Development and Application of a Multidimensional Database for the Detection of Quaternary Ammonium Compounds and Their Phase I Hepatic Metabolites in Humans.

The COVID-19 pandemic has led to significantly increased human exposure to the widely used disinfectants quaternary ammonium compounds (QACs). Xenobiotic metabolism serves a critical role in the clearance of environmental molecules, yet limited data are available on the routes of QAC metabolism or metabolite levels in humans. To address this gap and to advance QAC biomonitoring capabilities, we analyzed 19 commonly used QACs and their phase I metabolites by liquid chromatography-ion mobility-tandem mass spectrometry (LC-IM-MS/MS). In vitro generation of QAC metabolites by human liver microsomes produced a series of oxidized metabolites, with metabolism generally occurring on the alkyl chain group, as supported by MS/MS fragmentation. Discernible trends were observed in the gas-phase IM behavior of QAC metabolites, which, despite their increased mass, displayed smaller collision cross-section (CCS) values than those of their respective parent compounds. We then constructed a multidimensional reference SQLite database consisting of m/z, CCS, retention time (rt), and MS/MS spectra for 19 parent QACs and 81 QAC metabolites. Using this database, we confidently identified 13 parent QACs and 35 metabolites in de-identified human fecal samples. This is the first study to integrate in vitro metabolite biosynthesis with LC-IM-MS/MS for the simultaneous monitoring of parent QACs and their metabolites in humans.

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.

PBPK modeling of CYP3A and P-gp substrates to predict drug-drug interactions in patients undergoing Roux-en-Y gastric bypass surgery.

Roux-en-Y gastric bypass surgery (RYGBS) is an effective surgical intervention to reduce mortality in morbidly obese patients. Following RYGBS, the disposition of drugs may be affected by anatomical alterations and changes in intestinal and hepatic drug metabolizing enzyme activity. The aim of this study was to better understand the drug-drug interaction (DDI) potential of CYP3A and P-gp inhibitors. The impacts of RYGBS on the absorption and metabolism of midazolam, acetaminophen, digoxin, and their major metabolites were simulated using physiologically-based pharmacokinetic (PBPK) modeling. PBPK models for verapamil and posaconazole were built to evaluate CYP3A- and P-gp-mediated DDIs pre- and post-RYGBS. The simulations suggest that for highly soluble drugs, such as verapamil, the predicted bioavailability was comparable pre- and post-RYGBS. For verapamil inhibition, RYGBS did not affect the fold-change of the predicted inhibited-to-control plasma AUC ratio or predicted inhibited-to-control peak plasma concentration ratio for either midazolam or digoxin. In contrast, the predicted bioavailability of posaconazole, a poorly soluble drug, decreased from 12% pre-RYGBS to 5% post-RYGBS. Compared to control, the predicted posaconazole-inhibited midazolam plasma AUC increased by 2.0-fold pre-RYGBS, but only increased by 1.6-fold post-RYGBS. A similar trend was predicted for pre- and post-RYGBS inhibited-to-control midazolam peak plasma concentration ratios (2.0- and 1.6-fold, respectively) following posaconazole inhibition. Absorption of highly soluble drugs was more rapid post-RYGBS, resulting in higher predicted midazolam peak plasma concentrations, which was further increased following inhibition by verapamil or posaconazole. To reduce the risk of a drug-drug interaction in patients post-RYGBS, the dose or frequency of object drugs may need to be decreased when administered with highly soluble inhibitor drugs, especially if toxicities are associated with plasma peak concentrations.

Selection of Priority Natural Products for Evaluation as Potential Precipitants of Natural Product-Drug Interactions: A NaPDI Center Recommended Approach.

Pharmacokinetic interactions between natural products (NPs) and conventional medications (prescription and nonprescription) are a longstanding but understudied problem in contemporary pharmacotherapy. Consequently, there are no established methods for selecting and prioritizing commercially available NPs to evaluate as precipitants of NP-drug interactions (NPDIs). As such, NPDI discovery remains largely a retrospective, bedside-to-bench process. This Recommended Approach, developed by the Center of Excellence for Natural Product Drug Interaction Research (NaPDI Center), describes a systematic method for selecting NPs to evaluate as precipitants of potential clinically significant pharmacokinetic NPDIs. Guided information-gathering tools were used to score, rank, and triage NPs from an initial list of 47 candidates. Triaging was based on the presence and/or absence of an NPDI identified in a clinical study (≥20% or <20% change in the object drug area under the concentration vs. time curve, respectively), as well as mechanistic and descriptive in vitro and clinical data. A qualitative decision-making tool, termed the fulcrum model, was developed and applied to 11 high-priority NPs for rigorous study of NPDI risk. Application of this approach produced a final list of five high-priority NPs, four of which are currently under investigation by the NaPDI Center.

Perspective: 4ß-hydroxycholesterol as an emerging endogenous biomarker of hepatic CYP3A.

A key goal in the clinical development of a new molecular entity is to quickly identify whether it has the potential for drug-drug interactions. In particular, confirmation of in vitro data in the early stage of clinical development would facilitate the decision making and inform future clinical pharmacology study designs. Plasma 4ß-hydroxycholesterol (4ß-HC) is considered as an emerging endogenous biomarker for cytochrome P450 3A (CYP3A), one of the major drug metabolizing enzymes. Although there are increasing reports of the use of 4ß-HC in academic- and industry-sponsored clinical studies, a thorough review, summary and consideration of the advantages and challenges of using 4ß-HC to evaluate changes in CYP3A activity has not been attempted. Herein, we review the biology of 4ß-HC, its response to treatment with CYP3A inducers, inhibitors and mixed inducer/inhibitors in healthy volunteers and patients, the association of 4ß-HC with other probes of CYP3A activity (e.g. midazolam, urinary cortisol ratios), and present predictive pharmacokinetic models. We provide recommendations for studying hepatic CYP3A activity in clinical pharmacology studies utilizing 4ß-HC at different stages of drug development.

Pregnancy Increases the Renal Secretion of N1-methylnicotinamide, an Endogenous Probe for Renal Cation Transporters, in Patients Prescribed Metformin.

N1-methylnicotinamide (1-NMN) has been investigated as an endogenous probe for the renal transporter activity of organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins 1 and 2-K (MATE1 and MATE2-K). As pregnancy increased the renal secretion of metformin, a substrate for OCT2, MATE1, and MATE2-K, we hypothesized that the renal secretion of 1-NMN would be similarly affected. Blood and urine samples collected from women prescribed metformin for type 2 diabetes, gestational diabetes, and polycystic ovarian syndrome during early, mid, and late pregnancy (n = 34 visits) and postpartum (n = 14 visits) were analyzed for 1-NMN using liquid chromatography-mass spectrometry. The renal clearance and secretion clearance, using creatinine clearance to correct for glomerular filtration, were estimated for 1-NMN and correlated with metformin renal clearance. 1-NMN renal clearance was higher in both mid (504 ± 293 ml/min, P < 0.01) and late pregnancy (557 ± 305 ml/min, P < 0.01) compared with postpartum (240 ± 106 ml/min). The renal secretion of 1-NMN was 3.5-fold higher in mid pregnancy (269± 267, P < 0.05) and 4.5-fold higher in late pregnancy compared with postpartum (342 ± 283 versus 76 ± 92 ml/min, P < 0.01). Because creatinine is also a substrate of OCT2, MATE1, and MATE2-K, creatinine clearance likely overestimates filtration clearance, whereas the calculated 1-NMN secretion clearance is likely underestimated. Metformin renal clearance and 1-NMN renal clearance were positively correlated (rs = 0.68, P < 0.0001). 1-NMN renal clearance increases during pregnancy due to increased glomerular filtration and net secretion by renal transporters.

Interindividual Variability in Cytochrome P450-Mediated Drug Metabolism.

The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug-drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals.

In Vivo Imaging of Human MDR1 Transcription in the Brain and Spine of MDR1-Luciferase Reporter Mice.

P-glycoprotein (Pgp) [the product of the MDR1 (ABCB1) gene] at the blood-brain barrier (BBB) limits central nervous system (CNS) entry of many prescribed drugs, contributing to the poor success rate of CNS drug candidates. Modulating Pgp expression could improve drug delivery into the brain; however, assays to predict regulation of human BBB Pgp are lacking. We developed a transgenic mouse model to monitor human MDR1 transcription in the brain and spinal cord in vivo. A reporter construct consisting of ∼10 kb of the human MDR1 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line (MDR1-luc). Fluorescence in situ hybridization localized the MDR1-luciferase transgene on chromosome 3. Reporter gene expression was monitored with an in vivo imaging system following D-luciferin injection. Basal expression was detectable in the brain, and treatment with activators of the constitutive androstane, pregnane X, and glucocorticoid receptors induced brain and spinal MDR1-luc transcription. Since D-luciferin is a substrate of ABCG2, the feasibility of improving D-luciferin brain accumulation (and luciferase signal) was tested by coadministering the dual ABCB1/ABCG2 inhibitor elacridar. The brain and spine MDR1-luc signal intensity was increased by elacridar treatment, suggesting enhanced D-luciferin brain bioavailability. There was regional heterogeneity in MDR1 transcription (cortex > cerebellum) that coincided with higher mouse Pgp protein expression. We confirmed luciferase expression in brain vessel endothelial cells by ex vivo analysis of tissue luciferase protein expression. We conclude that the MDR1-luc mouse provides a unique in vivo system to visualize MDR1 CNS expression and regulation.

Estimated GFR and circulating 24,25-dihydroxyvitamin D3 concentration: a participant-level analysis of 5 cohort studies and clinical trials.

BACKGROUND: Decreased glomerular filtration rate (GFR) leads to reduced production of 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3 (25[OH]D3). Effects of low GFR on vitamin D catabolism are less well understood. We tested associations of estimated GFR (eGFR) with the circulating concentration of 24,25-dihydroxyvitamin D3 (24,25[OH]2D3), the most abundant product of 25(OH)D3 catabolism, across populations with a wide range of GFRs. STUDY DESIGN: Cross-sectional study. SETTING & PARTICIPANTS: 9,596 participants in 5 cohort studies and clinical trials: the Diabetes Control and Complications Trial (N=1,193), Multi-Ethnic Study of Atherosclerosis (N=6,470), Cardiovascular Health Study (N=932), Seattle Kidney Study (N=289), and Hemodialysis Study (N=712). PREDICTOR: eGFR. OUTCOME: Circulating 24,25(OH)2D3 concentration. MEASUREMENTS: GFR was estimated from serum creatinine using the Chronic Kidney Disease Epidemiology Collaboration equation. Vitamin D metabolites were measured by mass spectrometry. RESULTS: Circulating 24,25(OH)2D3 concentration was correlated with circulating 25(OH)D3 concentration (Pearson r range, 0.64-0.88). This correlation was weaker with lower eGFRs. Moreover, the increment in 24,25(OH)2D3 concentration associated with higher 25(OH)D3 concentration (slope) was lower with lower eGFRs: 2.06 (95% CI, 2.01-2.10), 1.77 (95% CI, 1.74-1.81), 1.55 (95% CI, 1.48-1.62), 1.17 (95% CI, 1.05-1.29), 0.92 (95% CI, 0.74-1.10), 0.61 (95% CI, 0.22-1.00), and 0.37 (95% CI, 0.35-0.39) ng/mL of 24,25(OH)2D3 per 10 ng/mL of 25(OH)D3 for eGFRs≥90, 60-89, 45-59, 30-44, 15-29, and <15 mL/min/1.73 m2 and end-stage renal disease treated with hemodialysis, respectively. As a result, at a 25(OH)D3 concentration of 20 ng/mL, mean 24,25(OH)2D3 concentrations were 2.92 (95% CI, 2.87-2.96), 2.68 (95% CI, 2.64-2.72), 2.35 (95% CI, 2.26-2.45), 1.92 (95% CI, 1.74-2.10), 1.69 (95% CI, 1.43-1.95), 1.14 (95% CI, 0.62-1.66), and 1.04 (95% CI,1.02-1.07) ng/mL for each category, respectively. This interaction was independent of other relevant clinical characteristics. Race, diabetes, urine albumin excretion, and circulating parathyroid hormone and fibroblast growth factor 23 concentrations more modestly modified the association of 24,25(OH)2D3 with 25(OH)D3. LIMITATIONS: Lack of direct pharmacokinetic measurements of vitamin D catabolism. CONCLUSIONS: Lower eGFR is associated strongly with reduced vitamin D catabolism, as measured by circulating 24,25(OH)2D3 concentration.

Herb-drug interactions: challenges and opportunities for improved predictions.

Supported by a usage history that predates written records and the perception that "natural" ensures safety, herbal products have increasingly been incorporated into Western health care. Consumers often self-administer these products concomitantly with conventional medications without informing their health care provider(s). Such herb-drug combinations can produce untoward effects when the herbal product perturbs the activity of drug metabolizing enzymes and/or transporters. Despite increasing recognition of these types of herb-drug interactions, a standard system for interaction prediction and evaluation is nonexistent. Consequently, the mechanisms underlying herb-drug interactions remain an understudied area of pharmacotherapy. Evaluation of herbal product interaction liability is challenging due to variability in herbal product composition, uncertainty of the causative constituents, and often scant knowledge of causative constituent pharmacokinetics. These limitations are confounded further by the varying perspectives concerning herbal product regulation. Systematic evaluation of herbal product drug interaction liability, as is routine for new drugs under development, necessitates identifying individual constituents from herbal products and characterizing the interaction potential of such constituents. Integration of this information into in silico models that estimate the pharmacokinetics of individual constituents should facilitate prospective identification of herb-drug interactions. These concepts are highlighted with the exemplar herbal products milk thistle and resveratrol. Implementation of this methodology should help provide definitive information to both consumers and clinicians about the risk of adding herbal products to conventional pharmacotherapeutic regimens.

Identification, replication, and functional fine-mapping of expression quantitative trait loci in primary human liver tissue.

The discovery of expression quantitative trait loci ("eQTLs") can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (n = 206) and Illumina (n = 60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (n = 266). We found that ∼30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3'UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits.

Metabolism and pharmacokinetics of vitamin D in patients with cystic fibrosis.

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)D2 and 25(OH)D3, 1α,25-dihydroxyvitamins D2 and D3 (1α,25(OH)2D2 and 1α,25(OH)2D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), 4ß,25-dihydroxyvitamin D3 (4ß,25(OH)2D3), 25-hydroxyvitamin D3-3-sulfate (25(OH)D3-S), and 25-hydroxyvitamin D3-3-glucuronide (25(OH)D3-G). In a 56-day prospective pharmacokinetic study, ∼25 µg deuterium-labeled 25(OH)D3 (d6-25(OH)D3) was administered intravenously to participants (N = 5 with CF, N = 5 control subjects). Serum was analyzed for d6-25(OH)D3 and d6-24,25(OH)2D3, 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)2D (43.6 [12.7] vs. 50.7 [13.0] pg/mL), 4ß,25(OH)2D3 (52.1 [38.9] vs. 79.9 [60.2] pg/mL), and 25(OH)D3-S (17.7 [11.6] vs. 30.1 [12.3] ng/mL) (p < 0.001 for all). The pharmacokinetics of d6-25(OH)D3 and d6-24,25(OH)D3 did not differ between groups. In summary, although 25(OH)D concentrations were comparable, participants with CF had lower 1α,25(OH)2D, 4ß,25(OH)2D3, and 25(OH)D3-S concentrations than healthy controls. Neither 25(OH)D3 clearance, nor formation of 24,25(OH)2D3, 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.

An inducible cytochrome P450 3A4-dependent vitamin D catabolic pathway.

Vitamin D(3) is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D(3) (25OHD(3)), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD(3) was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD(3) hydroxylation by human liver microsomes, with the formation of 4ß,25-dihydroxyvitamin D(3) [4ß,25(OH)(2)D(3)] dominating (V(max)/K(m) = 0.85 ml · min(-1) · nmol enzyme(-1)). 4ß,25(OH)(2)D(3) was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D(3), and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4ß,25(OH)(2)D(3) in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4ß,25(OH)(2)D(3), but not CYP24A1-dependent 24R,25-dihydroxyvitamin D(3) formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD(3) metabolism may play an important role in the regulation of vitamin D(3) in vivo and in the etiology of drug-induced osteomalacia.

In vitro hepatotoxicity and cytochrome P450 induction and inhibition characteristics of carnosic acid, a dietary supplement with antiadipogenic properties.

Carnosic acid is a phenolic diterpene isolated from rosemary (Rosmarinus officinalis), which may have anticancer, antiadipogenic, and anti-inflammatory properties. Recently, carnosic acid was shown to prevent weight gain and hepatic steatosis in a mouse model of obesity and type II diabetes. Based on these results, carnosic acid has been suggested as a potential treatment for obesity and nonalcoholic fatty liver disease; however, little is known about the safety of carnosic acid at doses needed to elicit a pharmacological effect. For this reason, hepatotoxicity and cytochrome P450 inhibition and induction studies were performed in primary human hepatocytes and microsomes. Measuring cellular ATP, carnosic acid showed a dose-dependent increase in hepatotoxicity with an EC(50) value of 94.8 ± 36.7 µM in three human hepatocyte donors without a concurrent increase in the apoptosis markers caspase-3/7. In human liver microsomes, carnosic acid did not exhibit significant time-dependent inhibition for any of the cytochrome P450 enzymes investigated, although it did inhibit CYP2C9- and CYP3A4-catalyzed reactions with K(i) values of 9.2 and 4.3 µM, respectively. Carnosic acid also induced CYP2B6 and CYP3A4 mRNA and enzyme activity in a dose-dependent manner. At 10 µM, carnosic acid increased CYP2B6 enzyme activity 61.6 and 49.3% in two donors compared with phenobarbital, and it increased CYP3A enzyme activity 82.6 and 142% compared with rifampicin. These results indicate the potential for drug interactions with carnosic acid and illustrate the need for an appropriate safety assessment before being used as a weight loss supplement.

Pharmacokinetics of 38 Percent Silver Diamine Fluoride in Children.

PURPOSE: The purpose of this study was to measure serum levels and characterize the pharmacokinetics of silver and fluoride in healthy children receiving silver diamine fluoride (SDF) treatment for dental caries lesions. METHODS: Children (three to 13 years old with at least one caries lesion) were recruited at the University of California, San Francisco Pediatric Dental Clinic from August 2019 through March 2020. Blood was obtained at one randomly selected timepoint up to 168 hours after SDF application. Serum fluoride and silver were measured, and population pharmacokinetic modeling was used to estimate pharmacokinetic parameters and simulate silver concentration versus time profiles in cohorts of children (15 to 50 kg). RESULTS: Fifty-five children completed the study. Serum fluoride had no discernable temporal pattern. Silver concentra- tions were best described by a one-compartment model with first-order absorption and elimination, and weight as a covariate. Simulated 15 kg children had higher predicted peak silver concentrations than simulated 50 kg children (22.0 ng/mL [95 percent confidence interval {95 percent CI} equals 19.4 to 24.6] versus 12.8 ng/mL [95 percent CI equals 11.3 to 14.3]), and a longer predicted silver half-life (15.5 days [95 percent CI equals 12.5 to 18.5] versus 4.0 days [95 percent CI equals 2.7 to 5.3]). CONCLUSIONS: Evidence presented indicate that topical silver diamine fluoride application in children is safe, and serum concentrations of fluoride and silver pose little risk of toxicity.

A longitudinal study of cytochrome P450 2D6 (CYP2D6) activity during adolescence.

CYP2D6 substrates are among the most highly prescribed medications in teenagers and also commonly associated with serious adverse events. To investigate the relative contributions of genetic variation, growth, and development on CYP2D6 activity during puberty, healthy children and adolescents 7-15 years of age at enrollment participated in a longitudinal phenotyping study involving administration of 0.3 mg/kg dextromethorphan (DM) and 4-h urine collection every 6 months for 3 years (7 total visits). At each visit, height, weight, and sexual maturity were recorded, and CYP2D6 activity was determined as the urinary molar ratio of DM to its metabolite dextrorphan (DX). A total of 188 participants completed at least one visit, and 102 completed all seven study visits. Following univariate analysis, only CYP2D6 activity score (p < 0.001), urinary pH (p < 0.001), weight (p = 0.018), and attention-deficit/hyperactivity disorder (ADHD) diagnosis (p < 0.001) were significantly correlated with log(DM/DX). Results of linear mixed model analysis with random intercept, random slope covariance structure revealed that CYP2D6 activity score had the strongest effect on log(DM/DX), with model-estimated average log(DM/DX) being 3.8 SDs higher for poor metabolizers than for patients with activity score 3. A moderate effect on log(DM/DX) was observed for sex, and smaller effects were observed for ADHD diagnosis and urinary pH. The log(DM/DX) did not change meaningfully with age or pubertal development. CYP2D6 genotype remains the single, largest determinant of variability in CYP2D6 activity during puberty. Incorporation of genotype-based dosing guidelines should be considered for CYP2D6 substrates given the prevalent use of these agents in this pediatric age group.

Validation of the 24,25-dihydroxyvitamin D3 to 25-hydroxyvitamin D3 ratio as a biomarker of 25-hydroxyvitamin D3 clearance.

The formation of 24,25-dihydroxyvitamin D (24,25(OH)2D) 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)2D3 to 25(OH)D3 in blood (vitamin D metabolite ratio, VDMR) is postulated to be a marker of 25(OH)D3 clearance, however this has never been tested. We measured baseline 24,25(OH)2D3 and 25(OH)D3 concentrations in 87 participants by liquid chromatography-tandem mass spectrometry. Following an infusion of deuterated 25(OH)D3, blood samples for each participant were collected over 56 days and analyzed for deuterated vitamin D metabolites. 25(OH)D3 clearance and the deuterated metabolite-to-parent AUC ratio (ratio of the AUC of deuterated 24,25(OH)2D3 to that of deuterated 25(OH)D3) 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)D3 clearance and the deuterated metabolite-to-parent AUC ratio (r = 0.51 and 0.76, respectively). Adjusting for 25(OH)D3 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)D3 clearance. This relationship was biased by characteristics including race and kidney disease, which warrant consideration in studies assessing the VDMR.

Simultaneous measurement of plasma vitamin D(3) metabolites, including 4ß,25-dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry.

Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that permits the quantification of major circulating vitamin D(3) metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid-liquid extraction, and Diels-Alder derivatization procedure prior to LC-MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D(3) peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] concentrations. This interfering metabolite has been identified as 4ß,25-dihydroxyvitamin D(3) [4ß,25(OH)(2)D(3)] and was found at concentrations comparable to 1α,25(OH)(2)D(3). Quantification of 1α,25(OH)(2)D(3) in plasma required complete chromatographic separation of 1α,25(OH)(2)D(3) from 4ß,25(OH)(2)D(3). An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD(3), 24R,25(OH)(2)D(3), 1α,25(OH)(2)D(3), and 4ß,25(OH)(2)D(3) in healthy individuals. The LC-MS/MS method developed and described here could result in considerable improvement in quantifying 1α,25(OH)(2)D(3) as well as monitoring the newly identified circulating metabolite, 4ß,25(OH)(2)D(3).

Sources of Interindividual Variability.

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, Cmax, and/or Cmin) 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.

Cardiovascular Safety and Hemostatic Efficacy of Topical Epinephrine in Children Receiving Zirconia Crowns.

OBJECTIVE: The primary aim of this study was to determine the cardiovascular safety of topical racemic epinephrine pellets by measuring heart rate, systolic blood pressure, diastolic blood pressure, and mean arterial pressure in children receiving dental care under general anesthesia. The secondary aim was to assess clinical efficacy by measuring time to reach adequate hemostasis. METHODS: For this pilot study utilizing a split-mouth randomized design, 13 patients requiring prefabricated zirconia crowns on both primary maxillary first molars were recruited. Patients received continuous infusions of propofol and remifentanil with 50-70% inhaled nitrous oxide and oxygen. After randomization and tooth preparation, either saline pellets (control) or racemic epinephrine pellets (experimental) were applied directly to gingival tissue. Vital signs were recorded for 5 minutes. The procedure was repeated on the contralateral side using the alternative (control or experimental) treatment. RESULTS: Topical racemic epinephrine compared to saline produced a significantly larger decrease in mean diastolic blood pressure (-11.1% vs -3.9%; P < .01) and mean arterial pressure (-8.1% vs -2.1%; P < .01), although all noted decreases in cardiovascular variables were clinically insignificant. All experimental treatment teeth achieved adequate hemostasis after 2.2 minutes. Only 5 of the 13 control treatment teeth achieved adequate hemostasis during the 5-minute observation period (1.6 vs 4.2 minutes; P = .01). CONCLUSION: Overall, we conclude that use of topical racemic epinephrine pellets did not result in adverse cardiovascular effects and hemostasis was reached more quickly and predictably compared to saline pellets.