The INGENIOUS trial: Impact of pharmacogenetic testing on adverse events in a pragmatic clinical trial.

Adverse drug events (ADEs) account for a significant mortality, morbidity, and cost burden. Pharmacogenetic testing has the potential to reduce ADEs and inefficacy. The objective of this INGENIOUS trial (NCT02297126) analysis was to determine whether conducting and reporting pharmacogenetic panel testing impacts ADE frequency. The trial was a pragmatic, randomized controlled clinical trial, adapted as a propensity matched analysis in individuals (N = 2612) receiving a new prescription for one or more of 26 pharmacogenetic-actionable drugs across a community safety-net and academic health system. The intervention was a pharmacogenetic testing panel for 26 drugs with dosage and selection recommendations returned to the health record. The primary outcome was occurrence of ADEs within 1 year, according to modified Common Terminology Criteria for Adverse Events (CTCAE). In the propensity-matched analysis, 16.1% of individuals experienced any ADE within 1-year. Serious ADEs (CTCAE level ≥ 3) occurred in 3.2% of individuals. When combining all 26 drugs, no significant difference was observed between the pharmacogenetic testing and control arms for any ADE (Odds ratio 0.96, 95% CI: 0.78-1.18), serious ADEs (OR: 0.91, 95% CI: 0.58-1.40), or mortality (OR: 0.60, 95% CI: 0.28-1.21). However, sub-group analyses revealed a reduction in serious ADEs and death in individuals who underwent pharmacogenotyping for aripiprazole and serotonin or serotonin-norepinephrine reuptake inhibitors (OR 0.34, 95% CI: 0.12-0.85). In conclusion, no change in overall ADEs was observed after pharmacogenetic testing. However, limitations incurred during INGENIOUS likely affected the results. Future studies may consider preemptive, rather than reactive, pharmacogenetic panel testing.

Stereoselective Metabolism of Bupropion to Active Metabolites in Cellular Fractions of Human Liver and Intestine.

Striking stereoselective disposition of the antidepressant and smoking cessation aid bupropion (BUP) and its active metabolites observed clinically influence patients' response to BUP therapy and its clinically important drug-drug interactions (DDI) with CYP2D6 substrates. However, understanding of the biochemical mechanisms responsible is incomplete. This study comprehensively examined hepatic and extrahepatic stereoselective metabolism of BUP in vitro Racemic-, R-, and S-BUP were incubated separately with pooled cellular fractions of human liver [microsomes (HLMs), S9 fractions (HLS9s), and cytosols (HLCs)] and intestinal [microsomes (HIMs), S9 fractions (HIS9s), and cytosols (HICs)] and cofactors. Formations of diastereomers of 4-hydroxyBUP (OHBUP), threohydroBUP (THBUP), and erythrohydroBUP (EHBUP) were quantified using a novel chiral ultra-high performance liquid chromatography/tandem mass spectrometry method. Racemic BUP (but not R- or S-BUP) was found suitable to determine stereoselective metabolism of BUP; both enantiomers showed complete racemization. Compared with that of RR-THBUP, the in vitro intrinsic clearance (Clint) for the formation of SS-THBUP was 42-, 19-, and 8.3-fold higher in HLMs, HLS9 fractions, and HLCs, respectively; Clint for the formation of SS-OHBUP and RS-EHBUP was also higher (2.7- to 3.9-fold) than their R-derived counterparts. In cellular fractions of human intestine, ≥ 95% of total reduction was accounted by the formation of RR-THBUP. Ours is the first to demonstrate marked stereoselective reduction of BUP in HLCs, HIMs, HIS9 fractions, and HICs, providing the first evidence for tissue- and cellular fraction-dependent stereoselective metabolism of BUP. These data may serve as the first critical step toward understanding factors dictating BUP's stereoselective disposition, effects, and DDI risks. SIGNIFICANCE STATEMENT: This work provides a deeper insight into bupropion (BUP) stereoselective oxidation and reduction to active metabolites in cellular fractions of human liver and intestine tissues. The results demonstrate tissue- and cellular fraction-dependent stereospecific metabolism of BUP. These data may improve prediction of BUP stereoselective disposition and understanding of BUP's effects and CYP2D6-dependent drug-drug interaction in vivo.

Influence of CYP2B6 Pharmacogenetics on Stereoselective Inhibition and Induction of Bupropion Metabolism by Efavirenz in Healthy Volunteers.

We investigated the acute and chronic effects of efavirenz, a widely used antiretroviral drug, and CYP2B6 genotypes on the disposition of racemic and stereoisomers of bupropion (BUP) and its active metabolites, 4-hydroxyBUP, threohydroBUP and erythrohydroBUP. The primary objective of this study was to test how multiple processes unique to the efavirenz-CYP2B6 genotype interaction influence the extent of efavirenz-mediated drug-drug interaction (DDI) with the CYP2B6 probe substrate BUP. In a three-phase, sequential, open-label study, healthy volunteers (N=53) were administered a single 100 mg oral dose of BUP alone (control phase), with a single 600 mg oral efavirenz dose (inhibition phase), and after 17-days pretreatment with efavirenz (600 mg/day) (induction phase). Compared to the control phase, we show for the first time that efavirenz significantly decreases and chronically increases the exposure of hydroxyBUP and its diastereomers, respectively, and these interactions were CYP2B6 genotype dependent. Chronic efavirenz enhances the elimination of racemic BUP and its enantiomers as well as of threo- and erythro-hydroBUP and their diastereomers, suggesting additional novel mechanisms underlying efavirenz interaction with BUP. The effects of efavirenz and genotypes were nonstereospecific. In conclusion, acute and chronic administration of efavirenz inhibits and induces CYP2B6 activity. Efavirenz-BUP interaction is complex involving time- and CYP2B6 genotype-dependent inhibition and induction of primary and secondary metabolic pathways. Our findings highlight important implications to the safety and efficacy of BUP, study design considerations for future efavirenz interactions, and individualized drug therapy based on CYP2B6 genotypes. Significance Statement The effects of acute and chronic doses of efavirenz on the disposition of racemic and stereoisomers of BUP and its active metabolites were investigated in healthy volunteers. Efavirenz causes an acute inhibition, but chronic induction of CYP2B6 in a genotype dependent manner. Chronic efavirenz induces BUP reduction and the elimination of BUP active metabolites. Efavirenz's effects were non-stereospecific. These data reveal novel mechanisms underlying efavirenz DDI with BUP and provide important insights into time- and CYP2B6 genotype dependent DDIs.

Genome-wide association study of aromatase inhibitor discontinuation due to musculoskeletal symptoms.

OBJECTIVE: Aromatase inhibitors (AIs) are commonly used to treat hormone receptor positive (HR +) breast cancer. AI-induced musculoskeletal syndrome (AIMSS) is a common toxicity that causes AI treatment discontinuation. The objective of this genome-wide association study (GWAS) was to identify genetic variants associated with discontinuation of AI therapy due to AIMSS and attempt to replicate previously reported associations. METHODS: In the Exemestane and Letrozole Pharmacogenetics (ELPh) study, postmenopausal patients with HR + non-metastatic breast cancer were randomized to letrozole or exemestane. Genome-wide genotyping of germline DNA was conducted followed by imputation. Each imputed variant was tested for association with time-to-treatment discontinuation due to AIMSS using a Cox proportional hazards model assuming additive genetic effects and adjusting for age, baseline pain score, prior taxane treatment, and AI arm. Secondary analyses were conducted within each AI arm and analyses of candidate variants previously reported to be associated with AIMSS risk. RESULTS: Four hundred ELPh participants were included in the combined analysis. Two variants surpassed the genome-wide significance level in the primary analysis (p value < 5 × 10-8), an intronic variant (rs79048288) within CCDC148 (HR = 4.42, 95% CI: 2.67-7.33) and an intergenic variant (rs912571) upstream of PPP1R14C (HR = 0.30, 95% CI: 0.20-0.47). In the secondary analysis, rs74418677, which is known to be associated with expression of SUPT20H, was significantly associated with discontinuation of letrozole therapy due to AIMSS (HR = 5.91, 95% CI: 3.16-11.06). We were able to replicate associations for candidate variants previously reported to be associated with AIMSS in this cohort, but were not able to replicate associations for any other variants previously reported in other patient cohorts. CONCLUSIONS: Our GWAS findings identify several candidate variants that may be associated with AIMSS risk from AI generally or letrozole specifically. Validation of these associations in independent cohorts is needed before translating these findings into clinical practice to improve treatment outcomes in patients with HR + breast cancer.

Genome-wide association study of letrozole plasma concentrations identifies non-exonic variants that may affect CYP2A6 metabolic activity.

OBJECTIVES: Letrozole is a nonsteroidal aromatase inhibitor used to treat hormone-receptor-positive breast cancer. Variability in letrozole efficacy and toxicity may be partially attributable to variable systemic drug exposure, which may be influenced by germline variants in the enzymes responsible for letrozole metabolism, including cytochrome P450 2A6 (CYP2A6). The objective of this genome-wide association study (GWAS) was to identify polymorphisms associated with steady-state letrozole concentrations. METHODS: The Exemestane and Letrozole Pharmacogenetics (ELPh) Study randomized postmenopausal patients with hormone-receptor-positive nonmetastatic breast cancer to letrozole or exemestane treatment. Germline DNA was collected pretreatment and blood samples were collected after 1 or 3 months of treatment to measure steady-state letrozole (and exemestane) plasma concentrations via HPLC/MS. Genome-wide genotyping was conducted on the Infinium Global Screening Array (>650 000 variants) followed by imputation. The association of each germline variant with age- and BMI-adjusted letrozole concentrations was tested in self-reported white patients via linear regression assuming an additive genetic model. RESULTS: There were 228 patients who met the study-specific inclusion criteria and had both DNA and letrozole concentration data for this GWAS. The association for one genotyped polymorphism (rs7937) with letrozole concentration surpassed genome-wide significance (P = 5.26 × 10-10), explaining 13% of the variability in untransformed steady-state letrozole concentrations. Imputation around rs7937 and in silico analyses identified rs56113850, a variant in the CYP2A6 intron that may affect CYP2A6 expression and activity. rs7937 was associated with age- and BMI-adjusted letrozole levels even after adjusting for genotype-predicted CYP2A6 metabolic phenotype (P = 3.86 × 10-10). CONCLUSION: Our GWAS findings confirm that steady-state letrozole plasma concentrations are partially determined by germline polymorphisms that affect CYP2A6 activity, including variants near rs7937 such as the intronic rs56113850 variant. Further research is needed to confirm whether rs56113850 directly affects CYP2A6 activity and to integrate nonexonic variants into CYP2A6 phenotypic activity prediction systems.

Influence of Uridine Diphosphate Glucuronosyltransferase Family 1 Member A1 and Solute Carrier Organic Anion Transporter Family 1 Member B1 Polymorphisms and Efavirenz on Bilirubin Disposition in Healthy Volunteers.

Chronic administration of efavirenz is associated with decreased serum bilirubin levels, probably through induction of UGT1A1 We assessed the impact of efavirenz monotherapy and UGT1A1 phenotypes on total, conjugated, and unconjugated serum bilirubin levels in healthy volunteers. Healthy volunteers were enrolled into a clinical study designed to address efavirenz pharmacokinetics, drug interactions, and pharmacogenetics. Volunteers received multiple oral doses (600 mg/day for 17 days) of efavirenz. Serum bilirubin levels were obtained at study entry and 1 week after completion of the study. DNA genotyping was performed for UGT1A1 [*80 (C>T), *6 (G>A), *28 (TA7), *36 (TA5), and *37 (TA8)] and for SLCO1B1 [*5 (521T>C) and *1b (388A>G] variants. Diplotype predicted phenotypes were classified as normal, intermediate, and slow metabolizers. Compared with bilirubin levels at screening, treatment with efavirenz significantly reduced total, conjugated, and unconjugated bilirubin. After stratification by UGT1A1 phenotypes, there was a significant decrease in total bilirubin among all phenotypes, conjugated bilirubin among intermediate metabolizers, and unconjugated bilirubin among normal and intermediate metabolizers. The data also show that UGT1A1 genotype predicts serum bilirubin levels at baseline, but this relationship is lost after efavirenz treatment. SLCO1B1 genotypes did not predict bilirubin levels at baseline or after efavirenz treatment. Our data suggest that efavirenz may alter bilirubin disposition mainly through induction of UGT1A1 metabolism and efflux through multidrug resistance-associated protein 2. SIGNIFICANCE STATEMENT: Efavirenz likely alters the pharmacokinetics of coadministered drugs, potentially causing lack of efficacy or increased adverse effects, as well as the disposition of endogenous compounds relevant in homeostasis through upregulation of UGT1A1 and multidrug resistance-associated protein 2. Measurement of unconjugated and conjugated bilirubin during new drug development may provide mechanistic understanding regarding enzyme and transporters modulated by the new drug.

Mechanistic Assessment of Extrahepatic Contributions to Glucuronidation of Integrase Strand Transfer Inhibitors.

Integrase strand transfer inhibitor (INSTI)-based regimens dominate initial human immunodeficiency virus treatment. Most INSTIs are metabolized predominantly via UDP-glucuronosyltransferases (UGTs). For drugs predominantly metabolized by UGTs, including INSTIs, in vitro data recovered from human liver microsomes (HLMs) alone often underpredict human oral clearance. While several factors may contribute, extrahepatic glucuronidation may contribute to this underprediction. Thus, we comprehensively characterized the kinetics for the glucuronidation of INSTIs (cabotegravir, dolutegravir, and raltegravir) using pooled human microsomal preparations from liver (HLMs), intestine (HIMs), and kidney (HKMs) tissues; human embryonic kidney 293 cells expressing individual UGTs; and recombinant UGTs. In vitro glucuronidation of cabotegravir (HLMs≈HKMs>>>HIMs), dolutegravir (HLMs>HIMs>>HKMs), and raltegravir (HLMs>HKMs>> HIMs) occurred in hepatic and extrahepatic tissues. The kinetic data from expression systems suggested the major enzymes in each tissue: hepatic UGT1A9 > UGT1A1 (dolutegravir and raltegravir) and UGT1A1 (cabotegravir), intestinal UGT1A3 > UGT1A8 > UGT1A1 (dolutegravir) and UGT1A8 > UGT1A1 (raltegravir), and renal UGT1A9 (dolutegravir and raltegravir). Enzymes catalyzing cabotegravir glucuronidation in the kidney and intestine could not be identified unequivocally. Using data from dolutegravir glucuronidation as a prototype, a "bottom-up" physiologically based pharmacokinetic model was developed in a stepwise approach and predicted dolutegravir oral clearance within 4.5-fold (hepatic data only), 2-fold (hepatic and intestinal data), and 32% (hepatic, intestinal, and renal data). These results suggest clinically meaningful glucuronidation of dolutegravir in tissues other than the liver. Incorporation of additional novel mechanistic and physiologic underpinnings of dolutegravir metabolism along with in silico approaches appears to be a powerful tool to accurately predict the clearance of dolutegravir from in vitro data.

Comprehensive assessment of cytochromes P450 and transporter genetics with endoxifen concentration during tamoxifen treatment.

OBJECTIVES: Tamoxifen bioactivation to endoxifen is mediated primarily by CYP2D6; however, considerable variability remains unexplained. Our aim was to perform a comprehensive assessment of the effect of genetic variation in tamoxifen-relevant enzymes and transporters on steady-state endoxifen concentrations. PATIENTS AND METHODS: Comprehensive genotyping of CYP enzymes and transporters was performed using the iPLEX ADME PGx Pro Panel in 302 tamoxifen-treated breast cancer patients. Predicted activity phenotype for 19 enzymes and transporters were analyzed for univariate association with endoxifen concentration, and then adjusted for CYP2D6 and clinical covariates. RESULTS: In univariate analysis, higher activity of CYP2C8 (regression ß=0.22, P=0.020) and CYP2C9 (ß=0.20, P=0.04), lower body weight (ß=-0.014, P<0.0001), and endoxifen measurement during winter (each ß<-0.39, P=0.002) were associated with higher endoxifen concentrations. After adjustment for the CYP2D6 diplotype, weight, and season, CYP2C9 remained significantly associated with higher concentrations (P=0.02), but only increased the overall model R by 1.3%. CONCLUSION: Our results further support a minor contribution of CYP2C9 genetic variability toward steady-state endoxifen concentrations. Integration of clinician and genetic variables into individualized tamoxifen dosing algorithms would marginally improve their accuracy and potentially enhance tamoxifen treatment outcomes.

Population Pharmacokinetic Modeling To Estimate the Contributions of Genetic and Nongenetic Factors to Efavirenz Disposition.

Efavirenz pharmacokinetics is characterized by large between-subject variability, which determines both therapeutic response and adverse effects. Some of the variability in efavirenz pharmacokinetics has been attributed to genetic variability in cytochrome P450 genes that alter efavirenz metabolism, such as CYP2B6 and CYP2A6 While the effects of additional patient factors have been studied, such as sex, weight, and body mass index, the extent to which they contribute to variability in efavirenz exposure is inconsistently reported. The aim of this analysis was to develop a pharmacometric model to quantify the contribution of genetic and nongenetic factors to efavirenz pharmacokinetics. A population-based pharmacokinetic model was developed using 1,132 plasma efavirenz concentrations obtained from 73 HIV-seronegative volunteers administered a single oral dose of 600 mg efavirenz. A two-compartment structural model with absorption occurring by zero- and first-order processes described the data. Allometric scaling adequately described the relationship between fat-free mass and apparent oral clearance, as well as fat mass and apparent peripheral volume of distribution. Inclusion of fat-free mass and fat mass in the model mechanistically accounted for correlation between these disposition parameters and sex, weight, and body mass index. Apparent oral clearance of efavirenz was reduced by 25% and 51% in subjects predicted to have intermediate and slow CYP2B6 metabolizer status, respectively. The final pharmacokinetic model accounting for fat-free mass, fat mass, and CYP2B6 metabolizer status was consistent with known mechanisms of efavirenz disposition, efavirenz physiochemical properties, and pharmacokinetic theory. (This study has been registered at ClinicalTrials.gov under identifier NCT00668395.).

Variable aromatase inhibitor plasma concentrations do not correlate with circulating estrogen concentrations in post-menopausal breast cancer patients.

PURPOSE: The aromatase inhibitors (AI) exemestane (EXE), letrozole (LET), and anastrozole suppress estrogen biosynthesis, and are effective treatments for estrogen receptor (ER)-positive breast cancer. Prior work suggests that anastrozole blood concentrations are associated with the magnitude of estrogen suppression. The objective of this study was to determine whether the magnitude of estrogen suppression, as determined by plasma estradiol (E2) concentrations, in EXE or LET treated patients is associated with plasma AI concentrations. METHODS: Five hundred post-menopausal women with ER-positive breast cancer were enrolled in the prospective Exemestane and Letrozole Pharmacogenetic (ELPh) Study conducted by the COnsortium on BReast cancer phArmacogomics (COBRA) and randomly assigned to either drug. Estrogen concentrations were measured at baseline and after 3 months of AI treatment and drug concentrations were measured after 1 or 3 months. EXE or LET concentrations were compared with 3-month E2 concentration or the change from baseline to 3 months using several complementary statistical procedures. RESULTS: Four-hundred patients with on-treatment E2 and AI concentrations were evaluable (EXE n = 200, LET n = 200). Thirty (7.6%) patients (EXE n = 13, LET n = 17) had 3-month E2 concentrations above the lower limit of quantification (LLOQ) (median: 4.75; range: 1.42-63.8 pg/mL). EXE and LET concentrations were not associated with on-treatment E2 concentrations or changes in E2 concentrations from baseline (all p > 0.05). CONCLUSIONS: Steady-state plasma AI concentrations do not explain variability in E2 suppression in post-menopausal women receiving EXE or LET therapy, in contrast with prior evidence in anastrozole treated patients.

Genome-wide discovery of drug-dependent human liver regulatory elements.

Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.

Inhibition of Cytochrome P450 2B6 Activity by Voriconazole Profiled Using Efavirenz Disposition in Healthy Volunteers.

Cytochrome P450 2B6 (CYP2B6) metabolizes clinically important drugs and other compounds. Its expression and activity vary widely among individuals, but quantitative estimation is hampered by the lack of safe and selective in vivo probes of CYP2B6 activity. Efavirenz, a nonnucleoside HIV-1 reverse transcriptase inhibitor, is mainly cleared by CYP2B6, an enzyme strongly inhibited in vitro by voriconazole. To test efavirenz metabolism as an in vivo probe of CYP2B6 activity, we quantified the inhibition of CYP2B6 activity by voriconazole in 61 healthy volunteers administered a single 100-mg oral dose of efavirenz with and without voriconazole administration. The kinetics of efavirenz metabolites demonstrated formation rate-limited elimination. Compared to control, voriconazole prolonged the elimination half-life (t1/2) and increased both the maximum concentration of drug in serum (Cmax) and the area under the concentration-time curve from 0 h to t (AUC0-t) of efavirenz (mean change of 51%, 36%, and 89%, respectively) (P < 0.0001) with marked intersubject variability (e.g., the percent change in efavirenz AUC0-t ranged from 0.4% to ∼224%). Voriconazole decreased efavirenz 8-hydroxylation by greater than 60% (P < 0.0001), whereas its effect on 7-hydroxylation was marginal. The plasma concentration ratio of efavirenz to 8-hydroxyefavirenz, determined 1 to 6 h after dosing, was significantly increased by voriconazole and correlated with the efavirenz AUC0-t (Pearson r = >0.8; P < 0.0001). This study demonstrates the mechanisms of voriconazole-efavirenz interaction, establishes the use of a low dose of efavirenz as a safe and selective in vivo probe for phenotyping CYP2B6 activity, and identifies several easy-to-use indices that should enhance understanding of the mechanisms of CYP2B6 interindividual variability. (This study is registered at ClinicalTrials.gov under identifier NCT01104376.).

Tamoxifen Dose Escalation in Patients With Diminished CYP2D6 Activity Normalizes Endoxifen Concentrations Without Increasing Toxicity.

BACKGROUND: Polymorphic CYP2D6 is primarily responsible for metabolic activation of tamoxifen to endoxifen. We previously reported that by increasing the daily tamoxifen dose to 40 mg/day in CYP2D6 intermediate metabolizer (IM), but not poor metabolizer (PM), patients achieve endoxifen concentrations similar to those of extensive metabolizer patients on 20 mg/day. We expanded enrollment to assess the safety of CYP2D6 genotype-guided dose escalation and investigate concentration differences between races. METHODS: PM and IM breast cancer patients currently receiving tamoxifen at 20 mg/day were enrolled for genotype-guided escalation to 40 mg/day. Endoxifen was measured at baseline and after 4 months. Quality-of-life data were collected using the Functional Assessment of Cancer Therapy-Breast (FACT-B) and Breast Cancer Prevention Trial Menopausal Symptom Scale at baseline and after 4 months. RESULTS: In 353 newly enrolled patients, genotype-guided dose escalation eliminated baseline concentration differences in IM (p = .08), but not PM (p = .009), patients. Endoxifen concentrations were similar in black and white patients overall (p = .63) and within CYP2D6 phenotype groups (p > .05). In the quality-of-life analysis of 480 patients, dose escalation did not meaningfully diminish quality of life; in fact, improvements were seen in several measures including the FACT Breast Cancer subscale (p = .004) and limitations in range of motion (p < .0001) in IM patients. CONCLUSION: Differences in endoxifen concentration during treatment can be eliminated by doubling the tamoxifen dose in IM patients, without an appreciable effect on quality of life. Validation of the association between endoxifen concentration and efficacy or prospective demonstration of improved efficacy is necessary to warrant clinical uptake of this personalized treatment strategy. IMPLICATIONS FOR PRACTICE: This secondary analysis of a prospective CYP2D6 genotype-guided tamoxifen dose escalation study confirms that escalation to 40 mg/day in patients with low-activity CYP2D6 phenotypes (poor or intermediate metabolizers) increases endoxifen concentrations without any obvious increases in treatment-related toxicity. It remains unknown whether endoxifen concentration is a useful predictor of tamoxifen efficacy, and thus, there is no current role in clinical practice for CYP2D6 genotype-guided tamoxifen dose adjustment. If future studies confirm the importance of endoxifen concentrations for tamoxifen efficacy and report a target concentration, this study provides guidance for a dose-adjustment approach that could maximize efficacy while maintaining patient quality of life.

Chiral Plasma Pharmacokinetics and Urinary Excretion of Bupropion and Metabolites in Healthy Volunteers.

Bupropion, widely used as an antidepressant and smoking cessation aid, undergoes complex metabolism to yield numerous metabolites with unique disposition, effect, and drug-drug interactions (DDIs) in humans. The stereoselective plasma and urinary pharmacokinetics of bupropion and its metabolites were evaluated to understand their potential contributions to bupropion effects. Healthy human volunteers (n = 15) were administered a single oral dose of racemic bupropion (100 mg), which was followed by collection of plasma and urine samples and determination of bupropion and metabolite concentrations using novel liquid chromatography-tandem mass spectrometry assays. Time-dependent, elimination rate-limited, stereoselective pharmacokinetics were observed for all bupropion metabolites. Area under the plasma concentration-time curve from zero to infinity ratios were on average approximately 65, 6, 6, and 4 and Cmax ratios were approximately 35, 6, 3, and 0.5 for (2R,3R)-/(2S,3S)-hydroxybupropion, R-/S-bupropion, (1S,2R)-/(1R,2S)-erythrohydrobupropion, and (1R,2R)-/(1S,2S)-threohydrobupropion, respectively. The R-/S-bupropion and (1R,2R)-/(1S,2S)-threohydrobupropion ratios are likely indicative of higher presystemic metabolism of S- versus R-bupropion by carbonyl reductases. Interestingly, the apparent renal clearance of (2S,3S)-hydroxybupropion was almost 10-fold higher than that of (2R,3R)-hydroxybupropion. The prediction of steady-state pharmacokinetics demonstrated differential stereospecific accumulation [partial area under the plasma concentration-time curve after the final simulated bupropion dose (300-312 hours) from 185 to 37,447 nM⋅h] and elimination [terminal half-life of approximately 7-46 hours] of bupropion metabolites, which may explain observed stereoselective differences in bupropion effect and DDI risk with CYP2D6 at steady state. Further elucidation of bupropion and metabolite disposition suggests that bupropion is not a reliable in vivo marker of CYP2B6 activity. In summary, to our knowledge, this is the first comprehensive report to provide novel insight into mechanisms underlying bupropion disposition by detailing the stereoselective pharmacokinetics of individual bupropion metabolites, which will enhance clinical understanding of bupropion's effects and DDIs with CYP2D6.

Efavirenz Inhibits the Human Ether-A-Go-Go Related Current (hERG) and Induces QT Interval Prolongation in CYP2B6*6*6 Allele Carriers.

BACKGROUND: Efavirenz (EFV) has been associated with torsade de pointes despite marginal QT interval lengthening. Since EFV is metabolized by the cytochrome P450 (CYP) 2B6 enzyme, we hypothesized that EFV would lengthen the rate-corrected QT (QTcF) interval in carriers of the CYP2B6*6 decreased functional allele. OBJECTIVE: The primary objective of this study was to evaluate EFV-associated QT interval changes with regard to CYP2B6 genotype and to explore mechanisms of QT interval lengthening. METHODS: EFV was administered to healthy volunteers (n = 57) as a single 600 mg dose followed by multiple doses to steady-state. Subjects were genotyped for known CYP2B6 alleles and ECGs and EFV plasma concentrations were obtained serially. Whole-cell, voltage-clamp experiments were performed on cells stably expressing hERG and exposed to EFV in the presence and absence of CYP2B6 expression. RESULTS: EFV demonstrated a gene-dose effect and exceeded the FDA criteria for QTcF interval prolongation in CYP2B6*6/*6 carriers. The largest mean time-matched differences ∆∆QTcF were observed at 6 hours (14 milliseconds; 95% CI [1; 27]), 12 hours (18 milliseconds; 95% CI [-4; 40]), and 18 hours (6 milliseconds; 95% CI [-1; 14]) in the CYP2B6*6/*6 genotype. EFV concentrations exceeding 0.4 µg/mL significantly inhibited outward hERG tail currents (P < 0.05). CONCLUSIONS: This study demonstrates that homozygous carriers of CYP2B6*6 allele may be at increased risk for EFV-induced QTcF interval prolongation via inhibition of hERG.

Stereoselective Glucuronidation of Bupropion Metabolites In Vitro and In Vivo.

Bupropion is a widely used antidepressant and smoking cessation aid in addition to being one of two US Food and Drug Administration-recommended probe substrates for evaluation of cytochrome P450 2B6 activity. Racemic bupropion undergoes oxidative and reductive metabolism, producing a complex profile of pharmacologically active metabolites with relatively little known about the mechanisms underlying their elimination. A liquid chromatography-tandem mass spectrometry assay was developed to simultaneously separate and detect glucuronide metabolites of (R,R)- and (S,S)-hydroxybupropion, (R,R)- and (S,S)-hydrobupropion (threo) and (S,R)- and (R,S)-hydrobupropion (erythro), in human urine and liver subcellular fractions to begin exploring mechanisms underlying enantioselective metabolism and elimination of bupropion metabolites. Human liver microsomal data revealed marked glucuronidation stereoselectivity [Cl(int), 11.4 versus 4.3 µl/min per milligram for the formation of (R,R)- and (S,S)-hydroxybupropion glucuronide; and Cl(max), 7.7 versus 1.1 µl/min per milligram for the formation of (R,R)- and (S,S)-hydrobupropion glucuronide], in concurrence with observed enantioselective urinary elimination of bupropion glucuronide conjugates. Approximately 10% of the administered bupropion dose was recovered in the urine as metabolites with glucuronide metabolites, accounting for approximately 40%, 15%, and 7% of the total excreted hydroxybupropion, erythro-hydrobupropion, and threo-hydrobupropion, respectively. Elimination pathways were further characterized using an expressed UDP-glucuronosyl transferase (UGT) panel with bupropion enantiomers (both individual and racemic) as substrates. UGT2B7 catalyzed the stereoselective formation of glucuronides of hydroxybupropion, (S,S)-hydrobupropion, (S,R)- and (R,S)-hydrobupropion; UGT1A9 catalyzed the formation of (R,R)-hydrobupropion glucuronide. These data systematically describe the metabolic pathways underlying bupropion metabolite disposition and significantly expand our knowledge of potential contributors to the interindividual and intraindividual variability in therapeutic and toxic effects of bupropion in humans.

Associations between genetic variants and the effect of letrozole and exemestane on bone mass and bone turnover.

Adjuvant therapy for hormone receptor (HR) positive postmenopausal breast cancer patients includes aromatase inhibitors (AI). While both the non-steroidal AI letrozole and the steroidal AI exemestane decrease serum estrogen concentrations, there is evidence that exemestane may be less detrimental to bone. We hypothesized that single nucleotide polymorphisms (SNP) predict effects of AIs on bone turnover. Early stage HR-positive breast cancer patients were enrolled in a randomized trial of exemestane versus letrozole. Effects of AI on bone mineral density (BMD) and bone turnover markers (BTM), and associations between SNPs in 24 candidate genes and changes in BMD or BTM were determined. Of the 503 enrolled patients, paired BMD data were available for 123 and 101 patients treated with letrozole and exemestane, respectively, and paired BTM data were available for 175 and 173 patients, respectively. The mean change in lumbar spine BMD was significantly greater for letrozole-treated (-3.2 %) compared to exemestane-treated patients (-1.0 %) (p = 0.0016). Urine N-telopeptide was significantly increased in patients treated with exemestane (p = 0.001) but not letrozole. Two SNPs (rs4870061 and rs9322335) in ESR1 and one SNP (rs10140457) in ESR2 were associated with decreased BMD in letrozole-treated patients. In the exemestane-treated patients, SNPs in ESR1 (Rs2813543) and CYP19A1 (Rs6493497) were associated with decreased bone density. Exemestane had a less negative impact on bone density compared to letrozole, and the effects of AI therapy on bone may be impacted by genetic variants in the ER pathway.

In Vitro Metabolism of Montelukast by Cytochrome P450s and UDP-Glucuronosyltransferases.

Montelukast has been recommended as a selective in vitro and in vivo probe of cytochrome P450 (P450) CYP2C8 activity, but its selectivity toward this enzyme remains unclear. We performed detailed characterization of montelukast metabolism in vitro using human liver microsomes (HLMs), expressed P450s, and uridine 5'-diphospho-glucuronosyltransferases (UGTs). Kinetic and inhibition experiments performed at therapeutically relevant concentrations reveal that CYP2C8 and CYP2C9 are the principal enzymes responsible for montelukast 36-hydroxylation to 1,2-diol. CYP3A4 was the main catalyst of montelukast sulfoxidation and stereoselective 21-hydroxylation, and multiple P450s participated in montelukast 25-hydroxylation. We confirmed direct glucuronidation of montelukast to an acyl-glucuronide. We also identified a novel peak that appears consistent with an ether-glucuronide. Kinetic analysis in HLMs and experiments in expressed UGTs indicate that both metabolites were exclusively formed by UGT1A3. Comparison of in vitro intrinsic clearance in HLMs suggest that direct glucuronidation may play a greater role in the overall metabolism of montelukast than does P450-mediated oxidation, but the in vivo contribution of UGT1A3 needs further testing. In conclusion, our in vitro findings provide new insight toward montelukast metabolism. The utility of montelukast as a probe of CYP2C8 activity may be compromised owing to involvement of multiple P450s and UGT1A3 in its metabolism.

In vivo assessment of the metabolic activity of CYP2D6 diplotypes and alleles.

AIMS: A prospectively enrolled patient cohort was used to assess whether the prediction of CYP2D6 phenotype activity from genotype data could be improved by reclassification of diplotypes or alleles. METHODS: Three hundred and fifty-five patients receiving tamoxifen 20 mg were genotyped for CYP2D6 and tamoxifen metabolite concentrations were measured. The endoxifen : N-desmethly-tamoxifen metabolic ratio, as a surrogate of CYP2D6 activity, was compared across four diplotypes (EM/IM, EM/PM, IM/IM, IM/PM) that are typically collapsed into an intermediate metabolizer (IM) phenotype. The relative metabolic activity of each allele type (UM, EM, IM, and PM) and each EM and IM allele was estimated for comparison with the activity scores typically assigned, 2, 1, 0.5 and 0, respectively. RESULTS: Each of the four IM diplotypes have distinct CYP2D6 activity from each other and from the EM and PM phenotype groups (each P < 0.05). Setting the activity of an EM allele at 1.0, the relative activities of a UM, IM and PM allele were 0.85, 0.67 and 0.52, respectively. The activity of the EM alleles were statistically different (P < 0.0001), with the CYP2D6*2 allele (scaled activity = 0.63) closer in activity to an IM than an EM allele. The activity of the IM alleles were also statistically different (P = 0.014). CONCLUSION: The current systems for translating CYP2D6 genotype into phenotype are not optimally calibrated, particularly in regards to IM diplotypes and the *2 allele. Additional research is needed to improve the prediction of CYP2D6 activity from genetic data for individualized dosing of CYP2D6 dependent drugs.

Platelet factor XIIIa release during platelet aggregation and plasma clot strength measured by thrombelastography in patients with coronary artery disease treated with clopidogrel.

It has been estimated that up to half of circulating factor XIIIa (FXIIIa) is stored in platelets. The release of FXIIIa from platelets upon stimulation with adenosine diphosphate (ADP) in patients with coronary artery disease treated with dual antiplatelet therapy has not been previously examined. Samples from 96 patients with established coronary artery disease treated with aspirin and clopidogrel were examined. Platelet aggregation was performed by light transmittance aggregometry in platelet-rich plasma (PRP), with platelet-poor plasma (PPP) as reference, and ADP 5 µM as agonist. Kaolin-activated thrombelastography (TEG) was performed in citrate PPP. PRP after aggregation was centrifuged and plasma supernatant (PSN) collected. FXIIIa was measured in PPP and PSN. Platelet aggregation after stimulation with ADP 5 µM resulted in 24% additional FXIIIa release in PSN as compared to PPP (99.3 ± 27 vs. 80.3 ± 24%, p < 0.0001). FXIIIa concentration in PSN correlated with maximal plasma clot strength (TEG-G) (r = 0.48, p < 0.0001), but not in PPP (r = 0.15, p = 0.14). Increasing quartiles of platelet-derived FXIIIa were associated with incrementally higher TEG-G (p = 0.012). FXIIIa release was similar between clopidogrel responders and non-responders (p = 0.18). In summary, platelets treated with aspirin and clopidogrel release a significant amount of FXIIIa upon aggregation by ADP. Platelet-derived FXIIIa may contribute to differences in plasma TEG-G, and thus, in part, provide a mechanistic explanation for high clot strength observed as a consequence of platelet activation. Variability in clopidogrel response does not significantly influence FXIIIa release from platelets.