Marianne K. DeGorter

Clinical and Pharmacogenetic Predictors of Circulating Atorvastatin and Rosuvastatin Concentrations in Routine Clinical Care

Background—A barrier to statin therapy is myopathy associated with elevated systemic drug exposure. Our objective was to examine the association between clinical and pharmacogenetic variables and statin concentrations in patients. Methods and Results—In total, 299 patients taking atorvastatin or rosuvastatin were prospectively recruited at an outpatient referral center. The contribution of clinical variables and transporter gene polymorphisms to statin concentration was assessed using multiple linear regression. We observed 45-fold variation in statin concentration among patients taking the same dose. After adjustment for sex, age, body mass index, ethnicity, dose, and time from last dose, SLCO1B1 c.521T>C (P<0.001) and ABCG2 c.421C>A (P<0.01) were important to rosuvastatin concentration (adjusted R2=0.56 for the final model). Atorvastatin concentration was associated with SLCO1B1 c.388A>G (P<0.01) and c.521T>C (P<0.05) and 4&bgr;-hydroxycholesterol, a CYP3A activity marker (adjusted R2=0.47). A second cohort of 579 patients from primary and specialty care databases were retrospectively genotyped. In this cohort, genotypes associated with statin concentration were not differently distributed among dosing groups, implying providers had not yet optimized each patient’s risk–benefit ratio. Nearly 50% of patients in routine practice taking the highest doses were predicted to have statin concentrations greater than the 90th percentile. Conclusions—Interindividual variability in statin exposure in patients is associated with uptake and efflux transporter polymorphisms. An algorithm incorporating genomic and clinical variables to avoid high atorvastatin and rosuvastatin levels is described; further study will determine whether this approach reduces incidence of statin myopathy.

Clarifying the importance of CYP2C19 and PON1 in the mechanism of clopidogrel bioactivation and in vivo antiplatelet response

AIMS It is thought that clopidogrel bioactivation and antiplatelet response are related to cytochrome P450 2C19 (CYP2C19). However, a recent study challenged this notion by proposing CYP2C19 as wholly irrelevant, while identifying paraoxonase-1 (PON1) and its Q192R polymorphism as the major driver of clopidogrel bioactivation and efficacy. The aim of this study was to systematically elucidate the mechanism and relative contribution of PON1 in comparison to CYP2C19 to clopidogrel bioactivation and antiplatelet response. METHODS AND RESULTS First, the influence of CYP2C19 and PON1 polymorphisms and plasma paraoxonase activity on clopidogrel active metabolite (H4) levels and antiplatelet response was assessed in a cohort of healthy subjects (n = 21) after administration of a single 75 mg dose of clopidogrel. There was a remarkably good correlation between H4 AUC (0-8 h) and antiplatelet response (r2 = 0.78). Furthermore, CYP2C19 but not PON1 genotype was predictive of H4 levels and antiplatelet response. There was no correlation between plasma paraoxonase activity and H4 levels. Secondly, metabolic profiling of clopidogrel in vitro confirmed the role of CYP2C19 in bioactivating clopidogrel to H4. However, heterologous expression of PON1 in cell-based systems revealed that PON1 cannot generate H4, but mediates the formation of another thiol metabolite, termed Endo. Importantly, Endo plasma levels in humans are nearly 20-fold lower than H4 and was not associated with any antiplatelet response. CONCLUSION Our results demonstrate that PON1 does not mediate clopidogrel active metabolite formation or antiplatelet action, while CYP2C19 activity and genotype remains a predictor of clopidogrel pharmacokinetics and antiplatelet response.

Interaction of three regiospecific amino acid residues is required for OATP1B1 gain of OATP1B3 substrate specificity.

The human organic anion-transporting polypeptides OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) are liver-enriched membrane transporters of major importance to hepatic uptake of numerous endogenous compounds, including bile acids, steroid conjugates, hormones, and drugs, including the 3-hydroxy-3-methylglutaryl Co-A reductase inhibitor (statin) family of cholesterol-lowering compounds. Despite their remarkable substrate overlap, there are notable exceptions: in particular, the gastrointestinal peptide hormone cholecystokinin-8 (CCK-8) is a high affinity substrate for OATP1B3 but not OATP1B1. We utilized homologous recombination of linear DNA by E. coli to generate a library of cDNA containing monomer size chimeric OATP1B1-1B3 and OATP1B3-1B1 transporters with randomly distributed chimeric junctions to identify three discrete regions of the transporter involved in conferring CCK-8 transport activity. Site-directed mutagenesis of three key residues in OATP1B1 transmembrane helices 1 and 10, and extracellular loop 6, to the corresponding residues in OATP1B3, resulted in a gain of CCK-8 transport by OATP1B1. The residues appear specific to CCK-8, as the mutations did not affect transport of the shared OATP1B substrate atorvastatin or the OATP1B1-specific substrate estrone sulfate. Regions involved in gain of CCK-8 transport by OATP1B1, when mapped to the crystal structures of bacterial transporters from the major facilitator superfamily, are positioned to suggest these regions could readily interact with drug substrates. Accordingly, our data provide new insight into the molecular determinants of the substrate specificity of these hepatic uptake transporters with relevance to targeted drug design and prediction of drug-drug interactions.

Efficacy and Plasma Drug Concentrations With Nondaily Dosing of Rosuvastatin

BACKGROUND Nondaily statin dosing is an alternative for patients unable to tolerate daily dosing. The higher potency and longer half-life of rosuvastatin lends itself to this regimen. The basis of this improved tolerability is not understood, but might be related to lower plasma drug concentrations. We examined the efficacy of nondaily rosuvastatin in previously statin-intolerant patients and determined plasma drug concentrations at various dose regimens. METHODS A retrospective analysis at a specialty lipid clinic identified 58 patients eligible for evaluation after therapy with nondaily rosuvastatin. Plasma rosuvastatin levels were measured by liquid chromatography-mass spectrometry in 12 patients taking 10 mg nondaily rosuvastatin and in 11 and 12 age- and sex-matched patients taking 10 mg and 5 mg rosuvastatin daily, respectively. Whole body cholesterol synthesis was estimated from serum lathosterol measured by liquid chromatography-mass spectrometry. RESULTS In patients with a previous history of statin intolerance, nondaily rosuvastatin (average of 29.4 mg per week) lowered low-density lipoprotein cholesterol by 34.4 ± 21.3% (P < 0.001). Serum lathosterol levels were significantly higher in patients on nondaily regimens, as expected. However, mean plasma rosuvastatin levels of patients taking 10 mg nondaily did not significantly differ from those taking 10 mg daily. CONCLUSIONS In statin intolerant patients, nondaily rosuvastatin resulted in clinically relevant reductions in low-density lipoprotein cholesterol levels, with improved compliance. Whole body cholesterol synthesis was higher in patients taking nondaily rosuvastatin, but no differences in plasma drug concentrations were observed, suggesting that the improved tolerability was independent of plasma rosuvastatin levels.

Hepatic drug transporters, old and new: pharmacogenomics, drug response, and clinical relevance.

Interindividual variation in drug response continues to pose a considerable challenge to optimal drug therapy. Drug-metabolizing enzymes have been established as critical determinants of drug disposition and response, and drug transporters are becoming widely appreciated for their role in these processes. We now know that transport is required for many drugs to pass through the membrane, and it is a rate-limiting step governing drug absorption and entry into target tissue.1 As reported by Nies and colleagues2 in this issue of HEPATOLOGY, both genetic and environmental factors contribute to interindividual variation in the expression of hepatic cation transporters. Indeed, genetic polymorphisms have been identified in most transport proteins, and many are now recognized as significant contributors to interindividual variation in drug exposure. For most drug transporters, the complex interaction of clinical and genetic parameters affects the observed drug disposition profile and response phenotype.1,3,4 Drug transporters recognize structurally diverse compounds and may be broadly classified into two groups: the major facilitator superfamily and the adenosine triphosphate–binding cassette (ABC) superfamily. Transcriptional, translational, and posttranslational regulation can affect the amount, localization, and functional activity of the expressed protein. In the hepatocyte, transporters on the sinusoidal membrane include organic cation transporters [OCTs; solute carrier family 22 (SLC22)], organic anion-transporting polypeptides (SLCO), and organic anion transporters (SLC22). On the canalicular membrane, drug efflux is mediated primarily by ABC transporters1 (Fig. 1). Uptake of cationic drugs into the liver occurs largely by OCTs, whereas their efflux is facilitated by a recently identified multidrug and toxin extrusion transporter [multidrug and toxin extrusion 1 (MATE1)] and the widely studied P-glycoprotein [multidrug resistance 1 (MDR1)/ABCB1].5 OCTs mediate electrogenic, sodium-independent transport and may be inhibited by a number of compounds not transported. OCT substrates include the oral antihyperglycemic drug metformin, the histamine receptor antagonist cimetidine, and antivirals such as acyclovir. Endogenous substrates of OCTs include acetylcholine and catecholamines.6 In this issue of HEPATOLOGY, Nies and colleagues2 report the expression of the two hepatic OCTs, OCT1 (SLC22A1) and OCT3 (SLC22A3), in 150 human liver samples. They found protein expression of OCT1 to be highly variable between individuals, although it was correlated with OCT1 messenger RNA (mRNA) levels. For OCT3, protein expression data were not obtained, but mRNA levels were also highly variable. The authors were able to correlate some of the observed variation to genetic polymorphisms in SLC22A1 and SLC22A3. Of 92 OCTrelated variants, one nonsynonymous coding variant, OCT1-Arg61Cys (rs12208357), was associated with reduced OCT1 protein and mRNA expression. In addition, three noncoding OCT3 variants (rs2048327, rs1810126, and rs3088442) in linkage disequilibrium and one synonymous coding variant (rs2292334) were associated with decreased OCT3 mRNA expression.2 OCT polymorphisms have been extensively studied in the context of metformin pharmacokinetics and response.3,5-14 Metformin is widely prescribed to treat diabetes mellitus type 2, with considerable variability in efficacy. Reduced-function OCT1 alleles—OCT1Arg61Cys, OCT1-Gly401Ser, OCT1-420del, and OCT1-Gly465Arg—have been demonstrated by Shu and colleagues8 to increase systemic exposure to metformin. This observation provides a mechanistic basis for the reduced response to metformin observed in healthy Abbreviations: ABC, adenosine triphosphate–binding cassette; ATP, adenosine triphosphate; BCRP, breast cancer resistance protein; BSEP, bile salt export pump; MATE1, multidrug and toxin extrusion 1; MDR, multidrug resistance; mRNA, messenger RNA; MRP, multidrug resistance–associated protein; NTCP, sodiumdependent taurocholate cotransporting polypeptide; OAT2, organic anion transporter 2; OATP, organic anion transporting polypeptide; OCT, organic cation transporter; SLC, solute carrier family. This work was supported in part by a grant from the Canadian Institutes for Health Research (MOP-89753) and by a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada (to Marianne K. DeGorter). Address reprint requests to: Richard B. Kim, M.D., Department of Medicine, London Health Sciences Centre, University Hospital, 339 Windermere Road, London, Ontario, Canada N6A 5A5. E-mail: richard.kim@lhsc.on.ca; fax: 519-663-3232. Copyright

Incremental Lowering of Low-Density Lipoprotein Cholesterol With Ezetimibe 20 mg vs 10 mg Daily in Patients Receiving Concomitant Statin Therapy

BACKGROUND Ezetimibe is typically administered at a dose of 10 mg daily, with few reports of use at other doses. We compared plasma concentrations of low-density lipoprotein (LDL) cholesterol and other lipid variables in patients with dyslipidemia who were receiving ezetimibe 10 mg and then 20 mg daily. METHODS A retrospective chart review identified 27 patients who received ezetimibe 10 mg and then 20 mg daily at different times; 15 participants were receiving stable statin therapy and 12 were not receiving concomitant statins. Plasma concentrations of lipids, creatine kinase (CK), and aspartate transaminase (AST) were determined. Plasma concentrations of ezetimibe and ezetimibe glucuronide were measured in a second group of patients. RESULTS Patients taking statins and ezetimibe 20 mg had further reductions in total and LDL cholesterol of 7.1% and 10.3%, respectively (both P < 0.05) than did those receiving the 10-mg dose. No difference between 20-mg and 10-mg dosing was seen among patients not receiving statins. Plasma concentrations of ezetimibe and its active metabolite were about 2-fold higher (P < 0.05) in patients taking ezetimibe 20 mg than in those receiving 10 mg daily. All patients tolerated ezetimibe 20 mg without side effects. CONCLUSIONS Ezetimibe 20 mg daily reduced total and LDL cholesterol further in patients receiving statin therapy compared with 10 mg daily. Prospective studies are required to show whether the higher plasma levels of ezetimibe and its active metabolite in patients taking the 20-mg dose have any detrimental effects. Increasing the ezetimibe dose to 20 mg daily might be an interesting potential approach for patients who fail to reach lipid targets on ezetimibe 10 mg daily along with maximally tolerated doses of statin.