the role of Gde1 by in vivo hepatic over-expression and shRNA knockdown studies. We hypothesize that Gde1 expression increases TG production by contributing to the production of glycerol-3-phosphate. Our multi-level data, including transcript levels, metabolite levels, and gut microbiota composition, provide a framework for understanding genetic and environmental interactions underlying hepatic steatosis.

Genetic architecture of insulin resistance in the mouse.

Insulin resistance (IR) is a complex trait with multiple genetic and environmental components. Confounded by large differences between the sexes, environment, and disease pathology, the genetic basis of IR has been difficult to dissect. Here we examine IR and related traits in a diverse population of more than 100 unique male and female inbred mouse strains after feeding a diet rich in fat and refined carbohydrates. Our results show dramatic variation in IR among strains of mice and widespread differences between sexes that are dependent on genotype. We uncover more than 15 genome-wide significant loci and validate a gene, Agpat5, associated with IR. We also integrate plasma metabolite levels and global gene expression from liver and adipose tissue to identify metabolite quantitative trait loci (mQTL) and expression QTL (eQTL), respectively. Our results provide a resource for analysis of interactions between diet, sex, and genetic background in IR.

P2Y6 receptor potentiates pro-inflammatory responses in macrophages and exhibits differential roles in atherosclerotic lesion development.

BACKGROUND: P2Y(6), a purinergic receptor for UDP, is enriched in atherosclerotic lesions and is implicated in pro-inflammatory responses of key vascular cell types and macrophages. Evidence for its involvement in atherogenesis, however, has been lacking. Here we use cell-based studies and three murine models of atherogenesis to evaluate the impact of P2Y(6) deficiency on atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Cell-based studies in 1321N1 astrocytoma cells, which lack functional P2Y(6) receptors, showed that exogenous expression of P2Y(6) induces a robust, receptor- and agonist-dependent secretion of inflammatory mediators IL-8, IL-6, MCP-1 and GRO1. P2Y(6)-mediated inflammatory responses were also observed, albeit to a lesser extent, in macrophages endogenously expressing P2Y(6) and in acute peritonitis models of inflammation. To evaluate the role of P2Y(6) in atherosclerotic lesion development, we used P2Y(6)-deficient mice in three mouse models of atherosclerosis. A 43% reduction in aortic arch plaque was observed in high fat-fed LDLR knockout mice lacking P2Y(6) receptors in bone marrow-derived cells. In contrast, no effect on lesion development was observed in fat-fed whole body P2Y(6)xLDLR double knockout mice. Interestingly, in a model of enhanced vascular inflammation using angiotensin II, P2Y(6) deficiency enhanced formation of aneurysms and exhibited a trend towards increased atherosclerosis in the aorta of LDLR knockout mice. CONCLUSIONS: P2Y(6) receptor augments pro-inflammatory responses in macrophages and exhibits a pro-atherogenic role in hematopoietic cells. However, the overall impact of whole body P2Y(6) deficiency on atherosclerosis appears to be modest and could reflect additional roles of P2Y(6) in vascular disease pathophysiologies, such as aneurysm formation.

Genetic regulation of human adipose microRNA expression and its consequences for metabolic traits.

The genetics of messenger RNA (mRNA) expression has been extensively studied in humans and other organisms, but little is known about genetic factors contributing to microRNA (miRNA) expression. We examined natural variation of miRNA expression in adipose tissue in a population of 200 men who have been carefully characterized for metabolic syndrome (MetSyn) phenotypes as part of the Metabolic Syndrome in Men (METSIM) study. We genotyped the subjects using high-density single-nucleotide polymorphism microarrays and quantified the mRNA abundance using genome-wide expression arrays and miRNA abundance using next-generation sequencing. We reliably quantified 356 miRNA species that were expressed in human adipose tissue, a limited number of which made up most of the expressed miRNAs. We mapped the miRNA abundance as an expression quantitative trait and determined cis regulation of expression for nine of the miRNAs and of the processing of one miRNA (miR-28). The degree of genetic variation of miRNA expression was substantially less than that of mRNAs. For the majority of the miRNAs, genetic regulation of expression was independent of the expression of mRNA from which the miRNA is transcribed. We also showed that for 108 miRNAs, mapped reads displayed widespread variation from the canonical sequence. We found a total of 24 miRNAs to be significantly associated with MetSyn traits. We suggest a regulatory role for miR-204-5p which was predicted to inhibit acetyl coenzyme A carboxylase ß, a key fatty acid oxidation enzyme that has been shown to play a role in regulating body fat and insulin resistance in adipose tissue.

11ß-hydroxysteroid dehydrogenase type 1 gene knockout attenuates atherosclerosis and in vivo foam cell formation in hyperlipidemic apoE⁻/⁻ mice.

BACKGROUND: Chronic glucocorticoid excess has been linked to increased atherosclerosis and general cardiovascular risk in humans. The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) increases active glucocorticoid levels within tissues by catalyzing the conversion of cortisone to cortisol. Pharmacological inhibition of 11ßHSD1 has been shown to reduce atherosclerosis in murine models. However, the cellular and molecular details for this effect have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: To examine the role of 11ßHSD1 in atherogenesis, 11ßHSD1 knockout mice were created on the pro-atherogenic apoE⁻/⁻ background. Following 14 weeks of Western diet, aortic cholesterol levels were reduced 50% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. 11ßHSD1⁺/⁺/apoE⁻/⁻ mice without changes in plasma cholesterol. Aortic 7-ketocholesterol content was reduced 40% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. In the aortic root, plaque size, necrotic core area and macrophage content were reduced ∼30% in 11ßHSD1⁻/⁻/apoE⁻/⁻mice. Bone marrow transplantation from 11ßHSD1⁻/⁻/apoE⁻/⁻ mice into apoE⁻/⁻ recipients reduced plaque area 39-46% in the thoracic aorta. In vivo foam cell formation was evaluated in thioglycollate-elicited peritoneal macrophages from 11ßHSD1⁺/⁺/apoE⁻/⁻ and 11ßHSD1⁻/⁻/apoE⁻/⁻ mice fed a Western diet for ∼5 weeks. Foam cell cholesterol levels were reduced 48% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. Microarray profiling of peritoneal macrophages revealed differential expression of genes involved in inflammation, stress response and energy metabolism. Several toll-like receptors (TLRs) were downregulated in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice including TLR 1, 3 and 4. Cytokine release from 11ßHSD1⁻/⁻/apoE⁻/⁻-derived peritoneal foam cells was attenuated following challenge with oxidized LDL. CONCLUSIONS: These findings suggest that 11ßHSD1 inhibition may have the potential to limit plaque development at the vessel wall and regulate foam cell formation independent of changes in plasma lipids. The diminished cytokine response to oxidized LDL stimulation is consistent with the reduction in TLR expression and suggests involvement of 11ßHSD1 in modulating binding of pro-atherogenic TLR ligands.

Hybrid mouse diversity panel: a panel of inbred mouse strains suitable for analysis of complex genetic traits.

We have developed an association-based approach using classical inbred strains of mice in which we correct for population structure, which is very extensive in mice, using an efficient mixed-model algorithm. Our approach includes inbred parental strains as well as recombinant inbred strains in order to capture loci with effect sizes typical of complex traits in mice (in the range of 5% of total trait variance). Over the last few years, we have typed the hybrid mouse diversity panel (HMDP) strains for a variety of clinical traits as well as intermediate phenotypes and have shown that the HMDP has sufficient power to map genes for highly complex traits with resolution that is in most cases less than a megabase. In this essay, we review our experience with the HMDP, describe various ongoing projects, and discuss how the HMDP may fit into the larger picture of common diseases and different approaches.

Systems genetics analysis of gene-by-environment interactions in human cells.

Gene by environment (GxE) interactions are clearly important in many human diseases, but they have proven to be difficult to study on a molecular level. We report genetic analysis of thousands of transcript abundance traits in human primary endothelial cell (EC) lines in response to proinflammatory oxidized phospholipids implicated in cardiovascular disease. Of the 59 most regulated transcripts, approximately one-third showed evidence of GxE interactions. The interactions resulted primarily from effects of distal-, trans-acting loci, but a striking example of a local-GxE interaction was also observed for FGD6. Some of the distal interactions were validated by siRNA knockdown experiments, including a locus involved in the regulation of multiple transcripts involved in the ER stress pathway. Our findings add to the understanding of the overall architecture of complex human traits and are consistent with the possibility that GxE interactions are responsible, in part, for the failure of association studies to more fully explain common disease variation.

Quantitative trait locus mapping and identification of Zhx2 as a novel regulator of plasma lipid metabolism.

BACKGROUND: We previously mapped a quantitative trait locus on chromosome 15 in mice contributing to high-density lipoprotein cholesterol and triglyceride levels and now report the identification of the underlying gene. METHODS AND RESULTS: We first fine-mapped the locus by studying a series of congenic strains derived from the parental strains BALB/cJ and MRL/MpJ. Analysis of gene expression and sequencing followed by transgenic complementation led to the identification of zinc fingers and homeoboxes 2 (Zhx2), a transcription factor previously implicated in the developmental regulation of alpha-fetoprotein. Reduced expression of the protein in BALB/cJ mice resulted in altered hepatic transcript levels for several genes involved in lipoprotein metabolism. Most notably, the Zhx2 mutation resulted in a failure to suppress expression of lipoprotein lipase, a gene normally silenced in the adult liver, and this was normalized in BALB/cJ mice carrying the Zhx2 transgene. CONCLUSIONS: We identified the gene underlying the chromosome 15 quantitative trait locus, and our results show that Zhx2 functions as a novel developmental regulator of key genes influencing lipoprotein metabolism.

Atherogenesis on the chopping block.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are now established features of the atherosclerotic plaque. In this issue, Thorp et al. (2009) provide initial insights into the causative relationship between UPR and the atherosclerotic disease process, specifically linking the proapoptotic mediator CHOP to plaque growth and necrosis.

CHAC1/MGC4504 is a novel proapoptotic component of the unfolded protein response, downstream of the ATF4-ATF3-CHOP cascade.

To understand pathways mediating the inflammatory responses of human aortic endothelial cells to oxidized phospholipids, we previously used a combination of genetics and genomics to model a coexpression network encompassing >1000 genes. CHAC1 (cation transport regulator-like protein 1), a novel gene regulated by ox-PAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine), was identified in a co-regulated group of genes enriched for components of the ATF4 (activating transcription factor 4) arm of the unfolded protein response pathway. Herein, we characterize the role of CHAC1 and validate the network model. We first define the activation of CHAC1 mRNA by chemical unfolded protein response-inducers, but not other cell stressors. We then define activation of CHAC1 by the ATF4-ATF3-CHOP (C/EBP homologous protein), and not parallel XBP1 (X box-binding protein 1) or ATF6 pathways, using siRNA and/or overexpression plasmids. To examine the subset of genes downstream of CHAC1, we used expression microarray analysis to identify a list of 227 differentially regulated genes. We validated the activation of TNFRSF6B (tumor necrosis factor receptor superfamily, member 6b), a FASL decoy receptor, in cells treated with CHAC1 small interfering RNA. Finally, we showed that CHAC1 overexpression enhanced apoptosis, while CHAC1 small interfering RNA suppressed apoptosis, as determined by TUNEL, PARP (poly(ADP-ribose) polymerase) cleavage, and AIF (apoptosis-inducing factor) nuclear translocation.