Effects of APOC3 Heterozygous Deficiency on Plasma Lipid and Lipoprotein Metabolism.

Objective- Apo (apolipoprotein) CIII inhibits lipoprotein lipase (LpL)-mediated lipolysis of VLDL (very-low-density lipoprotein) triglyceride (TG) and decreases hepatic uptake of VLDL remnants. The discovery that 5% of Lancaster Old Order Amish are heterozygous for the APOC3 R19X null mutation provided the opportunity to determine the effects of a naturally occurring reduction in apo CIII levels on the metabolism of atherogenic containing lipoproteins. Approach and Results- We conducted stable isotope studies of VLDL-TG and apoB100 in 5 individuals heterozygous for the null mutation APOC3 R19X (CT) and their unaffected (CC) siblings. Fractional clearance rates and production rates of VLDL-TG and apoB100 in VLDL, IDL (intermediate-density lipoprotein), LDL, apo CIII, and apo CII were determined. Affected (CT) individuals had 49% reduction in plasma apo CIII levels compared with CCs ( P<0.01) and reduced plasma levels of TG (35%, P<0.02), VLDL-TG (45%, P<0.02), and VLDL-apoB100 (36%, P<0.05). These changes were because of higher fractional clearance rates of VLDL-TG and VLDL-apoB100 with no differences in production rates. CTs had higher rates of the conversion of VLDL remnants to LDL compared with CCs. In contrast, rates of direct removal of VLDL remnants did not differ between the groups. As a result, the flux of apoB100 from VLDL to LDL was not reduced, and the plasma levels of LDL-cholesterol and LDL-apoB100 were not lower in the CT group. Apo CIII production rate was lower in CTs compared with CCs, whereas apo CII production rate was not different between the 2 groups. The fractional clearance rates of both apo CIII and apo CII were higher in CTs than CCs. Conclusions- These studies demonstrate that 50% reductions in plasma apo CIII, in otherwise healthy subjects, results in a significantly higher rate of conversion of VLDL to LDL, with little effect on direct hepatic uptake of VLDL. When put in the context of studies demonstrating significant protection from cardiovascular events in individuals with loss of function variants in the APOC3 gene, our results provide strong evidence that therapies which increase the efficiency of conversion of VLDL to LDL, thereby reducing remnant concentrations, should reduce the risk of cardiovascular disease.

Genome-wide association analysis of common genetic variants of resistant hypertension.

Resistant hypertension (RHTN), defined as uncontrolled blood pressure (BP) ≥ 140/90 using three or more drugs or controlled BP (<140/90) using four or more drugs, is associated with adverse outcomes, including decline in kidney function. We conducted a genome-wide association analysis in 1194 White and Hispanic participants with hypertension and coronary artery disease from the INternational VErapamil-SR Trandolapril STudy-GENEtic Substudy (INVEST-GENES). Top variants associated with RHTN at p < 10-4 were tested for replication in 585 White and Hispanic participants with hypertension and subcortical strokes from the Secondary Prevention of Subcortical Strokes GENEtic Substudy (SPS3-GENES). A genetic risk score for RHTN was created by summing the risk alleles of replicated RHTN signals. rs11749255 in MSX2 was associated with RHTN in INVEST (odds ratio (OR) (95% CI) = 1.50 (1.2-1.8), p = 7.3 × 10-5) and replicated in SPS3 (OR = 2.0 (1.4-2.8), p = 4.3 × 10-5), with genome-wide significance in meta-analysis (OR = 1.60 (1.3-1.9), p = 3.8 × 10-8). Other replicated signals were in IFLTD1 and PTPRD. IFLTD1 rs6487504 was associated with RHTN in INVEST (OR = 1.90 (1.4-2.5), p = 1.1 × 10-5) and SPS3 (OR = 1.70 (1.2-2.5), p = 4 × 10-3). PTPRD rs324498, a previously reported RHTN signal, was among the top signals in INVEST (OR = 1.60 (1.3-2.0), p = 3.4 × 10-5) and replicated in SPS3 (OR = 1.60 (1.1-2.4), one-sided p = 0.005). Participants with the highest number of risk alleles were at increased risk of RHTN compared to participants with a lower number (p-trend = 1.8 × 10-15). Overall, we identified and replicated associations with RHTN in the MSX2, IFLTD1, and PTPRD regions, and combined these associations to create a genetic risk score.

Genome-wide and candidate gene approaches of clopidogrel efficacy using pharmacodynamic and clinical end points-Rationale and design of the International Clopidogrel Pharmacogenomics Consortium (ICPC).

RATIONALE: The P2Y12 receptor inhibitor clopidogrel is widely used in patients with acute coronary syndrome, percutaneous coronary intervention, or ischemic stroke. Platelet inhibition by clopidogrel shows wide interpatient variability, and high on-treatment platelet reactivity is a risk factor for atherothrombotic events, particularly in high-risk populations. CYP2C19 polymorphism plays an important role in this variability, but heritability estimates suggest that additional genetic variants remain unidentified. The aim of the International Clopidogrel Pharmacogenomics Consortium (ICPC) is to identify genetic determinants of clopidogrel pharmacodynamics and clinical response. STUDY DESIGN: Based on the data published on www.clinicaltrials.gov, clopidogrel intervention studies containing genetic and platelet function data were identified for participation. Lead investigators were invited to share DNA samples, platelet function test results, patient characteristics, and cardiovascular outcomes to perform candidate gene and genome-wide studies. RESULTS: In total, 17 study sites from 13 countries participate in the ICPC, contributing individual patient data from 8,829 patients. Available adenosine diphosphate-stimulated platelet function tests included vasodilator-stimulated phosphoprotein assay, light transmittance aggregometry, and the VerifyNow P2Y12 assay. A proof-of-principle analysis based on genotype data provided by each group showed a strong and consistent association between CYP2C19*2 and platelet reactivity (P value=5.1 × 10-40). CONCLUSION: The ICPC aims to identify new loci influencing clopidogrel efficacy by using state-of-the-art genetic approaches in a large cohort of clopidogrel-treated patients to better understand the genetic basis of on-treatment response variability.

Pharmacogenetic Associations of ß1-Adrenergic Receptor Polymorphisms With Cardiovascular Outcomes in the SPS3 Trial (Secondary Prevention of Small Subcortical Strokes).

BACKGROUND AND PURPOSE: Functional polymorphisms (Ser49Gly and Arg389Gly) in ADRB1 have been associated with cardiovascular and ß-blocker response outcomes. Herein we examined associations of these polymorphisms with major adverse cardiovascular events (MACE), with and without stratification by ß-blocker treatment in patients with a history of stroke. METHODS: Nine hundred and twenty-six participants of the SPS3 trial's (Secondary Prevention of Small Subcortical Strokes) genetic substudy with hypertension were included. MACE included stroke, myocardial infarction, and all-cause death. Kaplan-Meier and multivariable Cox regression analyses were used. Because the primary component of MACE was ischemic stroke, we tested the association of Ser49Gly with ischemic stroke among 41 475 individuals of European and African ancestry in the NINDS (National Institute of Neurological Disorders and Stroke) SiGN (Stroke Genetics Network). RESULTS: MACE was higher in carriers of the Gly49 allele than in those with the Ser49Ser genotype (10.5% versus 5.4%, log-rank P=0.005). Gly49 carrier status was associated with MACE (hazard ratio, 1.62; 95% confidence interval, 1.00-2.68) and ischemic stroke (hazard ratio, 1.81; 95% confidence interval, 1.01-3.23) in SPS3 and with small artery ischemic stroke (odds ratio, 1.14; 95% confidence interval, 1.03-1.26) in SiGN. In SPS3, ß-blocker-treated Gly49 carriers had increased MACE versus non-ß-blocker-treated individuals and noncarriers (hazard ratio, 2.03; 95% confidence interval, 1.20-3.45). No associations were observed with the Arg389Gly polymorphism. CONCLUSION: Among individuals with previous small artery ischemic stroke, the ADRB1 Gly49 polymorphism was associated with MACE, particularly small artery ischemic stroke, a risk that may be increased among ß-blocker-treated individuals. Further research is needed to define ß-blocker benefit among ischemic stroke patients by ADRB1 genotype. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00059306.

Genome-wide analysis of clopidogrel active metabolite levels identifies novel variants that influence antiplatelet response.

Clopidogrel is one of the most commonly used therapeutics for the secondary prevention of cardiovascular events in patients with acute coronary syndromes. However, considerable interindividual variation in clopidogrel response has been documented, resulting in suboptimal therapy and an increased risk of recurrent events for some patients. In this investigation, we carried out the first genome-wide association study of circulating clopidogrel active metabolite levels in 513 healthy participants to directly measure clopidogrel pharmacokinetics. We observed that the CYP2C19 locus was the strongest genetic determinant of active metabolite formation (P=9.5×10). In addition, we identified novel genome-wide significant variants on chromosomes 3p25 (rs187941554, P=3.3×10) and 17q11 (rs80343429, P=1.3×10), as well as six additional loci that showed suggestive evidence of association (P≤1.0×10). Four of these loci showed nominal associations with on-clopidogrel ADP-stimulated platelet aggregation (P≤0.05). Evaluation of clopidogrel active metabolite concentration may help identify novel genetic determinants of clopidogrel response, which has implications for the development of novel therapeutics and improved antiplatelet treatment for at-risk patients in the future.

Null mutation in hormone-sensitive lipase gene and risk of type 2 diabetes.

BACKGROUND: Lipolysis regulates energy homeostasis through the hydrolysis of intracellular triglycerides and the release of fatty acids for use as energy substrates or lipid mediators in cellular processes. Genes encoding proteins that regulate energy homeostasis through lipolysis are thus likely to play an important role in determining susceptibility to metabolic disorders. METHODS: We sequenced 12 lipolytic-pathway genes in Old Order Amish participants whose fasting serum triglyceride levels were at the extremes of the distribution and identified a novel 19-bp frameshift deletion in exon 9 of LIPE, encoding hormone-sensitive lipase (HSL), a key enzyme for lipolysis. We genotyped the deletion in DNA from 2738 Amish participants and performed association analyses to determine the effects of the deletion on metabolic traits. We also obtained biopsy specimens of abdominal subcutaneous adipose tissue from 2 study participants who were homozygous for the deletion (DD genotype), 10 who were heterozygous (ID genotype), and 7 who were noncarriers (II genotype) for assessment of adipose histologic characteristics, lipolysis, enzyme activity, cytokine release, and messenger RNA (mRNA) and protein levels. RESULTS: Carriers of the mutation had dyslipidemia, hepatic steatosis, systemic insulin resistance, and diabetes. In adipose tissue from study participants with the DD genotype, the mutation resulted in the absence of HSL protein, small adipocytes, impaired lipolysis, insulin resistance, and inflammation. Transcription factors responsive to peroxisome-proliferator-activated receptor γ (PPAR-γ) and downstream target genes were down-regulated in adipose tissue from participants with the DD genotype, altering the regulation of pathways influencing adipogenesis, insulin sensitivity, and lipid metabolism. CONCLUSIONS: These findings indicate the physiological significance of HSL in adipocyte function and the regulation of systemic lipid and glucose homeostasis and underscore the severe metabolic consequences of impaired lipolysis. (Funded by the National Institutes of Health and others).

A systematic analysis and comparison of warfarin initiation strategies.

OBJECTIVE: Randomized trials have reported inconsistent evidence on the effectiveness of algorithms that use genotypes to initiate warfarin therapy. The Clarification of Optimal Anticoagulation through Genetics (COAG) trial initiated therapy on the basis of predicted maintenance doses, with a pharmacogenetic-guided algorithm in one study group and a clinically guided algorithm in the other. The European Pharmacogenetics of Anticoagulant Therapy (EU-PACT) consortium initiated therapy on the basis of loading doses, with an algorithm-based prediction in one study group and a fixed-dose regimen in the other. To understand the differences between these trials, we compared the initial doses between alternative dosing algorithms (the pharmacogenetic-guided and clinically guided algorithms developed by Gage and colleagues and those developed by the International Warfarin Pharmacogenetics Consortium) and between the COAG and EU-PACT dose-initiation strategies. METHODS: This was a secondary analysis of the COAG trial - a double-blind, randomized-controlled trial (2009-2013) - conducted at 18 clinical centers in the USA, which included 1010 adults initiating warfarin therapy, of whom 719 achieved maintenance dose. RESULTS: Among COAG participants, the distribution of initial doses differed between algorithms, but showed similar prediction accuracy for maintenance dose. However, had the COAG trial implemented the EU-PACT strategy, the 3-day initial dose would have been 4.8 mg greater among participants randomized to pharmacogenetic-guided dosing, but only 2.5 mg greater among participants randomized to clinically guided dosing (P<0.001). CONCLUSION: Compared with the COAG trial, the EU-PACT trial used systematically larger loading doses in the pharmacogenetic-guided group and might have inadequately adjusted for clinical variability in warfarin dose requirements in the fixed-dose group.

A pharmacogenetic versus a clinical algorithm for warfarin dosing.

BACKGROUND: The clinical utility of genotype-guided (pharmacogenetically based) dosing of warfarin has been tested only in small clinical trials or observational studies, with equivocal results. METHODS: We randomly assigned 1015 patients to receive doses of warfarin during the first 5 days of therapy that were determined according to a dosing algorithm that included both clinical variables and genotype data or to one that included clinical variables only. All patients and clinicians were unaware of the dose of warfarin during the first 4 weeks of therapy. The primary outcome was the percentage of time that the international normalized ratio (INR) was in the therapeutic range from day 4 or 5 through day 28 of therapy. RESULTS: At 4 weeks, the mean percentage of time in the therapeutic range was 45.2% in the genotype-guided group and 45.4% in the clinically guided group (adjusted mean difference, [genotype-guided group minus clinically guided group], -0.2; 95% confidence interval, -3.4 to 3.1; P=0.91). There also was no significant between-group difference among patients with a predicted dose difference between the two algorithms of 1 mg per day or more. There was, however, a significant interaction between dosing strategy and race (P=0.003). Among black patients, the mean percentage of time in the therapeutic range was less in the genotype-guided group than in the clinically guided group. The rates of the combined outcome of any INR of 4 or more, major bleeding, or thromboembolism did not differ significantly according to dosing strategy. CONCLUSIONS: Genotype-guided dosing of warfarin did not improve anticoagulation control during the first 4 weeks of therapy. (Funded by the National Heart, Lung, and Blood Institute and others; COAG ClinicalTrials.gov number, NCT00839657.).

Implementation of pharmacogenetics: the University of Maryland Personalized Anti-platelet Pharmacogenetics Program.

Despite a substantial evidence base, implementation of pharmacogenetics into routine patient care has been slow due to a number of non-trivial practical barriers. We implemented a Personalized Anti-platelet Pharmacogenetics Program (PAP3) for cardiac catheterization patients at the University of Maryland Medical Center and the Baltimore Veterans Administration Medical Center Patients' are offered CYP2C19 genetic testing, which is performed in our Clinical Laboratory Improvement Amendment (CLIA)-certified Translational Genomics Laboratory. Results are returned within 5 hr along with clinical decision support that includes interpretation of results and prescribing recommendations for anti-platelet therapy based on the Clinical Pharmacogenetics Implementation Consortium guidelines. Now with a working template for PAP3, implementation of other drug-gene pairs is in process. Lessons learned as described in this article may prove useful to other medical centers as they implement pharmacogenetics into patient care, a critical step in the pathway to personalized and genomic medicine.

The ABCG8 G574R variant, serum plant sterol levels, and cardiovascular disease risk in the Old Order Amish.

OBJECTIVE: To determine whether long-term exposure to moderate elevations in plasma plant sterol levels increases risk for atherosclerosis. METHODS AND RESULTS: In Old Order Amish participants aged 18 to 85 years, with (n=110) and without (n=181) 1 copy of the ABCG8 G574R variant, we compared mean plasma levels of plant sterols and cholesterol precursors and carotid intima-media wall thickness. Carriers of a single 574R allele had increased plant sterol levels (eg, 35%-37% higher plasma levels of sitosterol, campesterol, and stigmasterol) and increased plant sterol/cholesterol ratios (P<0.001 for all). 574R carriers had significantly decreased levels of lathosterol and lanosterol, precursors in a pathway for endogenous cholesterol synthesis, suggesting that plant sterols may alter regulation of genes involved in cholesterol synthesis. The G574R variant was not associated with high-density lipoprotein cholesterol or low-density lipoprotein cholesterol levels. Compared with noncarriers, 574R carriers had decreased carotid intima-media wall thickness (0.62 versus 0.66 mm; age- and sex-adjusted P=0.03). Adjustment for body weight, blood pressure, and standard lipid measures (high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides) did not alter this association. CONCLUSIONS: Although the G574R variant is associated with moderately elevated plant sterol levels, carriers of the 574R allele had modestly lower levels of carotid wall thickness compared with noncarriers.

The functional G143E variant of carboxylesterase 1 is associated with increased clopidogrel active metabolite levels and greater clopidogrel response.

INTRODUCTION: Carboxylesterase 1 (CES1) is the primary enzyme responsible for converting clopidogrel into biologically inactive carboxylic acid metabolites. METHODS: We genotyped a functional variant in CES1, G143E, in participants of the Pharmacogenomics of Anti-Platelet Intervention (PAPI) study (n=566) and in 350 patients with coronary heart disease treated with clopidogrel, and carried out an association analysis of bioactive metabolite levels, on-clopidogrel ADP-stimulated platelet aggregation, and cardiovascular outcomes. RESULTS: The levels of clopidogrel active metabolite were significantly greater in CES1 143E-allele carriers (P=0.001). Consistent with these findings, individuals who carried the CES1 143E-allele showed a better clopidogrel response as measured by ADP-stimulated platelet aggregation in both participants of the PAPI study (P=0.003) and clopidogrel-treated coronary heart disease patients (P=0.03). No association was found between this single nucleotide polymorphism and baseline measures of platelet aggregation in either cohort. CONCLUSION: Taken together, these findings suggest, for the first time, that genetic variation in CES1 may be an important determinant of the efficacy of clopidogrel.

Pharmacogenomics of anti-platelet and anti-coagulation therapy.

Arterial thrombosis is a major component of vascular disease, especially myocardial infarction (MI) and stroke. Current anti-thrombotic therapies such as warfarin and clopidogrel are effective in inhibiting cardiovascular events; however, there is great inter-individual variability in response to these medications. In recent years, it has been recognized that genetic factors play a significant role in drug response, and, subsequently, common variants in genes responsible for metabolism and drug action have been identified. These discoveries along with new diagnostic targets and therapeutic strategies hold promise for more effective individualized anti-coagulation and anti-platelet therapy.

Association of cytochrome P450 2C19 genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy.

CONTEXT: Clopidogrel therapy improves cardiovascular outcomes in patients with acute coronary syndromes and following percutaneous coronary intervention by inhibiting adenosine diphosphate (ADP)-dependent platelet activation. However, nonresponsiveness is widely recognized and is related to recurrent ischemic events. OBJECTIVE: To identify gene variants that influence clopidogrel response. DESIGN, SETTING, AND PARTICIPANTS: In the Pharmacogenomics of Antiplatelet Intervention (PAPI) Study (2006-2008), we administered clopidogrel for 7 days to 429 healthy Amish persons and measured response by ex vivo platelet aggregometry. A genome-wide association study was performed followed by genotyping the loss-of-function cytochrome P450 (CYP) 2C19*2 variant (rs4244285). Findings in the PAPI Study were extended by examining the relation of CYP2C19*2 genotype to platelet function and cardiovascular outcomes in an independent sample of 227 patients undergoing percutaneous coronary intervention. MAIN OUTCOME MEASURE: ADP-stimulated platelet aggregation in response to clopidogrel treatment and cardiovascular events. RESULTS: Platelet response to clopidogrel was highly heritable (h(2) = 0.73; P < .001). Thirteen single-nucleotide polymorphisms on chromosome 10q24 within the CYP2C18-CYP2C19-CYP2C9-CYP2C8 cluster were associated with diminished clopidogrel response, with a high degree of statistical significance (P = 1.5 x 10(-13) for rs12777823, additive model). The rs12777823 polymorphism was in strong linkage disequilibrium with the CYP2C19*2 variant, and was associated with diminished clopidogrel response, accounting for 12% of the variation in platelet aggregation to ADP (P = 4.3 x 10(-11)). The relation between CYP2C19*2 genotype and platelet aggregation was replicated in clopidogrel-treated patients undergoing coronary intervention (P = .02). Furthermore, patients with the CYP2C19*2 variant were more likely (20.9% vs 10.0%) to have a cardiovascular ischemic event or death during 1 year of follow-up (hazard ratio, 2.42; 95% confidence interval, 1.18-4.99; P = .02). CONCLUSION: CYP2C19*2 genotype was associated with diminished platelet response to clopidogrel treatment and poorer cardiovascular outcomes.

The genetic response to short-term interventions affecting cardiovascular function: rationale and design of the Heredity and Phenotype Intervention (HAPI) Heart Study.

BACKGROUND: The etiology of cardiovascular disease (CVD) is multifactorial. Efforts to identify genes influencing CVD risk have met with limited success to date, likely because of the small effect sizes of common CVD risk alleles and the presence of gene by gene and gene by environment interactions. METHODS: The HAPI Heart Study was initiated in 2002 to measure the cardiovascular response to 4 short-term interventions affecting cardiovascular risk factors and to identify the genetic and environmental determinants of these responses. The measurements included blood pressure responses to the cold pressor stress test and to a high salt diet, triglyceride excursion in response to a high-fat challenge, and response in platelet aggregation to aspirin therapy. RESULTS: The interventions were carried out in 868 relatively healthy Amish adults from large families. The heritabilities of selected response traits for each intervention ranged from 8% to 38%, suggesting that some of the variation associated with response to each intervention can be attributed to the additive effects of genes. CONCLUSIONS: Identifying these response genes may identify new mechanisms influencing CVD and may lead to individualized preventive strategies and improved early detection of high-risk individuals.

Multisite Investigation of Outcomes With Implementation of CYP2C19 Genotype-Guided Antiplatelet Therapy After Percutaneous Coronary Intervention.

OBJECTIVES: This multicenter pragmatic investigation assessed outcomes following clinical implementation of CYP2C19 genotype-guided antiplatelet therapy after percutaneous coronary intervention (PCI). BACKGROUND: CYP2C19 loss-of-function alleles impair clopidogrel effectiveness after PCI. METHODS: After clinical genotyping, each institution recommended alternative antiplatelet therapy (prasugrel, ticagrelor) in PCI patients with a loss-of-function allele. Major adverse cardiovascular events (defined as myocardial infarction, stroke, or death) within 12 months of PCI were compared between patients with a loss-of-function allele prescribed clopidogrel versus alternative therapy. Risk was also compared between patients without a loss-of-function allele and loss-of-function allele carriers prescribed alternative therapy. Cox regression was performed, adjusting for group differences with inverse probability of treatment weights. RESULTS: Among 1,815 patients, 572 (31.5%) had a loss-of-function allele. The risk for major adverse cardiovascular events was significantly higher in patients with a loss-of-function allele prescribed clopidogrel versus alternative therapy (23.4 vs. 8.7 per 100 patient-years; adjusted hazard ratio: 2.26; 95% confidence interval: 1.18 to 4.32; p = 0.013). Similar results were observed among 1,210 patients with acute coronary syndromes at the time of PCI (adjusted hazard ratio: 2.87; 95% confidence interval: 1.35 to 6.09; p = 0.013). There was no difference in major adverse cardiovascular events between patients without a loss-of-function allele and loss-of-function allele carriers prescribed alternative therapy (adjusted hazard ratio: 1.14; 95% confidence interval: 0.69 to 1.88; p = 0.60). CONCLUSIONS: These data from real-world observations demonstrate a higher risk for cardiovascular events in patients with a CYP2C19 loss-of-function allele if clopidogrel versus alternative therapy is prescribed. A future randomized study of genotype-guided antiplatelet therapy may be of value.

Acute-phase serum amyloid A: an inflammatory adipokine and potential link between obesity and its metabolic complications.

BACKGROUND: Obesity is associated with low-grade chronic inflammation, and serum markers of inflammation are independent risk factors for cardiovascular disease (CVD). However, the molecular and cellular mechanisms that link obesity to chronic inflammation and CVD are poorly understood. METHODS AND FINDINGS: Acute-phase serum amyloid A (A-SAA) mRNA levels, and A-SAA adipose secretion and serum levels were measured in obese and nonobese individuals, obese participants who underwent weight-loss, and persons treated with the insulin sensitizer rosiglitazone. Inflammation-eliciting activity of A-SAA was investigated in human adipose stromal vascular cells, coronary vascular endothelial cells and a murine monocyte cell line. We demonstrate that A-SAA was highly and selectively expressed in human adipocytes. Moreover, A-SAA mRNA levels and A-SAA secretion from adipose tissue were significantly correlated with body mass index (r = 0.47; p = 0.028 and r = 0.80; p = 0.0002, respectively). Serum A-SAA levels decreased significantly after weight loss in obese participants (p = 0.006), as well as in those treated with rosiglitazone (p = 0.033). The magnitude of the improvement in insulin sensitivity after weight loss was significantly correlated with decreases in serum A-SAA (r = -0.74; p = 0.034). SAA treatment of vascular endothelial cells and monocytes markedly increased the production of inflammatory cytokines, e.g., interleukin (IL)-6, IL-8, tumor necrosis factor alpha, and monocyte chemoattractant protein-1. In addition, SAA increased basal lipolysis in adipose tissue culture by 47%. CONCLUSIONS: A-SAA is a proinflammatory and lipolytic adipokine in humans. The increased expression of A-SAA by adipocytes in obesity suggests that it may play a critical role in local and systemic inflammation and free fatty acid production and could be a direct link between obesity and its comorbidities, such as insulin resistance and atherosclerosis. Accordingly, improvements in systemic inflammation and insulin resistance with weight loss and rosiglitazone therapy may in part be mediated by decreases in adipocyte A-SAA production.

Development of a physiology-directed population pharmacokinetic and pharmacodynamic model for characterizing the impact of genetic and demographic factors on clopidogrel response in healthy adults.

Clopidogrel (Plavix®), is a widely used antiplatelet agent, which shows high inter-individual variability in treatment response in patients following the standard dosing regimen. In this study, a physiology-directed population pharmacokinetic/pharmacodynamic (PK/PD) model was developed based on clopidogrel and clopidogrel active metabolite (clop-AM) data from the PAPI and the PGXB2B studies using a step-wise approach in NONMEM (version 7.2). The developed model characterized the in vivo disposition of clopidogrel, its bioactivation into clop-AM in the liver and subsequent platelet aggregation inhibition in the systemic circulation reasonably well. It further allowed the identification of covariates that significantly impact clopidogrel's dose-concentration-response relationship. In particular, CYP2C19 intermediate and poor metabolizers converted 26.2% and 39.5% less clopidogrel to clop-AM, respectively, compared to extensive metabolizers. In addition, CES1 G143E mutation carriers have a reduced CES1 activity (82.9%) compared to wild-type subjects, which results in a significant increase in clop-AM formation. An increase in BMI was found to significantly decrease clopidogrel's bioactivation, whereas increased age was associated with increased platelet reactivity. Our PK/PD model analysis suggests that, in order to optimize clopidogrel dosing on a patient-by-patient basis, all of these factors have to be considered simultaneously, e.g. by using quantitative clinical pharmacology tools.

The Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study: Variation in Platelet Response to Clopidogrel and Aspirin.

Clopidogrel and aspirin are commonly prescribed anti-platelet medications indicated for patients who have experienced, or are at risk for, ischemic cardiovascular events. The Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study was designed to characterize determinants of clopidogrel and dual anti-platelet therapy (DAPT) response in a healthy cohort of Old Order Amish from Lancaster, PA. Following a loading dose, clopidogrel was taken once a day for 7 days. One hour after the last dose of clopidogrel, 325 mg of aspirin was given. Ex vivo platelet aggregometry was performed at baseline, post-clopidogrel, and post-DAPT. Platelet aggregation measurements were significantly lower after both interventions for all agonists tested (p <0.05), although there was large inter-individual variation in the magnitude of anti-platelet response. Female sex and older age were associated with higher platelet aggregation at all three time-points. Change in aggregation was correlated among the various agonists at each time point. Heritability (h2) of change in platelet aggregation was significant for most traits at all time-points (range h2=0.14-0.57). Utilization of a standardized, short-term intervention provided a powerful approach to investigate sources of variation in platelet aggregation response due to drug therapy. Further, this short-term intervention approach may provide a useful paradigm for pharmacogenomics studies.

CYP2C19 metabolizer status and clopidogrel efficacy in the Secondary Prevention of Small Subcortical Strokes (SPS3) study.

BACKGROUND: The role of the CYP2C19 genotype on clopidogrel efficacy has been studied widely, with data suggesting reduced clopidogrel efficacy in loss-of-function variant carriers taking clopidogrel after percutaneous coronary intervention; however, data are limited regarding the association between CYP2C19 genetic variants and outcomes in stroke patients. We investigated whether CYP2C19 metabolizer status affects the risk of recurrent stroke or major bleeding in subcortical stroke patients taking dual antiplatelet therapy with aspirin and clopidogrel. METHODS AND RESULTS: CYP2C19*2 and CYP2C19*17 were genotyped in 522 patients treated with dual antiplatelet therapy from the Secondary Prevention of Small Subcortical Strokes (SPS3) study. CYP2C19 metabolizer status was inferred from genotype, and associations with the risk of recurrent stroke and major bleeding were assessed in the overall cohort and by race/ethnic group with logistic regression modeling. In the overall cohort, there were no differences in outcomes by CYP2C19 metabolizer status (recurrent stroke, odds ratio 1.81 [95% CI 0.76 to 4.30]; major bleeding, odds ratio 0.67 [95% CI 0.22 to 2.03]). In white participants, those with CYP2C19 intermediate or poor metabolizer status had higher odds of recurrent stroke (odds ratio 5.19 [95% CI 1.08 to 24.90]) than those with extensive or ultrarapid metabolizer status, but there was no evidence of difference in major bleeding. CONCLUSIONS: There were significant differences in recurrent stroke by CYP2C19 genotype-inferred metabolizer status in white subcortical stroke patients receiving dual antiplatelet therapy with aspirin and clopidogrel, consistent with cardiovascular studies on CYP2C19 and clopidogrel; however, the bleeding risk that led to early termination of the antiplatelet arm of the SPS3 trial does not appear to be explained by CYP2C19 genotype. This study was relatively underpowered; therefore, these findings should be interpreted with caution and warrant replication. CLINICAL TRIAL REGISTRATION: URL: www.clinicaltrials.gov. Unique identifier: NCT00059306.

Oxylipid Profile of Low-Dose Aspirin Exposure: A Pharmacometabolomics Study.

BACKGROUND: While aspirin is a well-established and generally effective anti-platelet agent, considerable inter-individual variation in drug response exists, for which mechanisms are not completely understood. Metabolomics allows for extensive measurement of small molecules in biological samples, enabling detailed mapping of pathways involved in drug response. METHODS AND RESULTS: We used a mass-spectrometry-based metabolomics platform to investigate the changes in the serum oxylipid metabolome induced by an aspirin intervention (14 days, 81 mg/day) in healthy subjects (n=156). We observed a global decrease in serum oxylipids in response to aspirin (25 metabolites decreased out of 30 measured) regardless of sex. This decrease was concomitant with a significant decrease in serum linoleic acid levels (-19%, P=1.3×10(-5)), one of the main precursors for oxylipid synthesis. Interestingly, several linoleic acid-derived oxylipids were not significantly associated with arachidonic-induced ex vivo platelet aggregation, a widely accepted marker of aspirin response, but were significantly correlated with platelet reactivity in response to collagen. CONCLUSIONS: Together, these results suggest that linoleic acid-derived oxylipids may contribute to the non-COX1 mediated variability in response to aspirin. Pharmacometabolomics allowed for more comprehensive interrogation of mechanisms of action of low dose aspirin and of variation in aspirin response.