This article is available online at http://www.jlr.org Proprotein convertase subtilisin kexin type 9 (PCSK9) has been recognized as a key regulator of serum low density lipoprotein cholesterol (LDL-C) levels ( 1-7 ). PCSK9 is a protease made and secreted by the liver into the plasma, which then binds to and degrades hepatic LDL receptors (LDLR) (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). The mechanism by which PCSK9 degrades LDLR is complex. Recent studies suggest that after self-cleavage and secretion, PCSK9 does not have to be enzymatically active to cause degradation of the LDLR ( 19-21 ). Rather, PCSK9 binds to the LDLR and subsequently targets it for lysosomal destruction within the hepatocyte ( 8,(19)(20)(21). This concept of how PCSK9 acts to decrease hepatic LDLR levels is supported by recent fi ndings that disruption of the binding of PCSK9 to the LDLR using anti-PCSK9 antibody results in preserved LDLR and decreased [22][23][24].Several different PCSK9 mutations have been reported in humans. Patients with gain-of-function mutations of PCSK9 present with severe familial hypercholesterolemia and accompanying increased cardiovascular risk (25)(26)(27)(28)(29). In contrast, individuals with loss-of-function mutations in PCSK9, including mutations which prevent the selfcleavage and secretion of the protein, have signifi cantly decreased levels of serum LDL-C and lower cardiovascular risk (30)(31)(32). Approximately 3% of African-Americans are heterozygous for such mutations ( 30 ). Of note, a compound heterozygote for PCSK9 loss-of-function mutations was recently described. The subject, a healthy 32-year-old female, had a serum LDL-C level of 14 mg/dl ( 32 ). A second Abstract Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of serum LDL-cholesterol (LDL-C) levels. PCSK9 is secreted by the liver into the plasma and binds the hepatic LDL receptor (LDLR), causing its subsequent degradation. We fi rst demonstrated that a moderate dose of atorvastatin (40 mg) increases PCSK9 serum levels, suggesting why increasing statin doses may have diminished effi cacy with regard to further LDL-C lowering. Since that initial observation, at least two other groups have reported statin-induced PCSK9 increases. To date, no analysis of the effect of high-dose atorvastatin (80 mg) on PCSK9 over time has been conducted. Therefore, we studied the time course of atorvastatin (80 mg) in human subjects. We measured PCSK9 and lipid levels during a 2-week lead-in baseline period and every 4 weeks thereafter for 16 weeks. We observed that atorvastatin (80 mg) caused a rapid 47% increase in serum PCSK9 at 4 weeks that was sustained throughout 16 weeks of dosing. Importantly, while PCSK9 levels were highly correlated with total cholesterol (TC), LDL-C, and triglyceride (TG) levels at baseline, atorvastatin (80 mg) completely abolished all of these correlations. Together, these results further suggest an explanation for why increasing doses of statins fail to achieve proportional LDL-C lowering. Abbreviations: HDL-...

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.

Carriers of two copies of the loss-of-function CYP2C19*2 variant convert less clopidogrel into its active metabolite, resulting in diminished anti-platelet responses and higher cardiovascular event rates. To evaluate whether increasing the daily clopidogrel dose in poor metabolizers (PM) overcomes the effect of the CYP2C19*2 variant, we enrolled 18 healthy participants in a genotype-stratified, multi-dose, three-period, fixed-sequence crossover study. Six participants with the *1/*1 extensive (EM), *1/*2 intermediate (IM), and *2/*2 poor metabolizer genotypes each received 75 mg, 150 mg, and 300 mg each for 8 days. In each period, maximal platelet aggregation 4 hours post-dose (MPA4) and active metabolite area under the curve (AUC) differed among genotype groups (p<0.05 for all). At day 8, PMs needed 300 mg daily and IMs needed 150 mg daily to attain a similar MPA4 as EMs on the 75 mg dose (32.6%, 33.2%, 31.3%, respectively). Similarly, PMs needed 300 mg daily to achieve active metabolite concentrations that were similar to EMs on 75 mg (AUC 37.7 and 33.5 ng.h/ml, respectively). These results suggest that quadrupling the usual clopidogrel dose might be necessary to overcome the effect of poor CYP2C19 metabolism.

No abstract

Pharmacogenetics, one of the cornerstones of personalized medicine, has the potential to change the way in which health care is offered by stratifying patients into various pretreatment categories, such as likely responders, likely non-responders or likely to experience adverse drug reactions. In order to advance drug development and regulatory science, regulatory agencies globally have promulgated guidelines on pharmacogenetics for nearly a decade. The aim of this article is to provide an overview of new guidelines for the implementation of pharmacogenetics in drug development from a multiregional regulatory perspective - encompassing Europe, the United States and Japan - with an emphasis on clinical pharmacokinetics.

No abstract

A sensitive, selective, and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) was developed for the simultaneous quantification of clopidogrel (Plavix ® ) and its derivatized active metabolite (CAMD) in human plasma. Derivatization of the active metabolite in blood with 2-bromo-3'-methoxy acetophenone (MPB) immediately after collection ensured metabolite stability during sample handling and storage. Following addition of ticlopidine as an internal standard and simple protein precipitation, the analytes were separated on a Waters Acquity UPLC™ sub-2µm-C 18 column via gradient elution before detection on a triplequadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was validated across the clinically-relevant concentration range of 0.01-50 ng/mL for parent clopidogrel and 0.1-150 ng/mL (r 2 = 0.99) for CAMD, with a fast run time of 1.5 min to support pharmacokinetic studies using 75, 150, or 300 mg oral doses of clopidogrel. The analytical method measured concentrations of clopidogrel and CAMD with accuracy (%DEV) < ±12% and precision (%CV) of < ±6%. The method was successfully applied to measure the plasma concentrations of clopidogrel and CAMD in three subjects administered single oral doses of 75, 150, and 300 mg clopidogrel. It was further demonstrated that the derivatizing agent (MPB) does not affect clopidogrel levels, thus from one aliquot of blood drawn clinically, this method can simultaneously quantify both clopidogrel and CAMD with sensitivity in the picogram per mL range.

SummaryThe U.S. Food and Drug Administration recently marked 10 years since first updating the labeling for warfarin (often referred to as the “poster child” of pharmacogenomics) to include information regarding the potential impact of CYP2C9 and VKORC1 genetic variation on warfarin dosing requirements and risks. Herein, we opine on the experience updating the warfarin labeling, highlighting more generally the enabling factors and challenges encountered when considering incorporation of pharmacogenomic information into the prescribing recommendations for already approved drugs. We also provide a historical perspective of implemented changes in regulatory policies related to personalized medicine.