A phase 1 study of triple-targeted therapy with BRAF, MEK, and AKT inhibitors for patients with BRAF-mutated cancers.

BACKGROUND: Aberrant PI3K/AKT signaling in BRAF-mutant cancers contributes to resistance to BRAF inhibitors. The authors examined dual MAPK and PI3K pathway inhibition in patients who had BRAF-mutated solid tumors (ClinicalTrials.gov identifier NCT01902173). METHODS: Patients with BRAF V600E/V600K-mutant solid tumors received oral dabrafenib at 150 mg twice daily with dose escalation of oral uprosertib starting at 50 mg daily, or, in the triplet cohorts, with dose escalation of both oral trametinib starting at 1.5 mg daily and oral uprosertib starting at 25 mg daily. Dose-limiting toxicities (DLTs) were assessed within the first 56 days of treatment. Radiographic responses were assessed at 8-week intervals. RESULTS: Twenty-seven patients (22 evaluable) were enrolled in parallel doublet and triplet cohorts. No DLTs were observed in the doublet cohorts (N = 7). One patient had a DLT at the maximum administered dose of triplet therapy (dabrafenib 150 mg twice daily and trametinib 2 mg daily plus uprosertib 75 mg daily). Three patients in the doublet cohorts had partial responses (including one who had BRAF inhibitor-resistant melanoma). Two patients in the triplet cohorts had a partial response, and one patient had an unconfirmed partial response. Pharmacokinetic data suggested reduced dabrafenib and dabrafenib metabolite exposure in patients who were also exposed to both trametinib and uprosertib, but not in whose who were exposed to uprosertib without trametinib. CONCLUSIONS: Concomitant inhibition of both the MAPK and PI3K-AKT pathways for the treatment of BRAF-mutated cancers was well tolerated, leading to objective responses, but higher level drug-drug interactions affected exposure to dabrafenib and its metabolites.

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.

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.

Mass balance and pharmacokinetics of an oral dose of 14 C-napabucasin in healthy adult male subjects.

This phase 1, open-label study assessed14 C-napabucasin absorption, metabolism, and excretion, napabucasin pharmacokinetics, and napabucasin metabolites (primary objectives); safety/tolerability were also evaluated. Eight healthy males (18-45 years) received a single oral 240-mg napabucasin dose containing ~100 µCi14 C-napabucasin. Napabucasin was absorbed and metabolized to dihydro-napabucasin (M1; an active metabolite [12.57-fold less activity than napabucasin]), the sole major circulating metabolite (median time to peak concentration: 2.75 and 2.25 h, respectively). M1 plasma concentration versus time profiles generally mirrored napabucasin; similar arithmetic mean half-lives (7.14 and 7.92 h, respectively) suggest M1 formation was rate limiting. Napabucasin systemic exposure (per Cmax and AUC) was higher than M1. The total radioactivity (TRA) whole blood:plasma ratio (AUClast : 0.376; Cmax : 0.525) indicated circulating drug-related compounds were essentially confined to plasma. Mean TRA recovery was 81.1% (feces, 57.2%; urine, 23.8%; expired air, negligible). Unlabeled napabucasin and M1 recovered in urine accounted for 13.9% and 11.0% of the dose (sum similar to urine TRA recovered); apparent renal clearance was 8.24 and 7.98 L/h. No uniquely human or disproportionate metabolite was quantified. Secondary glucuronide and sulfate conjugates were common urinary metabolites, suggesting napabucasin was mainly cleared by reductive metabolism. All subjects experienced mild treatment-emergent adverse events (TEAEs), the majority related to napabucasin. The most commonly reported TEAEs were gastrointestinal disorders. There were no clinically significant laboratory, vital sign, electrocardiogram, or physical examination changes. Napabucasin was absorbed, metabolized to M1 as the sole major circulating metabolite, and primarily excreted via feces. A single oral 240-mg dose was generally well tolerated.

Circulating miRNAs as Biomarkers for CYP2B6 Enzyme Activity.

The CYP2B6 gene is highly polymorphic and its activity shows wide interindividual variability. However, substantial variability in CYP2B6 activity remains unexplained by the known CYP2B6 genetic variations. Circulating, cell-free micro RNAs (miRNAs) may serve as biomarkers of hepatic enzyme activity. CYP2B6 activity in 72 healthy volunteers was determined using the disposition of efavirenz as a probe drug. Circulating miRNA expression was quantified from baseline plasma samples. A linear model consisting of the effects of miRNA expression, genotype-determined metabolizer status, and demographic information was developed to predict CYP2B6 activity. Expression of 2,510 miRNAs were quantified out of which 7 miRNAs, together with the CYP2B6-genotypic metabolizer status and demographics, was shown to be predictive markers for CYP2B6 activity. The reproducibility of the model was evaluated by cross-validation. The average Pearson's correlation (R) between the predicted and observed maximum plasma concentration (Cmax ) ratios of efavirenz and its metabolite-8-OH efavirenz using the linear model with all features (7 miRNA + metabolizer status + age + sex + race) was 0.6702. Similar results were also observed using area under the curve (AUC) ratios (Pearson correlation's R = 0.6035). Thus, at least 36% (R2 ) of the variability of in vivo CYP2B6 activity was explained using this model. This is a significant improvement over the models using only the genotype-based metabolizer status or the demographic information, which explained only 6% or less of the variability of in vivo CYP2B6 activity. Our results, therefore, demonstrate that circulating plasma miRNAs can be valuable biomarkers for in vivo CYP2B6 activity.

A New Data Repository for Pharmacokinetic Natural Product-Drug Interactions: From Chemical Characterization to Clinical Studies.

There are many gaps in scientific knowledge about the clinical significance of pharmacokinetic natural product-drug interactions (NPDIs) in which the natural product (NP) is the precipitant and a conventional drug is the object. The National Center for Complimentary and Integrative Health created the Center of Excellence for NPDI Research (NaPDI Center) (www.napdi.org) to provide leadership and guidance on the study of pharmacokinetic NPDIs. A key contribution of the Center is the first user-friendly online repository that stores and links pharmacokinetic NPDI data across chemical characterization, metabolomics analyses, and pharmacokinetic in vitro and clinical experiments (repo.napdi.org). The design is expected to help researchers more easily arrive at a complete understanding of pharmacokinetic NPDI research on a particular NP. The repository will also facilitate multidisciplinary collaborations, as the repository links all of the experimental data for a given NP across the study types. The current work describes the design of the repository, standard operating procedures used to enter data, and pharmacokinetic NPDI data that have been entered to date. To illustrate the usefulness of the NaPDI Center repository, more details on two high-priority NPs, cannabis and kratom, are provided as case studies. SIGNIFICANCE STATEMENT: The data and knowledge resulting from natural product-drug interaction (NPDI) studies is distributed across a variety of information sources, rendering difficulties to find, access, and reuse. The Center of Excellence for NPDI Research addressed these difficulties by developing the first user-friendly online repository that stores data from in vitro and clinical pharmacokinetic NPDI experiments and links them with study data from chemical characterization and metabolomics analyses of natural products that are also stored in the repository.

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.

Inhibitory Effects of Probenecid on Pharmacokinetics of Tenofovir Disoproxil Fumarate and Emtricitabine for On-Demand HIV Preexposure Prophylaxis.

In a randomized, crossover pharmacokinetic study in healthy volunteers (N = 14), a single dose of 2 g probenecid (PRO)-boosted 600 mg tenofovir disoproxil fumarate (TDF)/400 mg emtricitabine (FTC) (test (T) +PRO) was compared with the current on-demand HIV preexposure prophylaxis from the IPERGAY study (a 600 mg TDF/400 mg FTC on day 1 and 300 mg TDF/200 mg FTC on days 2 and 3) (control, C IPERGAY). PRO increased mean single-dose area under the plasma concentration-time curve extrapolated to infinity (AUC0-∞,SD ) of tenofovir (TFV) and FTC by 61% and 68%, respectively. The TFV-diphosphate (TFV-DP) concentrations in peripheral blood mononuclear cells were higher (~30%) at 24 hours in T +PRO but then fell significantly lower (~40%) at 72 hours compared with C IPERGAY. The interaction between FTC and PRO was unexpected and novel. Further study is needed to determine if this PRO-boosted TDF/FTC regimen would be clinically effective.

Probenecid-Boosted Tenofovir: A Physiologically-Based Pharmacokinetic Model-Informed Strategy for On-Demand HIV Preexposure Prophylaxis.

Multiple doses of tenofovir disoproxil fumarate (TDF) together with emtricitabine is effective for HIV preexposure prophylaxis (PrEP). TDF is converted to tenofovir (TFV) in circulation, which is subsequently cleared via tubular secretion by organic ion transporters (OATs; OAT1 and OAT3). Using in vitro kinetic parameters for TFV and the OAT1 and OAT3 inhibitor probenecid, a bottom-up physiologically-based pharmacokinetic model was successfully developed for the first time that accurately describes the probenecid-TFV interaction. This model predicted an increase in TFV plasma exposure by 60%, which was within 15% of the observed clinical pharmacokinetic data, and a threefold decrease in renal cells exposure following coadministration of a 600 mg TDF dose with 2 g probenecid. When compared with multiple-dose regimens, a single-dose probenecid-boosted TDF regimen may be effective for HIV PrEP and improve adherence and safety by minimizing TFV-induced nephrotoxicity by reducing TFV accumulation in renal cells.

Drug-gene and drug-drug interactions associated with tramadol and codeine therapy in the INGENIOUS trial.

Background: Tramadol and codeine are metabolized by CYP2D6 and are subject to drug-gene and drug-drug interactions. Methods: This interim analysis examined prescribing behavior and efficacy in 102 individuals prescribed tramadol or codeine while receiving pharmaco-genotyping as part of the INGENIOUS trial (NCT02297126). Results: Within 60 days of receiving tramadol or codeine, clinicians more frequently prescribed an alternative opioid in ultrarapid and poor metabolizers (odds ratio: 19.0; 95% CI: 2.8-160.4) as compared with normal or indeterminate metabolizers (p = 0.01). After adjusting the CYP2D6 activity score for drug-drug interactions, uncontrolled pain was reported more frequently in individuals with reduced CYP2D6 activity (odds ratio: 0.50; 95% CI: 0.25-0.94). Conclusion: Phenoconversion for drug-drug and drug-gene interactions is an important consideration in pharmacogenomic implementation; drug-drug interactions may obscure the potential benefits of genotyping.

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.

Rifampin modulation of xeno- and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes.

Rifampin is a pleiotropic inducer of multiple drug metabolizing enzymes and transporters. This work utilized a global approach to evaluate rifampin effects on conjugating enzyme gene expression with relevance to human xeno- and endo-biotic metabolism. Primary human hepatocytes from 7 subjects were treated with rifampin (10 µmol/L, 24 hours). Standard methods for RNA-seq library construction, EZBead preparation, and NextGen sequencing were used to measure UDP-glucuronosyl transferase UGT, sulfonyltransferase SULT, N acetyltransferase NAT, and glutathione-S-transferase GST mRNA expression compared to vehicle control (0.01% MeOH). Rifampin-induced (>1.25-fold) mRNA expression of 13 clinically important phase II drug metabolizing genes and repressed (>1.25-fold) the expression of 3 genes (P < .05). Rifampin-induced miRNA expression changes correlated with mRNA changes and miRNAs were identified that may modulate conjugating enzyme expression. NAT2 gene expression was most strongly repressed (1.3-fold) by rifampin while UGT1A4 and UGT1A1 genes were most strongly induced (7.9- and 4.8-fold, respectively). Physiologically based pharmacokinetic modeling (PBPK) was used to simulate the clinical consequences of rifampin induction of CYP3A4- and UGT1A4-mediated midazolam metabolism. Simulations evaluating isolated UGT1A4 induction predicted increased midazolam N-glucuronide exposure (~4-fold) with minimal reductions in parent midazolam exposure (~10%). Simulations accounting for simultaneous induction of both CYP3A4 and UGT1A4 predicted a ~10-fold decrease in parent midazolam exposure with only a ~2-fold decrease in midazolam N-glucuronide metabolite exposure. These data reveal differential effects of rifampin on the human conjugating enzyme transcriptome and potential associations with miRNAs that form the basis for future mechanistic studies to elucidate the interplay of conjugating enzyme regulatory elements.

Developing User Personas to Aid in the Design of a User-Centered Natural Product-Drug Interaction Information Resource for Researchers.

Pharmacokinetic interactions between natural products and conventional drugs can adversely impact patient outcomes. These complex interactions present unique challenges that require clear communication to researchers. We are creating a public information portal to facilitate researchers' access to credible evidence about these interactions. As part of a user-centered design process, three types of intended researchers were surveyed: drug-drug interaction scientists, clinical pharmacists, and drug compendium editors. Of the 23 invited researchers, 17 completed the survey. The researchers suggested a number of specific requirements for a natural product-drug interaction information resource, including specific information about a given interaction, the potential to cause adverse effects, and the clinical importance. Results were used to develop user personas that provided the development team with a concise and memorable way to represent information needs of the three main researcher types and a common basis for communicating the design's rationale.

Variants in the CYP2B6 3'UTR Alter In Vitro and In Vivo CYP2B6 Activity: Potential Role of MicroRNAs.

CYP2B6*6 and CYP2B6*18 are the most clinically important variants causing reduced CYP2B6 protein expression and activity. However, these variants do not account for all variability in CYP2B6 activity. Emerging evidence has shown that genetic variants in the 3'UTR may explain variable drug response by altering microRNA regulation. Five 3'UTR variants were associated with significantly altered efavirenz AUC0-48 (8-OH-EFV/EFV) ratios in healthy human volunteers. The rs70950385 (AG>CA) variant, predicted to create a microRNA binding site for miR-1275, was associated with a 33% decreased CYP2B6 activity among normal metabolizers (AG/AG vs. CA/CA (P < 0.05)). In vitro luciferase assays were used to confirm that the CA on the variant allele created a microRNA binding site causing an 11.3% decrease in activity compared to the AG allele when treated with miR-1275 (P = 0.0035). Our results show that a 3'UTR variant contributes to variability in CYP2B6 activity.

Clinical and educational impact of pharmacogenomics testing: a case series from the INGENIOUS trial.

Pharmacogenomic testing has become increasingly widespread. However, there remains a need to bridge the gap between test results and providers lacking the expertise required to interpret these results. The Indiana Genomics Implementation trial is underway at our institution to examine total healthcare cost and patient outcomes after genotyping in a safety-net healthcare system. As part of the study, trial investigators and clinical pharmacology fellows interpret genotype results, review patient histories and medication lists and evaluate potential drug-drug interactions. We present a case series of patients in whom pharmacogenomic consultations aided providers in appropriately applying pharmacogenomic results within the clinical context. Formal consultations not only provide valuable patient care information but educational opportunities for the fellows to cement pharmacogenomic concepts.

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.

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.

Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides.

Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary health care market. Mechanistic descriptions of the pharmacokinetics and pharmacodynamics of constituents composing these products remain nascent, particularly for metabolites produced following herbal product ingestion. Generation and characterization of authentic metabolite standards are essential to improve the quantitative mechanistic understanding of herbal product disposition in both in vitro and in vivo systems. Using the model herbal product, milk thistle, the objective of this work was to biosynthesize multimilligram quantities of glucuronides of select constituents (flavonolignans) to fill multiple knowledge gaps in the understanding of herbal product disposition and action. A partnership between clinical pharmacology and natural products chemistry expertise was leveraged to optimize reaction conditions for efficient glucuronide formation and evaluate alternate enzyme and reagent sources to improve cost effectiveness. Optimized reaction conditions used at least one-fourth the amount of microsomal protein (from bovine liver) and cofactor (UDP glucuronic acid) compared with typical conditions using human-derived subcellular fractions, providing substantial cost savings. Glucuronidation was flavonolignan-dependent. Silybin A, silybin B, isosilybin A, and isosilybin B generated five, four, four, and three monoglucuronides, respectively. Large-scale synthesis (40 mg of starting material) generated three glucuronides of silybin A: silybin A-7-O-ß-D-glucuronide (15.7 mg), silybin A-5-O-ß-D-glucuronide (1.6 mg), and silybin A-4´´-O-ß-D-glucuronide (11.1 mg). This optimized, cost-efficient method lays the foundation for a systematic approach to synthesize and characterize herbal product constituent glucuronides, enabling an improved understanding of mechanisms underlying herbal product disposition and action.

Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product-Drug Interaction.

Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 µM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4'- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27-66 µM; UGT1A1, 3.2-8.3 µM; UGT1A8, 19-73 µM; and UGT1A10, 65-120 µM) encompassed reported intestinal tissue concentrations (20-310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product-drug interactions in the context of cancer prevention.