Human Enteroid Monolayers: A Novel, Functionally-Stable Model for Investigating Oral Drug Disposition.

To further the development of an in vitro model which faithfully recapitulates drug disposition of orally administered drugs, we investigated the utility of human enteroid monolayers to simultaneously assess intestinal drug absorption and first-pass metabolism processes. We cultured human enteroid monolayers from three donors, derived via biopsies containing duodenal stem cells that were propagated and then differentiated atop permeable Transwell® inserts, and confirmed transformation into a largely enterocyte population via RNA-seq analysis and immunocytochemical (ICC) assays. Proper cell morphology was assessed and confirmed via bright field microscopy and ICC imaging of tight junction proteins and other apically and basolaterally localized proteins. Enteroid monolayer barrier integrity was demonstrated by elevated transepithelial electrical resistance (TEER) that stabilized after 10 days in culture and persisted for 42 days. These results were corroborated by low paracellular transport probe permeability at 7 and 21 days in culture. The activity of a prominent drug metabolizing enzyme, CYP3A, was confirmed at 7, 21, and 42 days culture under basal, 1α,25(OH)2 vitamin D3-induced, and 6',7'-dihydroxybergamottin-inhibited conditions. The duration of these experiments is particularly noteworthy, as this is the first study assessing drug metabolizing enzymes and transporters (DMET) expression/function for enteroids cultured for greater than 12 days. The sum of these results suggests enteroid monolayers are a promising ex vivo model to investigate and quantitatively predict an orally administered drug's intestinal absorption and/or metabolism. Significance Statement This study presents a novel ex vivo model of the human intestine, human intestinal organoid (enteroid) monolayers, that maintain barrier function and metabolic functionality for up to 42-days in culture. The incorporation of both barrier integrity and metabolic function over an extended period within the same model is an advancement over historically used in vitro systems, which either lack one or both of these attributes or have limited viability.

Identification of Sociodemographic, Clinical, and Genetic Factors to Aid Alaska Native and American Indian People to Successfully Quit Smoking.

INTRODUCTION: Alaska Native and American Indian (ANAI) people have a smoking prevalence of 23%. Nicotine metabolite ratio (NMR) and genetic testing may enable tailored selection of tobacco cessation medication. AIMS AND METHODS: The purpose of this study was to evaluate the relative contributions of NMR, cessation medication, demographics, and tobacco use history to cessation. Participants were recruited into an observational cohort study consisting of a baseline visit prior to their quit date and 6-week follow-up. Demographic and tobacco use surveys and blood, urine, and breath samples were collected at each visit. Electronic health records were queried for cessation medications. NMR was categorized into slow or normal nicotine metabolism phenotypes (<0.31 and ≥ 0.31, respectively). The main outcome was cessation at 6 weeks. Analyses consisted of descriptive statistics, medication and phenotype concordance, and estimates of relative risk (RR) of quitting. RESULTS: We enrolled 151 ANAI adults who smoked cigarettes daily. Two-thirds had normal nicotine metabolism phenotype. Retrospective medication and phenotype concordance was 39%. The overall quit rate was 25%. No demographic factors or tobacco use history were associated with quit success. Varenicline and bupropion increased the likelihood of quitting (RR = 2.93 [1.42, 6.03] and RR = 2.52 [1.12, 5.64], respectively) compared to nicotine replacement therapy. Non-optimal medication and phenotype concordance decreased likelihood of quit success (RR = 0.44 [0.22, 0.91]) compared to optimal concordance. CONCLUSIONS: This exploratory study found associations between quit success and tobacco cessation medication as well as medication and phenotype concordance. Additional research is needed to assess use of NMR for treatment selection among ANAI people. IMPLICATIONS: These results broadly support additional community-engaged research to improve medication and phenotype concordance in tribal health settings. Such future research on implementing meditcation and phenotype concordance holds promise to improve expectations, quit success, and health outcomes amongst individuals attempting to quit smoking.

Modeling Pharmacokinetic Natural Product-Drug Interactions for Decision-Making: A NaPDI Center Recommended Approach.

The popularity of botanical and other purported medicinal natural products (NPs) continues to grow, especially among patients with chronic illnesses and patients managed on complex prescription drug regimens. With few exceptions, the risk of a given NP to precipitate a clinically significant pharmacokinetic NP-drug interaction (NPDI) remains understudied or unknown. Application of static or dynamic mathematical models to predict and/or simulate NPDIs can provide critical information about the potential clinical significance of these complex interactions. However, methods used to conduct such predictions or simulations are highly variable. Additionally, published reports using mathematical models to interrogate NPDIs are not always sufficiently detailed to ensure reproducibility. Consequently, guidelines are needed to inform the conduct and reporting of these modeling efforts. This recommended approach from the Center of Excellence for Natural Product Drug Interaction Research describes a systematic method for using mathematical models to interpret the interaction risk of NPs as precipitants of potential clinically significant pharmacokinetic NPDIs. A framework for developing and applying pharmacokinetic NPDI models is presented with the aim of promoting accuracy, reproducibility, and generalizability in the literature. SIGNIFICANCE STATEMENT: Many natural products (NPs) contain phytoconstituents that can increase or decrease systemic or tissue exposure to, and potentially the efficacy of, a pharmaceutical drug; however, no regulatory agency guidelines exist to assist in predicting the risk of these complex interactions. This recommended approach from a multi-institutional consortium designated by National Institutes of Health as the Center of Excellence for Natural Product Drug Interaction Research provides a framework for modeling pharmacokinetic NP-drug interactions.

An Integrative Approach to Elucidate Mechanisms Underlying the Pharmacokinetic Goldenseal-Midazolam Interaction: Application of In Vitro Assays and Physiologically Based Pharmacokinetic Models to Understand Clinical Observations.

The natural product goldenseal is a clinical inhibitor of CYP3A activity, as evidenced by a 40%-60% increase in midazolam area under the plasma concentration versus time curve (AUC) after coadministration with goldenseal. The predominant goldenseal alkaloids berberine and (-)-ß-hydrastine were previously identified as time-dependent CYP3A inhibitors using human liver microsomes. Whether these alkaloids contribute to the clinical interaction, as well as the primary anatomic site (hepatic vs. intestinal) and mode of CYP3A inhibition (reversible vs. time-dependent), remain uncharacterized. The objective of this study was to mechanistically assess the pharmacokinetic goldenseal-midazolam interaction using an integrated in vitro-in vivo-in silico approach. Using human intestinal microsomes, (-)-ß-hydrastine was a more potent time-dependent inhibitor of midazolam 1'-hydroxylation than berberine (KI and kinact: 8.48 µM and 0.041 minutes-1, respectively, vs. >250 µM and ∼0.06 minutes-1, respectively). Both the AUC and Cmax of midazolam increased by 40%-60% after acute (single 3-g dose) and chronic (1 g thrice daily × 6 days) goldenseal administration to healthy adults. These increases, coupled with a modest or no increase (≤23%) in half-life, suggested that goldenseal primarily inhibited intestinal CYP3A. A physiologically based pharmacokinetic interaction model incorporating berberine and (-)-ß-hydrastine successfully predicted the goldenseal-midazolam interaction to within 20% of that observed after both chronic and acute goldenseal administration. Simulations implicated (-)-ß-hydrastine as the major alkaloid precipitating the interaction, primarily via time-dependent inhibition of intestinal CYP3A, after chronic and acute goldenseal exposure. Results highlight the potential interplay between time-dependent and reversible inhibition of intestinal CYP3A as the mechanism underlying natural product-drug interactions, even after acute exposure to the precipitant. SIGNIFICANCE STATEMENT: Natural products can alter the pharmacokinetics of an object drug, potentially resulting in increased off-target effects or decreased efficacy of the drug. The objective of this work was to evaluate fundamental mechanisms underlying the clinically observed goldenseal-midazolam interaction. Results support the use of an integrated approach involving established in vitro assays, clinical evaluation, and physiologically based pharmacokinetic modeling to elucidate the complex interplay between multiple phytoconstituents and various pharmacokinetic processes driving a drug interaction.

The Utility of Mixed Effects Models in the Evaluation of Complex Genomic Traits In Vitro.

In pharmacogenomic studies, the use of human liver microsomes as a model system to evaluate the impact of complex genomic traits (i.e., linkage-disequilibrium patterns, coding, and non-coding variation, etc.) on efficiency of drug metabolism is challenging. To accurately predict the true effect size of genomic traits requires large richly sampled datasets representative of the study population. Moreover, the acquisition of this data can be labor-intensive if the study design or bioanalytical methods are not high throughput, and it is potentially unfeasible if the abundance of sample needed for experiments is limited. To overcome these challenges, we developed a novel strategic approach using non-linear mixed effects models (NLME) to determine enzyme kinetic parameters for individual liver specimens using sparse data. This method can facilitate evaluation of the impact that complex genomic traits have on the metabolism of xenobiotics in vitro when tissue and other resources are limited. In addition to facilitating the accrual of data, it allows for rigorous testing of covariates as sources of kinetic parameter variability. In this in silico study, we present a practical application of such an approach using previously published in vitro cytochrome P450 (CYP) 2D6 data and explore the impact of sparse sampling, and experimental error on known kinetic parameter estimates of CYP2D6 mediated formation of 4-hydroxy-atomoxetine in human liver microsomes. SIGNIFICANCE STATEMENT: This study presents a novel non-linear mixed effects model (NLME)-based framework for evaluating the impact of complex genomic traits on saturable processes described by a Michaelis-Menten kinetics in vitro using sparse data. The utility of this approach extends beyond gene variant associations, including determination of covariate effects on in vitro kinetic parameters and reduced demand for precious experimental material.

Translating Kratom-Drug Interactions: From Bedside to Bench and Back.

Kratom is a botanical natural product belonging to the coffee family, with stimulant effects at low doses and opioid-like effects at higher doses. During the last two decades, kratom has been purported as a safer alternative to pharmaceutical and illicit drugs to self-manage pain and opioid withdrawal symptoms. Kratom alkaloids, typically mitragynine, have been detected in biologic samples from overdose deaths. These deaths are often observed in combination with other drugs and are suspected to result from polyintoxications. This review focuses on the potential for kratom to precipitate pharmacokinetic interactions with object drugs involved in these reported polyintoxications. The legal status, chemistry, pharmacology, and toxicology are also summarized. The aggregate in vitro and clinical data identified kratom and select kratom alkaloids as modulators of cytochrome P450 (P450) enzyme activity, notably as inhibitors of CYP2D6 and CYP3A, as well as P-glycoprotein-mediated efflux activity. These inhibitory effects could increase the systemic exposure to co-consumed object drugs, which may lead to adverse effects. Collectively, the evidence to date warrants further evaluation of potential kratom-drug interactions using an iterative approach involving additional mechanistic in vitro studies, well designed clinical studies, and physiologically based pharmacokinetic modeling and simulation. This critical information is needed to fill knowledge gaps regarding the safe and effective use of kratom, thereby addressing ongoing public health concerns. SIGNIFICANCE STATEMENT: The botanical kratom is increasingly used to self-manage pain and opioid withdrawal symptoms due to having opioid-like effects. The legal status, chemistry, pharmacology, toxicology, and drug interaction potential of kratom are reviewed. Kratom-associated polyintoxications and in vitro-in vivo extrapolations suggest that kratom can precipitate pharmacokinetic drug interactions by inhibiting CYP2D6, CYP3A, and P-glycoprotein. An iterative approach that includes clinical studies and physiologically based pharmacokinetic modeling and simulation is recommended for further evaluation of potential unwanted kratom-drug interactions.

Obesity-Associated Dyslipidemia Is Moderated by Habitual Intake of Marine-Derived n-3 Polyunsaturated Fatty Acids in Yup'ik Alaska Native People: A Cross-Sectional Mediation-Moderation Analysis.

BACKGROUND: Obesity leads to insulin resistance, altered lipoprotein metabolism, dyslipidemia, and cardiovascular disease. The relationship between long-term intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) and prevention of cardiometabolic disease remains unresolved. OBJECTIVES: The aim of this study was to explore direct and indirect pathways between adiposity and dyslipidemia, and the degree to which n-3 PUFAs moderate adiposity-induced dyslipidemia in a population with highly variable n-3 PUFA intake from marine foods. METHODS: In total, 571 Yup'ik Alaska Native adults (18-87 y) were enrolled in this cross-sectional study. The red blood cell (RBC) nitrogen isotope ratio (15N/14N, or NIR) was used as a validated objective measure of n-3 PUFA intake. EPA and DHA were measured in RBCs. Insulin sensitivity and resistance were estimated by the HOMA2 method. Mediation analysis was conducted to evaluate the contribution of the indirect causal path between adiposity and dyslipidemia mediated through insulin resistance. Moderation analysis was used to assess the influence of dietary n-3 PUFAs on the direct and indirect paths between adiposity and dyslipidemia. Outcomes of primary interest included plasma total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), non-HDL-C, and triglycerides (TG). RESULTS: In this Yup'ik study population, we found that up to 21.6% of the total effects of adiposity on plasma TG, HDL-C, and non-HDL-C are mediated through measures of insulin resistance or sensitivity. Moreover, RBC DHA and EPA moderated the positive association between waist circumference (WC) and TC or non-HDL-C, whereas only DHA moderated the positive association between WC and TG. However, the indirect path between WC and plasma lipids was not significantly moderated by dietary n-3 PUFAs. CONCLUSIONS: Intake of n-3 PUFAs may independently reduce dyslipidemia through the direct path resulting from excess adiposity in Yup'ik adults. NIR moderation effects suggest that additional nutrients contained in n-3 PUFA-rich foods may also reduce dyslipidemia.

Habitual Intake of Marine-Derived n-3 PUFAs is Inversely Associated with a Cardiometabolic Inflammatory Profile in Yup'ik Alaska Native People.

BACKGROUND: The relationship between dietary n-3 PUFAs and the prevention of cardiometabolic diseases, including type 2 diabetes, is unresolved. Examination of the association between n-3 PUFAs and chronic low-grade inflammation in a population where many individuals have had an extremely high intake of marine mammals and fish throughout their lifespan may provide important clues regarding the impact of n-3 PUFAs on health. OBJECTIVES: The aim of this study was to explore associations between concentrations of n-3 PUFAs resulting from habitual intake of natural food sources high in fish and marine mammals with immune biomarkers of metabolic inflammation and parameters of glucose regulation. METHODS: A total of 569 Yup'ik Alaska Native adults (18-87 years old) were enrolled in this cross-sectional study between December 2016 and November 2019. The RBC nitrogen isotope ratio (NIR; 15N/14N) was used as a validated measure of n-3 PUFA intake to select 165 participant samples from the first and fourth quartiles of n-3 PUFA intakes. Outcomes included 38 pro- and anti-inflammatory cytokines and 8 measures of glucose homeostasis associated with type 2 diabetes risks. These outcomes were evaluated for their associations with direct measurements of EPA, DHA, and arachidonic acid in RBCs. ANALYSIS: Linear regression was used to detect significant relationships with cytokines and n-3 PUFAs, adiposity, and glucose-related variables. RESULTS: The DHA concentration in RBC membranes was inversely associated with IL-6 (ß = -0.0066; P < 0.001); EPA was inversely associated with TNFα (ß = -0.4925; P < 0.001); and the NIR was inversely associated with Monocyte chemoattractant protein-1 (MCP-1) (ß = -0.8345; P < 0.001) and IL-10 (ß = -1.2868; P < 0.001). CONCLUSIONS: Habitual intake of marine mammals and fish rich in n-3 PUFAs in this study population of Yup'ik Alaska Native adults is associated with reduced systemic inflammation, which may contribute to the low prevalence of diseases in which inflammation plays an important role.

Differences in 25-Hydroxyvitamin D Clearance by eGFR and Race: A Pharmacokinetic Study.

BACKGROUND: Conversion of 25-hydroxyvitamin D (25[OH]D) to the active form of vitamin D occurs primarily in the kidney. Observational studies suggest 25(OH)D clearance from the circulation differs by kidney function and race. However, these potential variations have not been tested using gold-standard methods. METHODS: We administered intravenous, deuterated 25(OH)D3 (d-25[OH]D3) in a pharmacokinetic study of 87 adults, including 43 with normal eGFR (≥60 ml/min per 1.73 m2), 24 with nondialysis CKD (eGFR <60 ml/min per 1.73 m2), and 20 with ESKD treated with hemodialysis. We measured concentrations of d-25(OH)D3 and deuterated 24,25-dihydroxyvitamin D3 at 5 minutes and 4 hours after administration, and at 1, 4, 7, 14, 21, 28, 42, and 56 days postadministration. We calculated 25(OH)D clearance using noncompartmental analysis of d-25(OH)D3 concentrations over time. We remeasured 25(OH)D clearance in a subset of 18 participants after extended oral vitamin-D3 supplementation. RESULTS: The mean age of the study cohort was 64 years; 41% were female, and 30% were Black. Mean 25(OH)D clearances were 360 ml/d, 313 ml/d, and 263 ml/d in participants with normal eGFR, CKD, and kidney failure, respectively (P=0.02). After adjustment for age, sex, race, and estimated blood volume, lower eGFR was associated with reduced 25(OH)D clearance (ß=-17 ml/d per 10 ml/min per 1.73 m2 lower eGFR; 95% CI, -21 to -12). Black race was associated with higher 25(OH)D clearance in participants with normal eGFR, but not in those with CKD or kidney failure (P for interaction=0.05). Clearance of 25(OH)D before versus after vitamin-D3 supplementation did not differ. CONCLUSIONS: Using direct pharmacokinetic measurements, we show that 25(OH)D clearance is reduced in CKD and may differ by race. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Clearance of 25-hydroxyvitamin D in Chronic Kidney Disease (CLEAR), NCT02937350; Clearance of 25-hydroxyvitamin D3 During Vitamin D3 Supplementation (CLEAR-PLUS), NCT03576716.

Refined Prediction of Pharmacokinetic Kratom-Drug Interactions: Time-Dependent Inhibition Considerations.

Preparations from the leaves of the kratom plant (Mitragyna speciosa) are consumed for their opioid-like effects. Several deaths have been associated with kratom used concomitantly with some drugs. Pharmacokinetic interactions are potential underlying mechanisms of these fatalities. Accumulating in vitro evidence has demonstrated select kratom alkaloids, including the abundant indole alkaloid mitragynine, as reversible inhibitors of several cytochromes P450 (CYPs). The objective of this work was to refine the mechanistic understanding of potential kratom-drug interactions by considering both reversible and time-dependent inhibition (TDI) of CYPs in the liver and intestine. Mitragynine was tested against CYP2C9 (diclofenac 4'-hydroxylation), CYP2D6 (dextromethorphan O-demethylation), and CYP3A (midazolam 1'-hydroxylation) activities in human liver microsomes (HLMs) and CYP3A activity in human intestinal microsomes (HIMs). Comparing the absence to presence of NADPH during preincubation of mitragynine with HLMs or HIMs, an ∼7-fold leftward shift in IC50 (∼20 to 3 µM) toward CYP3A resulted, prompting determination of TDI parameters (HLMs: K I , 4.1 ± 0.9 µM; k inact , 0.068 ± 0.01 min-1; HIMs: K I , 4.2 ± 2.5 µM; k inact , 0.079 ± 0.02 min-1). Mitragynine caused no leftward shift in IC50 toward CYP2C9 (∼40 µM) and CYP2D6 (∼1 µM) but was a strong competitive inhibitor of CYP2D6 (K i , 1.17 ± 0.07 µM). Using a recommended mechanistic static model, mitragynine (2-g kratom dose) was predicted to increase dextromethorphan and midazolam area under the plasma concentration-time curve by 1.06- and 5.69-fold, respectively. The predicted midazolam area under the plasma concentration-time curve ratio exceeded the recommended cutoff (1.25), which would have been missed if TDI was not considered. SIGNIFICANCE STATEMENT: Kratom, a botanical natural product increasingly consumed for its opioid-like effects, may precipitate potentially serious pharmacokinetic interactions with drugs. The abundant kratom indole alkaloid mitragynine was shown to be a time-dependent inhibitor of hepatic and intestinal cytochrome P450 3A activity. A mechanistic static model predicted mitragynine to increase systemic exposure to the probe drug substrate midazolam by 5.7-fold, necessitating further evaluation via dynamic models and clinical assessment to advance the understanding of consumer safety associated with kratom use.

In Vivo Functional Effects of CYP2C9 M1L, a Novel and Common Variant in the Yup'ik Alaska Native Population.

Alaska Native people are under-represented in genetic research but have unique gene variation that may critically impact their response to pharmacotherapy. Full resequencing of CYP2C9 in a cross-section of this population identified CYP2C9 Met1Leu (M1L), a novel, relatively common single nucleotide polymorphism hypothesized to confer CYP2C9 poor metabolizer phenotype by disrupting the start codon. M1L is present at a minor allele frequency of 6.3% in Yup'ik Alaska Native people and thus can contribute to the risk of an adverse drug response from narrow-therapeutic-index CYP2C9 substrates such as (S)-warfarin. This study's objective was to characterize the catalytic efficiency of the Leu1 variant enzyme in vivo by evaluating the pharmacokinetic behavior of naproxen, a probe substrate for CYP2C9 activity, in genotyped Yup'ik participants. We first confirmed the selectivity of (S)-naproxen O-demethylation by CYP2C9 using activity-phenotyped human liver microsomes and selective cytochrome P450 inhibitors and then developed and validated a novel liquid chromatography mass spectrometry method for simultaneous quantification of (S)-naproxen, (S)-O-desmethylnaproxen, and naproxen acyl glucuronide in human urine. The average ratio of (S)-O-desmethylnaproxen to unchanged (S)-naproxen in urine was 18.0 ± 8.0 (n = 11) for the homozygous CYP2C9 Met1 reference group and 10.3 ± 6.6 (n = 11) for the Leu1 variant carrier group (P = 0.011). The effect of M1L variation on CYP2C9 function and its potential to alter the pharmacokinetics of drugs metabolized by the enzyme has clinical implications and should be included in a variant screening panel when pharmacogenetic testing in the Alaska Native population is warranted. SIGNIFICANCE STATEMENT: The novel CYP2C9 Met1Leu variant in Alaska Native people was recently identified. This study validated (S)-naproxen as a CYP2C9 probe substrate to characterize the in vivo functional activity of the CYP2C9 Leu1 variant. The results of this pharmacogenetic-pharmacokinetic study suggest that the CYP2C9 Leu1 variant exhibits loss of enzyme activity. This finding may be important to consider when administering narrow-therapeutic-index medications metabolized by CYP2C9 and also compels further investigation to characterize novel genetic variation in understudied populations.

Vitamin D Metabolism Is Dysregulated in Asthma and Chronic Obstructive Pulmonary Disease.

Rationale: Vitamin D deficiency is common in patients with asthma and chronic obstructive pulmonary disease (COPD). Low 25-hydroxyvitamin D (25[OH]D) levels may represent a cause or a consequence of these conditions.Objectives: To determine whether vitamin D metabolism is altered in asthma or COPD.Methods: We conducted a longitudinal study in 186 adults to determine whether the 25(OH)D response to six oral doses of 3 mg vitamin D3, administered over 1 year, differed between those with asthma or COPD versus control subjects. Serum concentrations of vitamin D3, 25(OH)D3, and 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3) were determined presupplementation and postsupplementation in 93 adults with asthma, COPD, or neither condition, and metabolite-to-parent compound molar ratios were compared between groups to estimate hydroxylase activity. Additionally, we analyzed 14 datasets to compare expression of 1α,25(OH)2D3-inducible gene expression signatures in clinical samples taken from adults with asthma or COPD versus control subjects.Measurements and Main Results: The mean postsupplementation 25(OH)D increase in participants with asthma (20.9 nmol/L) and COPD (21.5 nmol/L) was lower than in control subjects (39.8 nmol/L; P = 0.001). Compared with control subjects, patients with asthma and COPD had lower molar ratios of 25(OH)D3-to-vitamin D3 and higher molar ratios of 1α,25(OH)2D3-to-25(OH)D3 both presupplementation and postsupplementation (P ≤ 0.005). Intergroup differences in 1α,25(OH)2D3-inducible gene expression signatures were modest and variable if statistically significant.Conclusions: Attenuation of the 25(OH)D response to vitamin D supplementation in asthma and COPD associated with reduced molar ratios of 25(OH)D3-to-vitamin D3 and increased molar ratios of 1α,25(OH)2D3-to-25(OH)D3 in serum, suggesting that vitamin D metabolism is dysregulated in these conditions.

Heterologous Expression and Functional Characterization of Novel CYP2C9 Variants Identified in the Alaska Native People.

CYP2C9 is a major form of human liver cytochrome P450 that is responsible for the oxidative metabolism of several widely used low-therapeutic index drugs, including (S)-warfarin and phenytoin. In a cohort of Alaska Native people, ultrarare or novel CYP2C9 protein variants, M1L (rs114071557), N218I (rs780801862), and P279T (rs182132442, CYP2C9*29), are expressed with higher frequencies than the well characterized CYP2C9*2 and CYP2C9*3 alleles. We report here on their relative expression in lentivirus-infected HepG2 cells and the functional characterization of purified reconstituted enzyme variants expressed in Escherichia coli toward (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen. In the infected HepG2 cells, robust mRNA and protein expression were obtained for wild-type, N218I, and P279T variants, but as expected, the M1L variant protein was not translated in this liver-derived cell line. His-tagged wild-type protein and the N218I and P279T variants, but not M1L, expressed well in E. coli and were highly purified after affinity chromatography. Upon reconstitution with cytochrome P450 oxidoreductase and cytochrome b5, the N218I and P279T protein variants metabolized (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen to the expected monohydroxylated or O-demethylated metabolites. Steady-state kinetic analyses revealed that the relative catalytic efficiency ratios of (S)-warfarin metabolism by the P279T and N218I variants were 87% and 24%, respectively, of wild-type CYP2C9 protein. A similar rank ordering was observed for metabolism of phenytoin, flurbiprofen, and (S)-naproxen. We conclude that carriers of the variant N218I and, especially, the M1L alleles would be at risk of exacerbated therapeutic effects from drugs that rely on CYP2C9 for their metabolic clearance. SIGNIFICANCE STATEMENT: Novel gene variants of CYP2C9-M1L, and N218I, along with P279T (CYP2C9*29)-are expressed in Alaska Native people at relatively high frequencies. In vitro characterization of their functional effects revealed that each variant confers reduced catalytic efficiency toward several substrates, including the low-therapeutic index drugs (S)-warfarin and phenytoin. These data provide the first functional information for new, common CYP2C9 variants in this understudied population. The data may help guide dose adjustments in allele carriers, thus mitigating potential healthcare disparities.

Modulation of Major Human Liver Microsomal Cytochromes P450 by Component Alkaloids of Goldenseal: Time-Dependent Inhibition and Allosteric Effects.

Botanical and other natural products (NPs) are often coconsumed with prescription medications, presenting a risk for cytochrome P450 (P450)-mediated NP-drug interactions. The NP goldenseal (Hydrastis canadensis) has exhibited antimicrobial activities in vitro attributed to isoquinoline alkaloids contained in the plant, primarily berberine, (-)-ß-hydrastine, and to a lesser extent, hydrastinine. These alkaloids contain methylenedioxyphenyl rings, structural alerts with potential to inactivate P450s through formation of metabolic intermediate complexes. Time-dependent inhibition experiments were conducted to evaluate their ability to inhibit major P450 activities in human liver microsomes by using a cocktail of isozyme-specific substrate probes. Berberine inhibited CYP2D6 (dextromethorphan O-demethylation; K I = 2.7 µM, kinact = 0.065 minute-1) and CYP3A4/5 (midazolam 1'-hydroxylation; K I = 14.8 µM, kinact = 0.019 minute-1); (-)-ß-hydrastine inhibited CYP2C9 (diclofenac 4'-hydroxylation; K I = 49 µM, kinact = 0.036 minute-1), CYP2D6 (K I > 250 µM, kinact > 0.06 minute-1), and CYP3A4/5 (K I = 28 µM, kinact = 0.056 minute-1); and hydrastinine inhibited CYP2D6 (K I = 37 µM, kinact = 0.049 minute-1) activity. Berberine additionally exhibited allosteric effects on midazolam hydroxylation, showing both positive and negative heterotropic cooperativity. Experiments with recombinant isozymes showed that berberine activated midazolam 1'-hydroxylation by CYP3A5, lowering K m(app), but showed mixed inhibition and negative cooperativity toward this reaction when catalyzed by CYP3A4. Berberine inactivated CYP3A4 at a much faster rate than CYP3A5 and was a noncompetitive inhibitor of midazolam 4-hydroxylation by CYP3A4 but a strong mixed inhibitor of the CYP3A5 catalyzed reaction. These complex kinetics should be considered when extrapolating the risk for NP-drug interactions involving goldenseal. SIGNIFICANCE STATEMENT: Robust kinetic parameters were determined for the reversible and time-dependent inhibition of CYP2C9, CYP2D6, and CYP3A4/5 activities in human liver microsomes by major component isoquinoline alkaloids contained in the botanical natural product goldenseal. The alkaloid berberine also exhibited opposing, isozyme-specific allosteric effects on midazolam hydroxylation mediated by recombinant CYP3A4 (inhibition) and CYP3A5 (activation). These data will inform the development of a physiologically based pharmacokinetic model that can be used to predict potential clinically relevant goldenseal-drug interactions.

Regional Proteomic Quantification of Clinically Relevant Non-Cytochrome P450 Enzymes along the Human Small Intestine.

Current challenges in accurately predicting intestinal metabolism arise from the complex nature of the intestine, leading to limited applicability of available in vitro tools as well as knowledge deficits in intestinal physiology, including enzyme abundance. In particular, information on regional enzyme abundance along the small intestine is lacking, especially for non-cytochrome P450 enzymes such as carboxylesterases (CESs), UDP-glucuronosyltransferases (UGTs), and sulfotransferases (SULTs). We used cryopreserved human intestinal mucosa samples from nine donors as an in vitro surrogate model for the small intestine and performed liquid chromatography tandem mass spectrometry-based quantitative proteomics for 17 non-cytochrome P450 enzymes using stable isotope-labeled peptides. Relative protein quantification was done by normalization with enterocyte marker proteins, i.e., villin-1, sucrase isomaltase, and fatty acid binding protein 2, and absolute protein quantification is reported as picomoles per milligram of protein. Activity assays in glucuronidations and sequential metabolisms were conducted to validate the proteomics findings. Relative or absolute quantifications are reported for CES1, CES2, five UGTs, and four SULTs along the small intestine: duodenum, jejunum, and ileum for six donors and in 10 segments along the entire small intestine (A-J) for three donors. Relative quantification using marker proteins may be beneficial in further controlling for technical variabilities. Absolute quantification data will allow for scaling factor generation and in vivo extrapolation of intestinal clearance using physiologically based pharmacokinetic modeling. SIGNIFICANCE STATEMENT: Current knowledge gaps exist in intestinal protein abundance of non-cytochrome P450 enzymes. Here, we employ quantitative proteomics to measure non-cytochrome P450 enzymes along the human small intestine in nine donors using cryopreserved human intestinal mucosa samples. Absolute and relative abundances reported here will allow better scaling of intestinal clearance.

Effects of Pregnancy on the Pharmacokinetics of Metformin.

This study's primary objective was to fully characterize the pharmacokinetics of metformin in pregnant women with gestational diabetes mellitus (GDM) versus nonpregnant controls. Steady-state oral metformin pharmacokinetics in pregnant women with GDM receiving either metformin monotherapy (n = 24) or a combination with glyburide (n = 30) as well as in nonpregnant women with type 2 diabetes mellitus (T2DM) (n = 24) were determined utilizing noncompartmental techniques. Maternal and umbilical cord blood samples were collected at delivery from 38 women. With both 500- and 1000-mg doses, metformin bioavailability, volume of distribution beta (V ß ), clearance, and renal clearance were significantly increased during pregnancy. In addition, in the women receiving metformin 500 mg, significantly higher metformin apparent oral clearance (CL/F) (27%), weight-adjusted renal secretion clearance (64%), and apparent oral volume of distribution beta (V ß /F) (33%) were seen during pregnancy. Creatinine clearance was significantly higher during pregnancy. Increasing metformin dose from 500 to 1000 mg orally twice daily significantly increased V ß /F by 28%, weight-adjusted V ß /F by 32% and CL/F by 25%, and weight-adjusted CL/F by 28% during pregnancy. Mean metformin umbilical cord arterial-to-venous plasma concentration ratio was 1.0 ± 0.1, venous umbilical cord-to-maternal concentration ratio was 1.4 ± 0.5, and arterial umbilical cord-to-maternal concentration ratio was 1.5 ± 0.5. Systemic exposure after a 500-mg dose of metformin was lower during pregnancy compared with the nonpregnant women with T2DM. However, in patients receiving metformin 1000 mg, changes in estimated bioavailability during pregnancy offset the changes in clearance leading to no significant change in CL/F with the higher dose. SIGNIFICANCE STATEMENT: Gestational diabetes mellitus complicates 5%-13% of pregnancies and is often treated with metformin. Pregnant women undergo physiological changes that alter drug disposition. Preliminary data suggest that pregnancy lowers metformin concentrations, potentially affecting efficacy and safety. This study definitively describes pregnancy's effects on metformin pharmacokinetics and expands the mechanistic understanding of pharmacokinetic changes across the dosage range. Here we report the nonlinearity of metformin pharmacokinetics and the increase in bioavailability, clearance, renal clearance, and volume of distribution during pregnancy.

Pharmacogenomics of Nicotine Metabolism: Novel CYP2A6 and CYP2B6 Genetic Variation Patterns in Alaska Native and American Indian Populations.

INTRODUCTION: Alaska Native and American Indian (AN/AI) populations have higher tobacco use prevalence than other ethnic/racial groups. Pharmacogenetic testing to tailor tobacco cessation treatment may improve cessation rates. This study characterized polymorphic variations among AN/AI people in genes associated with metabolism of nicotine and drugs used for tobacco cessation. METHODS: Recruitment of AN/AI individuals represented six subgroups, five geographic subgroups throughout Alaska and a subgroup comprised of AIs from the lower 48 states living in Alaska. We sequenced the CYP2A6 and CYP2B6 genes to identify known and novel gain, reduced, and loss-of-function alleles, including structural variation (eg, gene deletions, duplications, and hybridizations). RESULTS: Variant allele frequencies differed substantially between AN/AI subgroups. The gene deletion CYP2A6*4 and reduced function CYP2A6*9 alleles were found at high frequency in Northern/Western subgroups and in Lower 48/Interior subgroups, respectively. The reduced function CYP2B6*6 allele was observed in all subgroups and a novel, predicted reduced function CYP2B6 variant was found at relatively high frequency in the Southeastern subgroup. CONCLUSIONS: Diverse CYP2A6 and CYP2B6 variation among the subgroups highlight the need for comprehensive pharmacogenetic testing to guide tobacco cessation therapy for AN/AI populations. IMPLICATIONS: Nicotine metabolism is largely determined by CYP2A6 genotype, and variation in CYP2A6 activity has altered the treatment success in other populations. These findings suggest pharmacogenetic-guided smoking cessation drug treatment could provide benefit to this unique population seeking tobacco cessation therapy.

Renal secretion of hydrochlorothiazide involves organic anion transporter 1/3, organic cation transporter 2, and multidrug and toxin extrusion protein 2-K.

Hydrochlorothiazide (HCTZ) is the most widely used thiazide diuretic for the treatment of hypertension either alone or in combination with other antihypertensives. HCTZ is mainly cleared by the kidney via tubular secretion, but the underlying molecular mechanisms are unclear. Using cells stably expressing major renal organic anion and cation transporters [human organic anion transporter 1 (hOAT1), human organic anion transporter 3 (hOAT3), human organic cation transporter 2 (hOCT2), human multidrug and toxin extrusion 1 (hMATE1), and human multidrug and toxin extrusion 2-K (hMATE2-K)], we found that HCTZ interacted with both organic cation and anion transporters. Uptake experiments further showed that HCTZ is transported by hOAT1, hOAT3, hOCT2, and hMATE2-K but not by hMATE1. Detailed kinetic analysis coupled with quantification of membrane transporter proteins by targeted proteomics revealed that HCTZ is an excellent substrate for hOAT1 and hOAT3. The apparent affinities (Km) for hOAT1 and hOAT3 were 112 ± 8 and 134 ± 13 µM, respectively, and the calculated turnover numbers (kcat) were 2.48 and 0.79 s-1, respectively. On the other hand, hOCT2 and hMATE2-K showed much lower affinity for HCTZ. The calculated transport efficiency (kcat/Km) at the single transporter level followed the rank order of hOAT1> hOAT3 > hOCT2 and hMATE2-K, suggesting a major role of organic anion transporters in tubular secretion of HCTZ. In vitro inhibition experiments further suggested that HCTZ is not a clinically relevant inhibitor for hOAT1 or hOAT3. However, strong in vivo inhibitors of hOAT1/3 may alter renal secretion of HCTZ. Together, our study elucidated the molecular mechanisms underlying renal handling of HCTZ and revealed potential pathways involved in the disposition and drug-drug interactions for this important antihypertensive drug in the kidney.

Stargazer: a software tool for calling star alleles from next-generation sequencing data using CYP2D6 as a model.

PURPOSE: Genotyping CYP2D6 is important for precision drug therapy because the enzyme it encodes metabolizes approximately 25% of drugs, and its activity varies considerably among individuals. Genotype analysis of CYP2D6 is challenging due to its highly polymorphic nature. Over 100 haplotypes (star alleles) have been defined for CYP2D6, some involving a gene conversion with its nearby nonfunctional but highly homologous paralog CYP2D7. We present Stargazer, a new bioinformatics tool that uses next-generation sequencing (NGS) data to call star alleles for CYP2D6 ( https://stargazer.gs.washington.edu/stargazerweb/ ). Stargazer is currently being extended for other pharmacogenes. METHODS: Stargazer identifies star alleles from NGS data by detecting single nucleotide variants, insertion-deletion variants, and structural variants. Stargazer detects structural variation, including gene deletions, duplications, and conversions, by calculating paralog-specific copy numbers from read depths. RESULTS: We applied Stargazer to the NGS data of 32 ethnically diverse HapMap trios that were genotyped by TaqMan assays, long-range polymerase chain reaction, quantitative multiplex polymerase chain reaction, high-resolution melting analysis, and/or Sanger sequencing. CYP2D6 genotyping by Stargazer was 99.0% concordant with the data obtained by these methods, and showed that 28.1% of the samples had structural variation including CYP2D6/CYP2D7 hybrids. CONCLUSION: Accurate genotyping of pharmacogenes with NGS and subsequent allele calling with Stargazer will aid the implementation of precision drug therapy.

Precision medicine and health disparities: The case of pediatric acute lymphoblastic leukemia.

BACKGROUND: Precision medicine has uncertain potential to address population health disparities. PURPOSE: Case study of disparities in pediatric acute lymphoblastic leukemia (ALL). METHOD: Literature-based evaluation of ALL in African American (AA) and European American (EA) children. FINDINGS: AA children have a lower incidence of ALL than EA children, experience higher relapse rates, and are more likely to be diagnosed with poor prognostic indicators. Environmental risk exposures for ALL have small effect sizes; data are insufficient to determine their contribution to differences in incidence and prognosis. Differences in prevalence of gene variants associated with treatment response contribute to higher relapse rates in AA children. However, higher relapse rates were not seen in a care setting that eliminated out of pocket costs, used risk-directed therapy, and included rigorous case management. DISCUSSION: Unequal access to effective treatment contributes to ALL disparities. Precision medicine can help to define effective treatment for diverse patient populations.