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The US Food and Drug Administration draft drug interaction guidance recommends that 400 mg ketoconazole (KTZ) be administered once daily for several days (QD400) for maximal CYP3A inhibition. Some investigators suggest that a single dose of 400 mg (SD400) KTZ is sufficient given its short half-life (t(1/2) approximately 3-5 hr). To determine the impact of KTZ regimens on CYP3A inhibition, we simulated AUC fold-change (AUCR) in the presence of SD400, QD400, or 200 mg twice-daily (BID200) KTZ for theoretical CYP3A substrates. Ratios of AUCR (AUCR(QD400)/AUCR(SD400) and AUCR(BID200) AUCR(QD400)) increase with increasing bioavailability and increasing substrate t(1/2). The SD400 KTZ regimen may provide maximal inhibition only for a subset of substrates (ie, low bioavailability and short t(1/2)). For substrates with t(1/2) longer than that of KTZ, multiple KTZ dosing is critical and BID200 appears to provide greater inhibition than QD400. Also, timing of KTZ administration should be optimized to allow maximal presystemic enzyme inhibition prior to substrate administration. [ABSTRACT FROM AUTHOR]

1 The effect of concurrent cimetidine administration on the disposition of theophylline was investigated in eight male patients (56-78 years) with chronic obstructive pulmonary disease (COPD). 2 The patients, who were taking oral theophylline preparations chronically (384-1020 mg/day), received a [15N], [13C]-labelled analogue of theophylline (10 mg i.v.) before and during cimetidine treatment (1200 mg/day p.o.). 3 During cimetidine treatment trough levels of theophylline increased 34% (6.4 +/- 0.8 to 8.6 +/- 1.0 micrograms/ml, P less than 0.05), half-life increased 48% (6.5 +/- 0.6 to 9.6 +/- 0.8 h, P less than 0.001), and total plasma clearance decreased 33% (3.88 +/- 0.46 to 2.59 +/- 0.33 l/h, P less than 0.001), without a significant change in volume of distribution or protein binding. 4 The effect of cimetidine on plasma levels of theophylline was maximal within 72 h. Levels returned to control values within 48 h after its discontinuation. 5 Although there was no correlation with mean plasma concentrations of cimetidine, the change in clearance of theophylline correlated with initial clearance values (r = 0.72). 6 Cimetidine reduced the plasma clearance of theophylline in patients with COPD to an extent similar to that reported in healthy volunteers. [ABSTRACT FROM AUTHOR]

Background: The management of warfarin therapy is complicated by a wide variation among patients in drug response. Variants in the gene encoding vitamin K epoxide reductase complex 1 (VKORC1) may affect the response to warfarin.Methods: We conducted a retrospective study of European-American patients receiving long-term warfarin maintenance therapy. Multiple linear-regression analysis was used to determine the effect of VKORC1 haplotypes on the warfarin dose. We determined VKORC1 haplotype frequencies in African-American, European-American, and Asian-American populations and VKORC1 messenger RNA (mRNA) expression in human liver samples.Results: We identified 10 common noncoding VKORC1 single-nucleotide polymorphisms and inferred five major haplotypes. We identified a low-dose haplotype group (A) and a high-dose haplotype group (B). The mean (+/-SE) maintenance dose of warfarin differed significantly among the three haplotype group combinations, at 2.7+/-0.2 mg per day for A/A, 4.9+/-0.2 mg per day for A/B, and 6.2+/-0.3 mg per day for B/B (P<0.001). VKORC1 haplotype groups A and B explained approximately 25 percent of the variance in dose. Asian Americans had a higher proportion of group A haplotypes and African Americans a higher proportion of group B haplotypes. VKORC1 mRNA levels varied according to the haplotype combination.Conclusions: VKORC1 haplotypes can be used to stratify patients into low-, intermediate-, and high-dose warfarin groups and may explain differences in dose requirements among patients of different ancestries. The molecular mechanism of this warfarin dose response appears to be regulated at the transcriptional level. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2024 American Society for Pharmacology and Experimental Therapeutics.)

The microgravity environment aboard the International Space Station (ISS) provides a unique stressor that can help understand underlying cellular and molecular drivers of pathological changes observed in astronauts with the ultimate goals of developing strategies to enable long- term spaceflight and better treatment of diseases on Earth. We used this unique environment to evaluate the effects of microgravity on kidney proximal tubule epithelial cell (PTEC) response to serum exposure and vitamin D biotransformation capacity. To test if microgravity alters the pathologic response of the proximal tubule to serum exposure, we treated PTECs cultured in a microphysiological system (PT-MPS) with human serum and measured biomarkers of toxicity and inflammation (KIM-1 and IL-6) and conducted global transcriptomics via RNAseq on cells undergoing flight (microgravity) and respective controls (ground). Given the profound bone loss observed in microgravity and PTECs produce the active form of vitamin D, we treated 3D cultured PTECs with 25(OH)D 3 (vitamin D) and monitored vitamin D metabolite formation, conducted global transcriptomics via RNAseq, and evaluated transcript expression of CYP27B1, CYP24A1, or CYP3A5 in PTECs undergoing flight (microgravity) and respective ground controls. We demonstrated that microgravity neither altered PTEC metabolism of vitamin D nor did it induce a unique response of PTECs to human serum, suggesting that these fundamental biochemical pathways in the kidney proximal tubule are not significantly altered by short-term exposure to microgravity. Given the prospect of extended spaceflight, more study is needed to determine if these responses are consistent with extended (>6 months) exposure to microgravity., (© 2024. The Author(s).)

This study evaluated the underlying mechanistic links between genetic variability in vitamin K metabolic pathway genes ( CYP4F2 and CYP4F11 ) and phylloquinone hydroxylation activity using genotype- and haplotype-based approaches. Specifically, we characterized genetic variability in the CYP4F2/CYP4F11 locus and compared common single allele genotypes and common haplotypes as predictors of hepatic gene expression, enzyme abundance, and phylloquinone (VK 1 ) ω-hydroxylation kinetics. We measured CYP4F2 and CYP4F11 mRNA levels, CYP4F2 and CYP4F11 protein abundances, and the VK 1 concentration-dependent ω-hydroxylation rate in matched human liver nucleic acid and microsome samples, utilizing a novel in vitro population modeling approach. Results indicate that accounting for the CYP4F2*3 allele alone is sufficient to capture most of the genetic-derived variability in the observed phenotypes. Additionally, our findings highlight the important contribution that CYP4F11 makes toward vitamin K metabolism in the human liver., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 (-)- β -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, (-)- β -hydrastine was a more potent time-dependent inhibitor of midazolam 1'-hydroxylation than berberine (K I and k inact : 8.48 μ M and 0.041 minutes -1 , respectively, vs. >250 μ M and ∼0.06 minutes -1 , respectively). Both the AUC and C max 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 (-)- β -hydrastine successfully predicted the goldenseal-midazolam interaction to within 20% of that observed after both chronic and acute goldenseal administration. Simulations implicated (-)- β -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., (Copyright © 2023 by The Author(s).)

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., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Green tea is a popular beverage worldwide. The abundant green tea catechin (-)-epigallocatechin gallate (EGCG) is a potent in vitro inhibitor of intestinal UDP-glucuronosyltransferase (UGT) activity (K i  ~2 μM). Co-consuming green tea with intestinal UGT drug substrates, including raloxifene, could increase systemic drug exposure. The effects of a well-characterized green tea on the pharmacokinetics of raloxifene, raloxifene 4'-glucuronide, and raloxifene 6-glucuronide were evaluated in 16 healthy adults via a three-arm crossover, fixed-sequence study. Raloxifene (60 mg) was administered orally with water (baseline), with green tea for 1 day (acute), and on the fifth day after daily green tea administration for 4 days (chronic). Unexpectedly, green tea decreased the geometric mean green tea/baseline raloxifene AUC 0-96h ratio to ~0.60 after both acute and chronic administration, which is below the predefined no-effect range (0.75-1.33). Lack of change in terminal half-life and glucuronide-to-raloxifene ratios indicated the predominant mechanism was not inhibition of intestinal UGT. One potential mechanism includes inhibition of intestinal transport. Using established transfected cell systems, a green tea extract normalized to EGCG inhibited 10 of 16 transporters tested (IC 50 , 0.37-12 μM). Another potential mechanism, interruption by green tea of gut microbe-mediated raloxifene reabsorption, prompted a follow-up exploratory clinical study to evaluate the potential for a green tea-gut microbiota-drug interaction. No clear mechanisms were identified. Overall, results highlight that improvements in current models and methods used to predict UGT-mediated drug interactions are needed. Informing patients about the risk of co-consuming green tea with raloxifene may be considered., (© 2023 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Patients with cystic fibrosis (CF) commonly have lower circulating concentrations of 25-hydroxyvitamin D (25(OH)D) than healthy populations. We comprehensively compared measures of vitamin D metabolism among individuals with CF and healthy control subjects. In a cross-sectional study, serum from participants with CF (N = 83) and frequency-matched healthy control subjects by age and race (N = 82) were analyzed for: 25(OH)D 2 and 25(OH)D 3 , 1α,25-dihydroxyvitamins D 2 and D 3 (1α,25(OH) 2 D 2 and 1α,25(OH) 2 D 3 ), 24,25-dihydroxyvitamin D 3 (24,25(OH) 2 D 3 ), 4β,25-dihydroxyvitamin D 3 (4β,25(OH) 2 D 3 ), 25-hydroxyvitamin D 3 -3-sulfate (25(OH)D 3 -S), and 25-hydroxyvitamin D 3 -3-glucuronide (25(OH)D 3 -G). In a 56-day prospective pharmacokinetic study, ∼25 μg deuterium-labeled 25(OH)D 3 (d 6 -25(OH)D 3 ) was administered intravenously to participants (N = 5 with CF, N = 5 control subjects). Serum was analyzed for d 6 -25(OH)D 3 and d 6 -24,25(OH) 2 D 3 , and pharmacokinetic parameters were estimated. In the cross-sectional study, participants with CF had similar mean (SD) total 25(OH)D concentrations as control subjects (26.7 [12.3] vs. 27.7 [9.9] ng/mL) and had higher vitamin D supplement use (53% vs. 22%). However, participants with CF had lower total 1α,25(OH) 2 D (43.6 [12.7] vs. 50.7 [13.0] pg/mL), 4β,25(OH) 2 D 3 (52.1 [38.9] vs. 79.9 [60.2] pg/mL), and 25(OH)D 3 -S (17.7 [11.6] vs. 30.1 [12.3] ng/mL) (p < 0.001 for all). The pharmacokinetics of d 6 -25(OH)D 3 and d 6 -24,25(OH)D 3 did not differ between groups. In summary, although 25(OH)D concentrations were comparable, participants with CF had lower 1α,25(OH) 2 D, 4β,25(OH) 2 D 3 , and 25(OH)D 3 -S concentrations than healthy controls. Neither 25(OH)D 3 clearance, nor formation of 24,25(OH) 2 D 3 , appears to account for these differences and alternative mechanisms for low 25(OH)D in CF (i.e., decreased formation, altered enterohepatic recirculation) should be explored., Competing Interests: Declarations of Competing Interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Understanding cannabis-drug interactions is critical given regulatory changes that have increased access to and use of cannabis. Cannabidiol (CBD) and Δ-9-tetrahydrocannabinol (Δ9-THC), the most abundant phytocannabinoids, are in vitro reversible and time-dependent (CBD only) inhibitors of several cytochrome P450 (CYP) enzymes. Cannabis extracts were used to evaluate quantitatively potential pharmacokinetic cannabinoid-drug interactions in 18 healthy adults. Participant received, in a randomized cross-over manner (separated by ≥ 1 week), a brownie containing (i) no cannabis extract (ethanol/placebo), (ii) CBD-dominant cannabis extract (640 mg CBD + 20 mg Δ9-THC), or (iii) Δ9-THC-dominant cannabis extract (20 mg Δ9-THC and no CBD). After 30 minutes, participants consumed a cytochrome P450 (CYP) drug cocktail consisting of caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A). Plasma and urine samples were collected (0-24 hours). The CBD + Δ9-THC brownie inhibited CYP2C19 > CYP2C9 > CYP3A > CYP1A2 (but not CYP2D6) activity, as evidenced by an increase in the geometric mean ratio of probe drug area under the plasma concentration-time curve (AUC) relative to placebo (AUC GMR ) of omeprazole, losartan, midazolam, and caffeine by 207%, 77%, 56%, and 39%, respectively. In contrast, the Δ9-THC brownie did not inhibit any of the CYPs. The CBD + Δ9-THC brownie increased Δ9-THC AUC GMR by 161%, consistent with CBD inhibiting CYP2C9-mediated oral Δ9-THC clearance. Except for caffeine, these interactions were well-predicted by our physiologically-based pharmacokinetic model (within 26% of observed interactions). Results can be used to help guide dose adjustment of drugs co-consumed with cannabis products and the dose of CBD in cannabis products to reduce interaction risk with Δ9-THC., (© 2023 The Authors. Clinical Pharmacology & Therapeutics © 2023 American Society for Clinical Pharmacology and Therapeutics.)

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., (Copyright © 2023 by The Author(s).)

Oral formulations prepared from the leaves of the kratom (Mitragyna speciosa) plant are increasingly used for their opioid-like effects to self-manage opioid withdrawal and pain. Calls to US poison centers involving kratom exposures increased >50-fold from 2011-2017, one-third of which reported concomitant use of kratom with drugs of abuse. Many of these drugs are eliminated primarily via cytochrome P450 (CYP) 3A and CYP2D6, raising concerns for potential adverse pharmacokinetic kratom-drug interactions. The impact of a single low dose of kratom tea (2 g) on the pharmacokinetics of the CYP3A probe midazolam (2.5 mg) and CYP2D6 probe dextromethorphan (30 mg) were assessed in 12 healthy adult participants after oral administration. Kratom showed no effect on dextromethorphan area under the plasma concentration time-curve (AUC) and maximum concentration (C max ; geometric mean ratio (90% confidence interval) 0.99 (0.83-1.19) and 0.96 (0.78-1.19), respectively) but a modest increase in midazolam AUC and C max (1.39 (1.23-1.57) and 1.50 (1.32-1.70), respectively). Lack of change in midazolam half-life (1.07 (0.98-1.17)) suggested that kratom primarily inhibited intestinal CYP3A. This inference was further supported by a physiologically based pharmacokinetic drug interaction model using the abundant alkaloid mitragynine, a relatively potent CYP3A time-dependent inhibitor in vitro (K I , ~4 μM; k inact , ~0.07 min -1 ). This work is the first to clinically evaluate the pharmacokinetic drug interaction potential of kratom. Co-consuming kratom with certain drugs extensively metabolized by CYP3A may precipitate serious interactions. These data fill critical knowledge gaps about the safe use of this increasingly popular natural product, thereby addressing ongoing public health concerns., (© 2023 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Circulating 25-hydroxyvitamin D [25(OH)D] concentration is used to monitor vitamin D status. Plasma protein binding may influence the 25(OH)D dose-response to vitamin D treatment through a direct relationship between the plasma unbound ("free") fraction and clearance of 25(OH)D. We previously evaluated 25(OH)D 3 clearance in relation to kidney function using intravenous administration of deuterium labeled 25(OH)D 3 . In this follow up study, we determined the free fraction of 25(OH)D 3 in plasma (i.e., percent free 25(OH)D 3 ) and the serum concentration and haplotype of vitamin D binding protein in these participants. We hypothesized that the percent free 25(OH)D 3 would be positively associated with 25(OH)D 3 clearance and would mediate associations between clearance and vitamin D binding protein (GC) haplotypes. Participants were mean (SD) age 64 (10) years and included 42 individuals with normal kidney function (controls), 24 individuals with chronic kidney disease, and 19 individuals with kidney failure on hemodialysis. Free plasma 25(OH)D 2 and 25(OH)D 3 concentrations were quantified with a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Because there is no reference measurement procedure for free 25(OH)D, we compared the new method with a widely-used predictive equation and a commercial immunoassay. The percent free 25(OH)D 3 determined by predictive equation was weakly associated with 25(OH)D 3 clearance (R = 0.27; P = 0.01). However, this association was absent when percent free 25(OH)D 3 was determined using LC-MS/MS-measured free and total 25(OH)D 3 concentrations. Method comparison uncovered a negative bias in immunoassay-measured free 25(OH)D concentrations among participants with kidney failure, so immunoassay results were not used to evaluate the association between percent free 25(OH)D 3 and clearance. GC2 haplotype carriage was associated with 25(OH)D 3 clearance. Among individuals with 2 relative to no GC2 alleles, clearance was 87 (95% CI: 15-158) mL/d greater. However, in contrast with the literature, GC2 carriage was not significantly related to DBP concentration or the percent free 25(OH)D 3 (either predicted or measured). In conclusion, the free fraction of 25(OH)D 3 is not strongly associated with 25(OH)D 3 clearance but may explain small differences in clearance according to GC haplotype., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 ( 15 N/ 14 N, 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., (Copyright © 2022 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Background: Early childhood rickets increased in Alaska Native children after decreases in vitamin D-rich subsistence diet in childbearing-aged women. We evaluated the impact of routine prenatal vitamin D supplementation initiated in Alaska’s Yukon Kuskokwim Delta in Fall 2016. Methods: We queried electronic health records of prenatal women with 25(OH) vitamin D testing during the period 2015−2019. We evaluated 25(OH)D concentrations, vitamin D3 supplement refills, and decayed, missing, and filled teeth (dmft) scores and rickets in offspring. Results: Mean 25(OH)D concentrations increased 36.5% from pre- to post-supplementation; the percentage with deficient 25(OH)D decreased by 66.4%. Women with ≥ 60 vitamin D3 refill days had higher late pregnancy 25(OH)D concentrations than those with no refill days (p < 0.0001). Women with late pregnancy insufficient 25(OH)D concentrations had offspring with higher dmft scores than those with sufficient 25(OH)D (RR 1.3, p < 0.0001). Three children were diagnosed with nutritional rickets during the period 2001−2021, and none after 2017. Conclusions: These findings suggest that prenatal vitamin D supplementation can improve childhood outcomes in high-risk populations with high rates of rickets.

Increasing use of the botanical kratom to self-manage opioid withdrawal and pain has led to increased kratom-linked overdose deaths. Despite these serious safety concerns, rigorous fundamental pharmacokinetic knowledge of kratom in humans remains lacking. We assessed the pharmacokinetics of a single low dose (2 g) of a well-characterized kratom product administered orally to six healthy participants. Median concentration-time profiles for the kratom alkaloids examined were best described by a two-compartment model with central elimination. Pronounced pharmacokinetic differences between alkaloids with the 3 S configuration (mitragynine, speciogynine, paynantheine) and alkaloids with the 3 R configuration (mitraciliatine, speciociliatine, isopaynantheine) were attributed to differences in apparent intercompartmental distribution clearance, volumes of distribution, and clearance. Based on noncompartmental analysis of individual concentration-time profiles, the 3 S alkaloids exhibited a shorter median time to maximum concentration (1-2 vs. 2.5-4.5 h), lower area under the plasma concentration-time curve (430-490 vs. 794-5120 nM × h), longer terminal half-life (24-45 vs. ~12-18 h), and higher apparent volume of distribution during the terminal phase (960-12,700 vs. ~46-130 L) compared to the 3 R alkaloids. Follow-up mechanistic in vitro studies suggested differential hepatic/intestinal metabolism, plasma protein binding, blood-to-plasma partitioning, and/or distribution coefficients may explain the pharmacokinetic differences between the two alkaloid types. This first comprehensive pharmacokinetic characterization of kratom alkaloids in humans provides the foundation for further research to establish safety and effectiveness of this emerging botanical product.

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 (β = -0.0066; P < 0.001); EPA was inversely associated with TNFα (β = -0.4925; P < 0.001); and the NIR was inversely associated with Monocyte chemoattractant protein-1 (MCP-1) (β = -0.8345; P < 0.001) and IL-10 (β = -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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

The formation of 24,25-dihydroxyvitamin D (24,25(OH) 2 D) from 25-hydroxyvitamin D (25(OH)D) is the primary mechanism for the metabolic clearance of 25(OH)D, and is regulated by tissue-level vitamin D activity. The ratio of 24,25(OH) 2 D 3 to 25(OH)D 3 in blood (vitamin D metabolite ratio, VDMR) is postulated to be a marker of 25(OH)D 3 clearance, however this has never been tested. We measured baseline 24,25(OH) 2 D 3 and 25(OH)D 3 concentrations in 87 participants by liquid chromatography-tandem mass spectrometry. Following an infusion of deuterated 25(OH)D 3 , blood samples for each participant were collected over 56 days and analyzed for deuterated vitamin D metabolites. 25(OH)D 3 clearance and the deuterated metabolite-to-parent AUC ratio (ratio of the AUC of deuterated 24,25(OH) 2 D 3 to that of deuterated 25(OH)D 3 ) were calculated. We compared the VDMR with these two measures using correlation coefficients and linear regression. Participants had a mean age of 64 ± 11years, 41 % were female, 30 % were self-described Black, 28 % had non-dialysis chronic kidney disease (CKD) and 23 % had kidney failure treated with hemodialysis. The VDMR was strongly correlated with 25(OH)D 3 clearance and the deuterated metabolite-to-parent AUC ratio (r = 0.51 and 0.76, respectively). Adjusting for 25(OH)D 3 clearance or the deuterated metabolite-to-parent AUC ratio in addition to clinical covariates, lower VDMR was observed in participants with CKD and kidney failure than in healthy controls; in Black than White participants; and in those with lower serum albumin. Our findings validate the VDMR as a measure of 25(OH)D 3 clearance. This relationship was biased by characteristics including race and kidney disease, which warrant consideration in studies assessing the VDMR., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pharmacokinetic drug interactions precipitated by botanical and other natural products (NPs) remain critically understudied. Investigating these complex interactions is fraught with difficulties due to the methodologic and technical challenges associated with the inherently complex chemistries and product variability of NPs. This knowledge gap is perpetuated by a continuing absence of a harmonized framework regarding the design of clinical pharmacokinetic studies of NPs and NP-drug interactions. Accordingly, this Recommended Approach, the fourth in a series of Recommended Approaches released by the Center of Excellence for Natural Product Drug Interaction Research (NaPDI Center), provides recommendations for the design of clinical pharmacokinetic studies involving NPs. Building on prior Recommended Approaches and data generated from the NaPDI Center, such a framework is presented for the design of (1) phase 0 studies to assess the pharmacokinetics of an NP and (2) clinical pharmacokinetic NP-drug interaction studies. Suggestions for NP sourcing, dosing, study design, participant selection, sampling periods, and data analysis are presented. With the intent to begin addressing the gap between regulatory agencies' guidance documents about drug-drug interactions and contemporary NPDI research, the objective of this Recommended Approach is to propose methods for the design of clinical pharmacokinetic studies of NPs and NP-drug interactions., (© 2021 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The intestine has important gate-keeping functions that can profoundly affect the systemic blood exposure of orally administered drugs. Thus, characterizing a new molecular entity's (NME) disposition within the intestine is of utmost importance in drug development. While currently used in vitro systems, such as Ussing chamber, precision-cut intestinal slices, immortalized cell lines, and primary enterocytes provide substantial knowledge about drug absorption and the intestinal first-pass effect, they remain sub-optimal for quantitatively predicting this process and the oral bioavailability of many drugs. Use of novel in vitro systems such as intestinal organoids and intestinal microphysiological systems have provided substantial advances over the past decade, expanding our understanding of intestinal physiology, pathology, and development. However, application of these emerging in vitro systems in the pharmaceutical science is in its infancy. Preliminary work has demonstrated that these systems more accurately recapitulate the physiology and biochemistry of the intact intestine, as it relates to oral drug disposition, and thus they hold considerable promise as preclinical testing platforms of the future. Here we review currently used and emerging in vitro models of the human intestine employed in pharmaceutical science research. We also highlight aspects of these emerging tools that require further study., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Context: The effect of daily vitamin D supplementation on the serum concentration of vitamin D (the parent compound) may offer insight into vitamin D disposition., Objective: To assess the total serum vitamin D response to vitamin D3 supplementation and whether it varies according to participant characteristics. To compare results with corresponding results for total serum 25-hydroxyvitamin D [25(OH)D], which is used clinically and measured in supplementation trials., Design: Exploratory study within a randomized trial., Intervention: 2000 International Units of vitamin D3 per day (or matching placebo)., Setting: Community-based., Participants: 161 adults (mean ± SD age 70 ± 6 years; 66% males) with type 2 diabetes., Main Outcome Measures: Changes in total serum vitamin D and total serum 25(OH)D concentrations from baseline to year 2., Results: At baseline, there was a positive, nonlinear relation between total serum vitamin D and total serum 25(OH)D concentrations. Adjusted effects of supplementation were a 29.2 (95% CI: 24.3, 34.1) nmol/L increase in serum vitamin D and a 33.4 (95% CI: 27.7, 39.2) nmol/L increase in serum 25(OH)D. Among those with baseline 25(OH)D < 50 compared with ≥ 50 nmol/L, the serum vitamin D response to supplementation was attenuated (15.7 vs 31.2 nmol/L; interaction P-value = 0.02), whereas the serum 25(OH)D response was augmented (47.9 vs 30.7 nmol/L; interaction P-value = 0.05)., Conclusions: Vitamin D3 supplementation increases total serum vitamin D and 25(OH)D concentrations with variation according to baseline 25(OH)D, which suggests that 25-hydroxylation of vitamin D3 is more efficient when serum 25(OH)D concentration is low., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Participant retention in longitudinal health research is necessary for generalizable results. Understanding factors that correlate with increased retention could improve retention in future studies. Here, we describe how participant and study process measures are associated with retention in a longitudinal tobacco cessation research study performed in Anchorage, Alaska. Specifically, we conducted a secondary analysis exploring retention among 151 Alaska Native and American Indian (ANAI) people and described our study processes using study retention categories from a recent meta-analysis. We found that our study processes influence retention among ANAI urban residents more than measures collected about the participant. For study process measures, calls where a participant answered and calls participants placed to the study team were associated with higher retention. Calls where the participant did not answer were associated with lower retention. For participant measures, only lower annual income was associated with lower retention at 6 weeks. Promoting communication from participants to the study team could improve retention, and alternative communication methods could be used after unsuccessful calls. Finally, categorizing our study retention strategies demonstrated that additional barrier-reduction strategies might be warranted.

CYP2A6 activity, phenotyped by the nicotine metabolite ratio (NMR), is a predictor of several smoking behaviors, including cessation and smoking-related disease risk. The heritability of the NMR is 60-80%, yet weighted genetic risk scores (wGRSs) based on common variants explain only 30-35%. Rare variants (minor allele frequency <1%) are hypothesized to explain some of this missing heritability. We present two targeted sequencing studies where rare protein-coding variants are functionally characterized in vivo, in silico, and in vitro to examine this hypothesis. In a smoking cessation trial, 1687 individuals were sequenced; characterization measures included the in vivo NMR, in vitro protein expression, and metabolic activity measured from recombinant proteins. In a human liver bank, 312 human liver samples were sequenced; measures included RNA expression, protein expression, and metabolic activity from extracted liver tissue. In total, 38 of 47 rare coding variants identified were novel; characterizations ranged from gain-of-function to loss-of-function. On a population level, the portion of NMR variation explained by the rare coding variants was small (~1%). However, upon incorporation, the accuracy of the wGRS was improved for individuals with rare protein-coding variants (i.e., the residuals were reduced), and approximately one-third of these individuals (12/39) were re-assigned from normal to slow metabolizer status. Rare coding variants can alter an individual's CYP2A6 activity; their integration into wGRSs through precise functional characterization is necessary to accurately assess clinical outcomes and achieve precision medicine for all. Investigation into noncoding variants is warranted to further explain the missing heritability in the NMR., (© 2021 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Prevalence of smoking is higher in Alaska Native and American Indian (ANAI) populations living in Alaska than the general US population. Genetic factors contribute to smoking and cessation rates. The objective of this study was to compare CYP2A6 genetic variation and CYP2A6 enzyme activity toward nicotine in an ANAI population. ANAI (N = 151) people trying to quit smoking were recruited. DNA samples were genotyped for CYP2A6 variants *1X2A, *1B, *2, *4, *9, *10, *12, and *35. Multiple nicotine metabolites were measured in plasma and urine samples, including cotinine and 3'-hydroxycotinine used to determine CYP2A6 activity (e.g., nicotine metabolite ratio [NMR]). We calculated summary statistics for all of the genotypes and metabolites and assigned CYP2A6 activity scores based on known information. We studied the association of CYP2A6 variants with the NMR and smoking histories. The overall frequency of the CYP2A6*1B gain of function allele was high in the ANAI versus non-ANAI populations in other studies. Both *4 null and *9 decrease of function alleles had frequencies similar to previous studies of ANAI populations. In a multivariate analysis, the genotype-inferred CYP2A6 activity score was associated with both plasma and urine NMR (p value = 8.56E-08 and 4.08E-13, respectively). Plasma NMR was also associated with duration of smoking (p value < 0.01) but not urinary total nicotine equivalents uncorrected for creatinine (TNE9 uc ) or biological sex. Urine NMR was significantly associated (p value < 0.01) with TNE9 uc . Variation in NMR in this ANAI population is explained in part by CYP2A6 genetic variation., (© 2021 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The frequencies of genetic variants in the CYP3A4 and CYP3A5 genes differ greatly across global populations, leading to profound differences in the metabolic activity of these enzymes and resulting drug metabolism rates, with important consequences for therapeutic safety and efficacy. Yet, the impact of genetic variants on enzyme activity are incompletely described, particularly in American Indian and Alaska Native (AIAN) populations. To characterize genetic variation in CYP3A4 and CYP3A5 and its effect on enzyme activity, we partnered with AIAN people living in two regions of Alaska: Yup'ik Alaska Native people living in the Yukon-Kuskokwim Delta region of rural southwest Alaska and AIAN people receiving care at the Southcentral Foundation in Anchorage, Alaska. We identified low frequencies of novel and known variation in CYP3A4 and CYP3A5, including low frequencies of the CYP3A4*1G and CYP3A5*1 variants, and linkage disequilibrium patterns that differed from those we previously identified in an American Indian population in western Montana. We also identified increased activity of the CYP3A4*1G allele in vitro and in vivo. We demonstrated that the CYP3A4*1G allele confers increased protein content in human lymphoblastoid cells and both increased protein content and increased activity in human liver microsomes. We confirmed enhanced CYP3A4-mediated 4β-vitamin D hydroxylation activity in Yup'ik people with the CYP3A4*1G allele. AIAN people in Alaska and Montana who carry the CYP3A4*1G allele-coupled with low frequency of the functional CYP3A5*1 variant-may metabolize CYP3A substrates more rapidly than people with the reference CYP3A4 allele., (© 2021 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

The botanical natural product goldenseal can precipitate clinical drug interactions by inhibiting cytochrome P450 (CYP) 3A and CYP2D6. Besides P-glycoprotein, effects of goldenseal on other clinically relevant transporters remain unknown. Established transporter-expressing cell systems were used to determine the inhibitory effects of a goldenseal extract, standardized to the major alkaloid berberine, on transporter activity. Using recommended basic models, the extract was predicted to inhibit the efflux transporter BCRP and uptake transporters OATP1B1/3. Using a cocktail approach, effects of the goldenseal product on BCRP, OATP1B1/3, OATs, OCTs, MATEs, and CYP3A were next evaluated in 16 healthy volunteers. As expected, goldenseal increased the area under the plasma concentration-time curve (AUC 0-inf ) of midazolam (CYP3A; positive control), with a geometric mean ratio (GMR) (90% confidence interval (CI)) of 1.43 (1.35-1.53). However, goldenseal had no effects on the pharmacokinetics of rosuvastatin (BCRP and OATP1B1/3) and furosemide (OAT1/3); decreased metformin (OCT1/2, MATE1/2-K) AUC 0-inf (GMR, 0.77 (0.71-0.83)); and had no effect on metformin half-life and renal clearance. Results indicated that goldenseal altered intestinal permeability, transport, and/or other processes involved in metformin absorption, which may have unfavorable effects on glucose control. Inconsistencies between model predictions and pharmacokinetic outcomes prompt further refinement of current basic models to include differential transporter expression in relevant organs and intestinal degradation/metabolism of the precipitant(s). Such refinement should improve in vitro-in vivo prediction accuracy, contributing to a standard approach for studying transporter-mediated natural product-drug interactions., (© 2020 The Authors. Clinical Pharmacology & Therapeutics © 2020 American Society for Clinical Pharmacology and Therapeutics.)

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., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 by The Author(s).)

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., Competing Interests: The authors have no financial conflicts of interest to disclose., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Serum 25-hydroxyvitamin D [25(OH)D] concentration is an indicator of vitamin D exposure, but it is also influenced by clinical characteristics that affect 25(OH)D production and clearance. Vitamin D is the precursor to 25(OH)D but is analytically challenging to measure in biological specimens., Objectives: We aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of vitamins D3 and D2 in serum and to explore the potential of circulating vitamin D as a biomarker of exposure in supplementation trials., Methods: The method was validated using guideline C62-A from the Clinical and Laboratory Standards Institute and was applied in 2 pilot clinical trials of oral vitamin D3 supplementation. Pilot study 1 included 22 adults randomly assigned to placebo or 2000 IU/d. Blood was collected at baseline, 1, 3, 6, and 12 mo. Pilot study 2 included 15 adults randomly assigned to 2000 or 4000 IU/d. Blood and subcutaneous (SUBQ) adipose tissue were collected at baseline and 3 mo., Results: In study 1, mean change (baseline to 3 mo) in serum vitamin D3 was -0.1 ng/mL in the placebo group and 6.8 ng/mL in the 2000 IU/d group (absolute difference: 6.9; 95% CI: 4.5, 9.3 ng/mL). In study 2, mean change (baseline to 3 mo) in serum vitamin D3 was 10.4 ng/mL in the 2000 IU/d group and 22.2 ng/mL in the 4000 IU/d group (fold difference: 2.15; 95% CI: 1.40, 3.37). Serum and adipose tissue vitamin D3 concentrations were correlated, and the dose-response of vitamin D3 in adipose mirrored that in serum., Conclusions: We validated a sensitive, robust, and high-throughput LC-MS/MS method to quantify vitamins D3 and D2 in serum. Serum and SUBQ adipose tissue vitamin D3 concentrations increased proportionally to dose with 3 mo of daily supplementation.These trials were registered at clinicaltrials.gov as NCT00552409 (pilot study 1) and NCT01477034 (pilot study 2)., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.)

The efficacy, safety, and tolerability of drugs are dependent on numerous factors that influence their disposition. A dose that is efficacious and safe for one individual may result in sub-therapeutic or toxic blood concentrations in others. A significant source of this variability in drug response is drug metabolism, where differences in presystemic and systemic biotransformation efficiency result in variable degrees of systemic exposure (e.g., AUC, C max , and/or C min ) following administration of a fixed dose.Interindividual differences in drug biotransformation have been studied extensively. It is recognized that both intrinsic factors (e.g., genetics, age, sex, and disease states) and extrinsic factors (e.g., diet , chemical exposures from the environment, and the microbiome) play a significant role. For drug-metabolizing enzymes, genetic variation can result in the complete absence or enhanced expression of a functional enzyme. In addition, upregulation and downregulation of gene expression, in response to an altered cellular environment, can achieve the same range of metabolic function (phenotype), but often in a less predictable and time-dependent manner. Understanding the mechanistic basis for variability in drug disposition and response is essential if we are to move beyond the era of empirical, trial-and-error dose selection and into an age of personalized medicine that will improve outcomes in maintaining health and treating disease., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

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 IC 50 (∼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 IC 50 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., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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)D 3 (d-25[OH]D 3 ) in a pharmacokinetic study of 87 adults, including 43 with normal eGFR (≥60 ml/min per 1.73 m 2 ), 24 with nondialysis CKD (eGFR <60 ml/min per 1.73 m 2 ), and 20 with ESKD treated with hemodialysis. We measured concentrations of d-25(OH)D 3 and deuterated 24,25-dihydroxyvitamin D 3 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)D 3 concentrations over time. We remeasured 25(OH)D clearance in a subset of 18 participants after extended oral vitamin-D 3 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 ( β =-17 ml/d per 10 ml/min per 1.73 m 2 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-D 3 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., (Copyright © 2021 by the American Society of Nephrology.)

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, (-)- β -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, k inact = 0.065 minute -1 ) and CYP3A4/5 (midazolam 1'-hydroxylation; K I = 14.8 μM, k inact = 0.019 minute -1 ); (-)- β -hydrastine inhibited CYP2C9 (diclofenac 4'-hydroxylation; K I = 49 μM, k inact = 0.036 minute -1 ), CYP2D6 ( K I > 250 μM, k inact > 0.06 minute -1 ), and CYP3A4/5 ( K I = 28 μM, k inact = 0.056 minute -1 ); and hydrastinine inhibited CYP2D6 ( K I = 37 μM, k inact = 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., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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 D 3 , administered over 1 year, differed between those with asthma or COPD versus control subjects. Serum concentrations of vitamin D 3 , 25(OH)D 3 , and 1α,25-dihydroxyvitamin D 3 (1α,25[OH] 2 D 3 ) 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) 2 D 3 -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)D 3 -to-vitamin D 3 and higher molar ratios of 1α,25(OH) 2 D 3 -to-25(OH)D 3 both presupplementation and postsupplementation ( P  ≤ 0.005). Intergroup differences in 1α,25(OH) 2 D 3 -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)D 3 -to-vitamin D 3 and increased molar ratios of 1α,25(OH) 2 D 3 -to-25(OH)D 3 in serum, suggesting that vitamin D metabolism is dysregulated in these conditions.

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., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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., Competing Interests: Conflicts of interest: CHIM is a commercial product of In Vitro ADMET Laboratories Inc., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Warfarin is a narrow therapeutic index anticoagulant drug and its use is associated with infrequent but significant adverse bleeding events. The international normalized ratio (INR) is the most commonly used biomarker to monitor and titrate warfarin therapy. However, INR is derived from a functional assay, which determines clotting efficiency at the time of measurement and is susceptible to technical variability. Protein induced by vitamin K antagonist-II (PIVKA-II) has been suggested as a biomarker of long-term vitamin K status, providing mechanistic insights about variation in the functional assay. However, the currently available antibody-based PIVKA-II assay does not inform on the position and number of des-carboxylation sites in prothrombin. The assay presented in this paper provides simultaneous quantification of carboxy and des-carboxy prothrombin that are essential for monitoring early changes in INR and, thus, serves as the superior tool for managing warfarin therapy. Additionally, this assay permits the quantification of total prothrombin level, which is affected by warfarin treatment. Prothrombin recovery from plasma was 95% and the liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was linear (r 2  = 0.98) with a dynamic range of 1-100 µg/mL. The assay interday precision was within 20%. A des-carboxy peptide of prothrombin (GNLER) was negatively correlated with active prothrombin (Pearson r = 0.99, P < 0.0001), whereas its association was positively linked with INR values (Pearson r = 0.75, P < 0.015). This novel LC-MS/MS assay for active and inactive prothrombin quantification can be applied to titrate anticoagulant therapy and to monitor the impact of diseases, such as hepatocellular carcinoma on clotting physiology., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

In gestational diabetes mellitus (GDM), women are unable to compensate for the increased insulin resistance during pregnancy. Data are limited regarding the pharmacodynamic effects of metformin and glyburide during pregnancy. This study characterized insulin sensitivity (SI), β-cell responsivity, and disposition index (DI) in women with GDM utilizing a mixed-meal tolerance test (MMTT) before and during treatment with glyburide monotherapy (GLY, n = 38), metformin monotherapy (MET, n = 34), or GLY and MET combination therapy (COMBO; n = 36). GLY significantly decreased dynamic β-cell responsivity (31%). MET and COMBO significantly increased SI (121% and 83%, respectively). Whereas GLY, MET, and COMBO improved DI, metformin (MET and COMBO) demonstrated a larger increase in DI (P = 0.05) and a larger decrease in MMTT peak glucose concentrations (P = 0.03) than subjects taking only GLY. Maximizing SI with MET followed by increasing β-cell responsivity with GLY or supplementing with insulin might be a more optimal strategy for GDM management than monotherapy., (© 2019 The Authors Clinical Pharmacology & Therapeutics © 2019 American Society for Clinical Pharmacology and Therapeutics.)