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A [[13]C]-dextromethorphan ([[13]C]-DM) breath test was evaluated to assess its feasibility as a rapid, phenotyping assay for CYP2D6 activity. [[13]C]-DM (0.5 mg/kg) was administered orally with water or potassium bicarbonate-sodium bicarbonate to 30 adult Caucasian volunteers (n = 1 each): CYP2D6 poor metabolizers (2 null alleles; PM-0) and extensive metabolizers with 1 (EM-1) or 2 functional alleles (EM-2). CYP2D6 phenotype was determined by [13]CO[2] enrichment measured by infrared spectrometry (delta-over-baseline [DOB] value) in expired breath samples collected before and up to 240 minutes after [[13]C]-DM ingestion and by 4-hour urinary metabolite ratio. The PM-0 group was readily distinguishable from either EM group by both the breath test and urinary metabolite ratio. Using a single point determination of phenotype at 40 minutes and defining PMs as subjects with a DOB

The expression of cytochrome P450 (CYP) enzymes is highly variable and associated with factors, such as age, genotype, sex, and disease states. In this study, quantification of metronidazole metabolizing CYP isoforms (CYP2A6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7) in human liver microsomes from 115 children and 35 adults was performed using a quantitative proteomics method. The data confirmed age-dependent increase in CYP2A6, CYP2E1, and CYP3A4 abundance, whereas, as expected, CYP3A7 abundance showed postnatal decrease with age. In particular, the fold difference (neonatal to adulthood levels) in the protein abundance of CYP2A6, CYP2E1, and CYP3A4 was 14, 11, and 20, respectively. In contrast, protein abundance of CYP3A7 was > 125-fold higher in the liver microsomes of neonates than of adults. The abundance of CYP2A6 and CYP3A5 was associated with genotypes, rs4803381 and rs776746, respectively. A proteomics-informed physiologically based pharmacokinetic (PBPK) model was developed to describe the pharmacokinetics of metronidazole and its primary metabolite, 2-hydroxymethylmetronidazole. The model revealed an increase in the metabolite-to-parent ratio with age and showed a strong correlation between CYP2A6 abundance and metabolite formation (r 2  = 0.75). Notably, the estimated contribution of CYP3A7 was ~ 75% in metronidazole clearance in neonates. These data suggest that variability in CYP2A6 and CYP3A7 in younger children poses the risk of variable pharmacokinetics of metronidazole and its active metabolite with a potential impact on drug efficacy and safety. No sex-dependent difference was observed in the protein abundance of the studied CYPs. The successful integration of hepatic CYP ontogeny data derived from a large liver bank into the pediatric PBPK model of metronidazole can be extended to other drugs metabolized by the studied CYPs., (© 2024 The Author(s). Clinical Pharmacology & Therapeutics © 2024 American Society for Clinical Pharmacology and Therapeutics.)

In this brief report, we provide an analysis of the influence of a novel CYP2C haplotype (CYP2C:TG) on proton pump inhibitor (PPI) pharmacokinetics (PK) in children. The CYP2C:TG haplotype has been proposed to be associated with increased CYP2C19 activity. We sought to determine if this CYP2C:TG haplotype resulted in similar alterations in metabolism for proton pump inhibitors, which are primarily metabolized by CYP2C19. In a cohort of 41 children aged 6-21 participating in a PPI pharmacokinetic study, effects of the CYP2C:TG allele were assessed by fitting two linear regression models for each of the six PK outcomes assessed, the second of which accounted for the presence of the CYP2C:TG allele. The difference in R 2 values between the two models was computed to quantify the variability in the outcome that could be accounted for by the CYP2C:TG allele after adjustment for the CYP2C19 genotype. We found the CYP2C:TG haplotype to have no measurable additive impact on CYP2C19-mediated metabolism of PPIs in vivo in older children and adolescents. The findings of this study do not support the clinical utility of routine testing for the CYP2C:TG haplotype to guide PPI dose adjustments in children., (© 2024 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Scaling factors are necessary for translating in vitro drug biotransformation data to in vivo clearance values in physiologically-based pharmacokinetic modeling and simulation. Values for microsomal protein per gram of liver are available from several sources for use as a scaling factor to estimate hepatic clearance from microsomal drug biotransformation data. However, data regarding the distribution of cytosolic protein per gram of liver (CPPGL) values across the lifespan are limited, and sparse pediatric data have been published to date. Thus, CPPGL was determined in 160 liver samples from pediatric ( n = 129) and adult ( n = 31) donors obtained from multiple sources: the University of Maryland Brain and Tissue Bank, tissue retrieval services at the University of Minnesota and University of Pittsburgh, and Sekisui-XenoTech. Tissues were homogenized and subjected to differential centrifugation to isolate cytosolic fractions. Cytosolic protein content was determined by BCA assay. CPPGL varied from two- to sixfold within each age group/developmental stage. Tissue source and sex did not contribute substantially to variability in protein content. Regression analyses revealed minimal change in CPPGL over the first two decades of life (logCPPGL increases 0.1 mg/g per decade). A mean ± S.D. CPPGL value of 44.4 ± 17.4 mg/g or median 41.0 mg/g is representative of values observed between birth and early adulthood (0-18 years, n = 129). SIGNIFICANCE STATEMENT: Cytosolic protein per gram of liver (CPPGL) is a scaling factor required for physiologically based pharmacokinetic modeling and simulation of drug biotransformation by cytosolic enzymes, but pediatric data are limited. Although CPPGL varies from two- to sixfold within developmental stages, a value of 44.4 ± 17.4 mg/g (mean ± S.D.) is representative of the pediatric period (0-18 years, n = 129)., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

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., (© 2022 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The 13 C-pantoprazole breath test (PAN-BT) is a safe, noninvasive, in vivo CYP2C19 phenotyping probe for adults. Our objective was to evaluate PAN-BT performance in children, with a focus on discriminating individuals who, according to guidelines from the Clinical Pharmacology Implementation Consortium (CPIC), would benefit from starting dose escalation versus reduction for proton pump inhibitors (PPIs). Children (n = 65, 6-17 years) genotyped for CYP2C19 variants *2, *3, *4, and *17 received a single oral dose of 13 C-pantoprazole. Plasma concentrations of pantoprazole and its metabolites, and changes in exhaled 13 CO 2 (termed delta-over-baseline or DOB), were measured 10 times over 8 h using high performance liquid chromatography with ultraviolet detection and spectrophotometry, respectively. Pharmacokinetic parameters of interest were generated and DOB features derived using feature engineering for the first 180 min postadministration. DOB features, age, sex, and obesity status were used to run bootstrap analysis at each timepoint (T i ) independently. For each iteration, stratified samples were drawn based on genotype prevalence in the original cohort. A random forest was trained, and predictive performance of PAN-BT was evaluated. Strong discriminating ability for CYP2C19 intermediate versus normal/rapid metabolizer phenotype was noted at DOB T30 min (mean sensitivity: 0.522, specificity: 0.784), with consistent model outperformance over a random or a stratified classifier approach at each timepoint (p < 0.001). With additional refinement and investigation, the test could become a useful and convenient dosing tool in clinic to help identify children who would benefit most from PPI dose escalation versus dose reduction, in accordance with CPIC guidelines., (© 2022 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

rs5758550 has been associated with enhanced transcription and suggested to be a useful marker of CYP2D6 activity. As there are limited and inconsistent data regarding the utility of this distant "enhancer" single nucleotide polymorphism (SNP), our goal was to further assess the impact of rs5758550 on CYP2D6 activity toward two probe substrates, atomoxetine (ATX) and dextromethorphan (DM), using in vivo urinary metabolite (DM; n = 188) and pharmacokinetic (ATX; n = 70) and in vitro metabolite formation (ATX and DM; n = 166) data. All subjects and tissues were extensively genotyped, the "enhancer" SNP phased with established CYP2D6 haplotypes either computationally or experimentally, and the impact on CYP2D6 activity investigated using several linear models of varying complexity to determine the proportion of variability in CYP2D6 activity captured by each model. For all datasets and models, the "enhancer" SNP had no or only a modest impact on CYP2D6 activity prediction. An increased effect, when present, was more pronounced for ATX than DM suggesting potential substate-dependency. In addition, CYP2D6*2 alleles with the "enhancer" SNP were associated with modestly higher metabolite formation rates in vitro, but not in vivo; no effect was detected for CYP2D6*1 alleles with "enhancer" SNP. In summary, it remains inconclusive whether the small effects detected in this investigation are indeed caused by the "enhancer" SNP or are rather due to its incomplete linkage with other variants within the gene. Taken together, there does not appear to be sufficient evidence to warrant the "enhancer" SNP be included in clinical CYP2D6 pharmacogenetic testing., (© 2021 The Authors. Clinical Pharmacology & Therapeutics © 2021 American Society for Clinical Pharmacology and Therapeutics.)

Naltrexone, an opioid antagonist primarily metabolized by aldo-keto reductase 1C4 (AKR1C4), treats pediatric conditions involving compulsiveness (e.g., autism spectrum, Prader-Willi, eating disorders, non-suicidal self-injury). Pharmacokinetic variability is apparent in adults, yet no data are available for children. This study aimed to examine the impact of age and genetic variation on naltrexone biotransformation. Human liver cytosol (HLC) samples ( n = 158) isolated from children and adult organ donors were incubated with therapeutically relevant concentrations of naltrexone (0.1, 1 µ M). Naltrexone biotransformation was determined by ultraperformance mass spectrometry quantification of the primary metabolite, 6-beta-naltrexol (6 β N), and 6 β N formation rates (pmol/mg protein/min) were calculated. HLCs from organ donors, age range 0-79 y (mean 16.0 ± 18.2 y), 37% ( n = 60) female, 20% ( n = 33) heterozygous and 1.2% ( n = 2) homozygous for co-occurring AKR1C4 variants (S145C/L311V) showed >200-fold range in 6 β N formation (0.37-76.5 pmol/mg protein/min). Source of donor samples was found to be a substantial contributor to variability. Model estimates for a trimmed data set of source-adjusted pediatric samples (aged 0-18 y) suggested that AKR1C4 genetic variation, age, and sex explained 36% of the variability in 6 β N formation. Although activity increased steadily from birth and peaked in middle childhood (2-5 years), genetic variation (S145C/L311V) demonstrated a greater effect on activity than did age. Naltrexone biotransformation is highly variable in pediatric and adult livers and can be partly accounted for by individual factors feasible to obtain (e.g., genetic variability, age, sex). These data may inform a precision therapeutics approach (e.g., exposure optimization) to further study Naltrexone responsiveness in children and adults. SIGNIFICANCE STATEMENT: Biotransformation of the commonly used opioid antagonist naltrexone is highly variable and may contribute to reduced therapeutic response. Age, sex, and genetic variation in the drug-metabolizing enzyme, AKR1C4, are potential factors contributing to this variability. In pediatric samples, genetic variation (S145C/L311V) demonstrates a greater impact on activity than age. Additionally, the source of donor samples was identified as an important contributor and must be accounted for to confidently elucidate the biological variables most impactful to drug biotransformation., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Microsomal protein per gram of liver (MPPGL) is an important scaling factor for bottom-up physiology-based pharmacokinetic modeling and simulation, but data in pediatrics are limited. Therefore, MPPGL was determined in 160 liver samples from pediatric ( n = 129) and adult ( n = 31) donors obtained from four sources: the University of Maryland Brain and Tissue Bank (UMBTB), tissue retrieval services at the University of Minnesota and University of Pittsburgh, and Sekisui-Xenotech. Tissues were homogenized and subjected to differential centrifugation to prepare microsomes, and cytochrome c reductase activities in tissue homogenates and microsomes were used to estimate cytochrome P450 reductase (POR) activity as a marker of microsomal recovery; microsomal POR content was also assessed by quantitative proteomics. MPPGL values varied 5- to 10-fold within various age groups/developmental stages, and tissue source was identified as a contributing factor. Using a "trimmed" dataset comprised of samples ranging from 3 to 18 years of age common to the four sources, POR protein abundance and activity in microsomes and POR activity in homogenates was lower in UMBTB samples (autopsy) compared with other sources (perfused/flash-frozen). Regression analyses revealed that the UMBTB samples were driving an apparent age effect as no effect of age on log-transformed MPPGL values was observed when the UMBTB samples were excluded. We conclude that a mean±SD MPPGL value of 30.4±1.7 mg/g is representative between one month postnatal age and early adulthood. Potential source effects should be considered for studies involving tissue samples from multiple sources with different procurement and processing procedures. SIGNIFICANCE STATEMENT: Microsomal protein per gram of liver (MPPGL) is an important scaling factor for bottom up PBPK modeling and simulation, but data in pediatrics are limited. Although MPPGL varies 5- to 10-fold at a given developmental stage, a value of 30.4 ± 1.7 mg/g (mean ± SD) is representative between one month postnatal age and early adulthood. However, when tissue samples are obtained from multiple sources, different procurement and processing procedures may influence the results and should be taken into consideration., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: WHO recommends simplified antibiotics for young infants with sepsis in countries where hospitalisation is not feasible. Amoxicillin provides safe, Gram-positive coverage. This study was done to determine pharmacokinetics, drug disposition and interpopulation variability of oral amoxicillin in this demographic., Methods: Young infants with signs of sepsis enrolled in an oral amoxicillin/intramuscular gentamicin treatment arm of a sepsis trial in Karachi, Pakistan, were studied. Limited pharmacokinetic (PK) sampling was performed at 0, 2-3 and 6-8 hours following an index dose of oral amoxicillin. Plasma concentrations were determined by high-performance liquid chromatography/mass spectrometry. Values of ≥2 mg/L were considered as the effect threshold, given the regional minimal inhibitory concentration (MIC) of resistant Streptococcus pneumoniae. RESULTS: Amoxicillin concentrations were determined in 129 samples from 60 young infants. Six of 44 infants had positive blood cultures with predominant Gram-positive organisms. Forty-four infants contributing blood at ≥2 of 3 specified timepoints were included in the analysis. Mean amoxicillin levels at 2-3 hours (11.6±9.5 mg/L, n=44) and 6-8 hours (16.4±9.3 mg/L, n=20) following the index dose exceeded the MIC for amoxicillin (2.0 mg/L) against resistant S. pneumoniae strains. Of 20 infants with three serum levels, 7 showed a classic dose-exposure profile and 13 showed increasing concentrations with time, implying delayed absorption or excretion., Conclusion: Amoxicillin concentrations in sera of young infants following oral administration at 75-100 mg/kg/day daily divided doses exceeds the susceptibility breakpoint for >50% of a 12-hour dosing interval.Oral amoxicillin may hold potential as a safe replacement of parenteral ampicillin in newborn sepsis regimens, including aminoglycosides, where hospitalisation is not feasible., Trial Registration Number: NCT01027429., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Cytosolic sulfotransferases (SULTs), including SULT1A, SULT1B, SULT1E, and SULT2A isoforms, play noteworthy roles in xenobiotic and endobiotic metabolism. We quantified the protein abundances of SULT1A1, SULT1A3, SULT1B1, and SULT2A1 in human liver cytosol samples ( n = 194) by liquid chromatography-tandem mass spectrometry proteomics. The data were analyzed for their associations by age, sex, genotype, and ethnicity of the donors. SULT1A1, SULT1B1, and SULT2A1 showed significant age-dependent protein abundance, whereas SULT1A3 was invariable across 0-70 years. The respective mean abundances of SULT1A1, SULT1B1, and SULT2A1 in neonatal samples was 24%, 19%, and 38% of the adult levels. Interestingly, unlike UDP-glucuronosyltransferases and cytochrome P450 enzymes, SULT1A1 and SULT2A1 showed the highest abundance during early childhood (1 to <6 years), which gradually decreased by approx. 40% in adolescents and adults. SULT1A3 and SULT1B1 abundances were significantly lower in African Americans compared with Caucasians. Multiple linear regression analysis further confirmed the association of SULT abundances by age, ethnicity, and genotype. To demonstrate clinical application of the characteristic SULT ontogeny profiles, we developed and validated a proteomics-informed physiologically based pharmacokinetic model of acetaminophen. The latter confirmed the higher fractional contribution of sulfation over glucuronidation in the metabolism of acetaminophen in children. The study thus highlights that the ontogeny-based age-dependent fractional contribution (f m ) of individual drug-metabolizing enzymes has better potential in prediction of drug-drug interactions and the effect of genetic polymorphisms in the pediatric population., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Aims: CYP2A6 is a genetically polymorphic enzyme resulting in differential substrate metabolism and health behaviours. Current phenotyping probes for CYP2A6 exhibit limitations related to procurement (deuterated cotinine), toxicity (coumarin), specificity (caffeine) and age-appropriate administration (nicotine, NIC). In vitro, CYP2A6 selectively forms 2-hydroxymetronidazole (2HM) from metronidazole (MTZ). The purpose of this study was to evaluate MTZ as a CYP2A6 phenotyping probe drug in healthy adults against the well-established method of measuring trans-3-hydroxycotinine (3HC)/cotinine (COT)., Methods: A randomized, cross-over, pharmacokinetic study was completed in 16 healthy, nonsmoking adults. Separated by a washout period of at least 2 weeks, MTZ 500 mg and NIC gum 2 mg were administered and plasma was sampled over 48 hours and 8 hours, respectively. Correlations of plasma metabolite/parent ratios (2HM/MTZ; 3HC/COT) were assessed by Pearson coefficient. CYP2A6 genotyping was conducted and incorporated as a variable of plasma ratio response., Results: Correlations between the plasma ratio 2HM/MTZ and 3HC/COT were ≥ 0.9 at multiple time points (P < 0.001), demonstrating a wide window during which 2HM/MTZ can be queried post-MTZ dose. CYP2A6 genotype had significant impacts on both MTZ and NIC phenotyping ratios with decreased activity predicted phenotypes demonstrating 2HM/MTZ ratios ≤58% and 3HC/COT ratios ≤56% compared with extensive activity predicted phenotypes at all time points examined in the study (P < 0.05). No adverse events were reported in the MTZ arm while 38% (n = 6) of participants reported mild adverse events in the NIC arm., Conclusions: Metronidazole via 2HM/MTZ performed well as a novel, safe phenotyping probe for CYP2A6 in healthy adults., (© 2019 The British Pharmacological Society.)

We have shown that an exchange dilution preparation method reduces the impact of surface adsorption of the target component in high-pressure gas mixtures used for underpinning measurements of amount-of-substance fraction. Gas mixtures are diluted in the same cylinder by releasing an aliquot of the parent mixture. Additional matrix gas is then added to the cylinder. This differs from conventional methods where dilutions are achieved by transferring the parent mixture to another cylinder, which then stores the final reference material. The benefit of this revolutionary approach is that losses due to adsorption to the walls of the cylinder and the valve are reduced as the parent mixture pacifies the surface with only a negligible relative change in amount-of-substance fraction. This development allows for preparation of gas reference materials with unprecedented uncertainties beyond the existing state of the art. It has significant implications for the preparation of high accuracy gas reference materials which underpin a broad range of requirements, particularly in atmospheric monitoring of carbon dioxide, where understanding the adsorption effects is the major obstacle to advancing the measurement science. It has the potential to remove the reliance on proprietary surface pretreatments as the method provides an in situ and consistent alternative.

The purity analysis of zero air is a significant contributor to the uncertainty in preparing reference materials of high impact greenhouse gases, limiting progress toward coherent and comparable measurements required to assess climate trends. We have produced a commutable synthetic zero air reference material with an oxygen, nitrogen, and argon matrix closely matching atmospheric composition. This is the critical step in preventing systematic biases from pressure broadening effects when using these reference materials to calibrate instruments based on optical spectroscopy. The amount fractions of carbon dioxide, methane, and carbon monoxide, which are present as minor impurities in the zero air reference material, have been accurately quantified using a novel dilution device that can generate gas mixtures of these components at trace amount fractions. These developments will have a significant impact on advancing the state of the art in high accuracy reference materials and for baseline calibration of spectroscopic instrumentation.

The ontogeny of hepatic uridine diphosphate-glucuronosyltransferases (UGTs) was investigated by determining their protein abundance in human liver microsomes isolated from 136 pediatric (0-18 years) and 35 adult (age >18 years) donors using liquid chromatography / tandem mass spectrometry (LC-MS/MS) proteomics. Microsomal protein abundances of UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 increased by ∼8, 55, 35, 33, 8, and 3-fold from neonates to adults, respectively. The estimated age at which 50% of the adult protein abundance is observed for these UGT isoforms was between 2.6-10.3 years. Measured in vitro activity was generally consistent with the protein data. UGT1A1 protein abundance was associated with multiple single nucleotide polymorphisms exhibiting noticeable ontogeny-genotype interplay. UGT2B15 rs1902023 (*2) was associated with decreased protein activity without any change in protein abundance. Taken together, these data are invaluable to facilitate the prediction of drug disposition in children using physiologically based pharmacokinetic modeling as demonstrated here for zidovudine and morphine., (© 2018, The American Society for Clinical Pharmacology and Therapeutics.)

Background: Children with obesity are more likely to suffer gastroesophageal reflux disease, requiring acid-suppression therapy with proton pump inhibitors (PPIs) and no guidelines regarding dosing., Objective: To prospectively evaluate lean-body-weight-based (LBW) dosing of the PPI pantoprazole for children with and without obesity., Methods: Methods: Sixty-two children (6-17 years) received a one-time oral dose of liquid pantoprazole (1.2 mg kg -1 LBW). Plasma pantoprazole concentrations were measured at 10 time points over 8 h and pharmacokinetic (PK) profiles generated using non-compartmental techniques, in order to compare PK parameters of interest between children with and without obesity, while accounting for CYP2C19 genotype., Results: Adjusted for milligram-per-kilogram total body weight (TBW) pantoprazole received, apparent drug clearance (CL/F) was reduced 50% in children with vs. without obesity (p=0.03). LBW-based dosing compensated for this reduction in CL/F (p = 0.15)., Conclusion: To achieve comparable systemic PPI exposures for children with and without obesity, we recommend using LBW, rather than TBW-based dosing for pantoprazole., (© 2018 World Obesity Federation.)

An ultra-performance liquid-chromatography mass-spectrometry (UPLC-MS/MS) method for simultaneous quantitation of metronidazole and 2-hydroxymetronidazole in human plasma was developed and validated. Metronidazole and 2-hydroxymetronidazole were extracted from a small volume of human plasma (10 μL) by hydrophilic lipophilic balanced solid phase extraction on 96-well μ-elution plates. Chromatographic separation of analytes was achieved on an Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) using gradient elution with a blend of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.25 mL/min. Mass spectrometric detection was achieved using multiple reaction monitoring (MRM) in positive-ion electrospray-ionization (ESI) mode. Ion transitions were optimized at m/z 171.85->127.9 for metronidazole and m/z 187.9->125.9 for 2-hydroxymetronidazole. The assay was linear for both analytes over the concentration range of 0.1-300 μM; intra- and inter-assay precisions and accuracies were <13%. Recoveries for metronidazole and 2-hydroxymetronidazole ranged from 88 to 99% and 78 to 86%, respectively. Matrix effects for metronidazole and 2-hydroxymetronidazole in plasma ranged from 102 to 105% and 99 to 106%, respectively. The method was successfully applied to determine metronidazole and 2-hydroxymetronidazole plasma concentrations in a pharmacokinetic study conducted in adults administered an oral dose of 500 mg metronidazole. Pharmacokinetic parameters were comparable to previously reported values. By design, this method is amenable to high sample throughput and has the potential to be automated., (Copyright © 2018 Elsevier B.V. All rights reserved.)

CYP2B6*6 and CYP2B6*18 are the most clinically important variants causing reduced CYP2B6 protein expression and activity. However, these variants do not account for all variability in CYP2B6 activity. Emerging evidence has shown that genetic variants in the 3'UTR may explain variable drug response by altering microRNA regulation. Five 3'UTR variants were associated with significantly altered efavirenz AUC 0-48 (8-OH-EFV/EFV) ratios in healthy human volunteers. The rs70950385 (AG>CA) variant, predicted to create a microRNA binding site for miR-1275, was associated with a 33% decreased CYP2B6 activity among normal metabolizers (AG/AG vs. CA/CA (P < 0.05)). In vitro luciferase assays were used to confirm that the CA on the variant allele created a microRNA binding site causing an 11.3% decrease in activity compared to the AG allele when treated with miR-1275 (P = 0.0035). Our results show that a 3'UTR variant contributes to variability in CYP2B6 activity., (© 2017 American Society for Clinical Pharmacology and Therapeutics.)

The major objective of this study was to investigate the association of genetic and nongenetic factors with variability in protein abundance and in vitro activity of the androgen-metabolizing enzyme UGT2B17 in human liver microsomes ( n = 455). UGT2B17 abundance was quantified by liquid chromatography-tandem mass spectrometry proteomics, and enzyme activity was determined by using testosterone and dihydrotestosterone as in vitro probe substrates. Genotyping or gene resequencing and mRNA expression were also evaluated. Multivariate analysis was used to test the association of UGT2B17 copy number variation, single nucleotide polymorphisms (SNPs), age, and sex with its mRNA expression, abundance, and activity. UGT2B17 gene copy number and SNPs (rs7436962, rs9996186, rs28374627, and rs4860305) were associated with gene expression, protein levels, and androgen glucuronidation rates in a gene dose-dependent manner. UGT2B17 protein (mean ± S.D. picomoles per milligram of microsomal protein) is sparsely expressed in children younger than 9 years (0.12 ± 0.24 years) but profoundly increases from age 9 years to adults (∼10-fold) with ∼2.6-fold greater abundance in males than in females (1.2 vs. 0.47). Association of androgen glucuronidation with UGT2B15 abundance was observed only in the low UGT2B17 expressers. These data can be used to predict variability in the metabolism of UGT2B17 substrates. Drug companies should include UGT2B17 in early phenotyping assays during drug discovery to avoid late clinical failures., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

We have shown that the amount fraction of carbon dioxide in a nitrogen or synthetic air matrix stored in cylinders increases as the pressure of the gas mixture reduces, while the amount fraction of methane remains unchanged. Our measurements show the initial amount fraction of carbon dioxide to be lower than the gravimetric value after preparation, which we attribute to the adsorption of a proportion of the molecules to active sites on the internal surface of the cylinder and the valve. As the mixture is consumed, the pressure in the cylinder reduces and the amount fraction of the component is observed to increase. The effect is less pronounced in the presence of water vapor. More dramatic effects have been observed for hydrogen chloride. These findings have significant implications for the preparation of high accuracy gaseous reference materials with unprecedented uncertainties which underpin a broad range of requirements, in particular atmospheric monitoring of high impact greenhouse gases.

Hepatic flavin-containing mono-oxygenase 3 (FMO3) metabolizes a broad array of nucleophilic heteroatom (e.g., N or S )-containing xenobiotics (e.g., amphetamine, sulindac, benzydamine, ranitidine, tamoxifen, nicotine, and ethionamide), as well as endogenous compounds (e.g., catecholamine and trimethylamine). To predict the effect of genetic and nongenetic factors on the hepatic metabolism of FMO3 substrates, we quantified FMO3 protein abundance in human liver microsomes (HLMs; n = 445) by liquid chromatography-tandem mass chromatography proteomics. Genotyping/gene resequencing, mRNA expression, and functional activity (with benzydamine as probe substrate) of FMO3 were also evaluated. FMO3 abundance increased 2.2-fold (13.0 ± 11.4 pmol/mg protein vs. 28.0 ± 11.8 pmol/mg protein) from neonates to adults. After 6 years of age, no significant difference in FMO3 abundance was found between children and adults. Female donors exhibited modestly higher mRNA fragments per kilobase per million reads values (139.9 ± 76.9 vs. 105.1 ± 73.1; P < 0.001) and protein FMO3 abundance (26.7 ± 12.0 pmol/mg protein vs. 24.1 ± 12.1 pmol/mg protein; P < 0.05) compared with males. Six single nucleotide polymorphisms (SNPs), including rs2064074, rs28363536, rs2266782 (E158K), rs909530 (N285N), rs2266780 (E308G), and rs909531, were associated with significantly decreased protein abundance. FMO3 abundance in individuals homozygous and heterozygous for haplotype 3 (H3), representing variant alleles for all these SNPs (except rs2066534), were 50.8% ( P < 0.001) and 79.5% ( P < 0.01), respectively, of those with the reference homozygous haplotype (H1, representing wild-type). In summary, FMO3 protein abundance is significantly associated with age, gender, and genotype. These data are important in predicting FMO3-mediated heteroatom-oxidation of xenobiotics and endogenous biomolecules in the human liver., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Hepatic cytosolic alcohol and aldehyde dehydrogenases (ADHs and ALDHs) catalyze the biotransformation of xenobiotics (e.g., cyclophosphamide and ethanol) and vitamin A. Because age-dependent hepatic abundance of these proteins is unknown, we quantified protein expression of ADHs and ALDH1A1 in a large cohort of pediatric and adult human livers by liquid chromatography coupled with tandem mass spectrometry proteomics. Purified proteins were used as calibrators. Two to three surrogate peptides per protein were quantified in trypsin digests of liver cytosolic samples and calibrator proteins under optimal conditions of reproducibility. Neonatal levels of ADH1A, ADH1B, ADH1C, and ALDH1A1 were 3-, 8-, 146-, and 3-fold lower than the adult levels, respectively. For all proteins, the abundance steeply increased during the first year of life, which mostly reached adult levels during early childhood (age between 1 and 6 years). Only for ADH1A protein abundance in adults (age > 18 year) was ∼40% lower relative to the early childhood group. Abundances of ADHs and ALDH1A1 were not associated with sex in samples with age > 1 year compared with males. Known single nucleotide polymorphisms had no effect on the protein levels of these proteins. Quantification of ADHs and ALDH1A1 protein levels could be useful in predicting disposition and response of substrates of these enzymes in younger children., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

The study of action selection in humans can present challenges of task design since our actions are usually defined by many degrees of freedom and therefore occupy a large action-space. While saccadic eye-movement offers a more constrained paradigm for investigating action selection, the study of reach-and-grasp in upper limbs has often been defined by more complex scenarios, not easily interpretable in terms of such selection. Here we present a novel motor behaviour task which addresses this by limiting the action space to a single degree of freedom in which subjects have to track (using a stylus) a vertical coloured target line displayed on a tablet computer, whilst ignoring a similarly oriented distractor line in a different colour. We ran this task with 55 subjects and showed that, in agreement with previous studies, the presence of the distractor generally increases the movement latency and directional error rate. Further, we used two distractor conditions according to whether the distractor's location changes asynchronously or synchronously with the location of the target. We found that the asynchronous distractor yielded poorer performance than its synchronous counterpart, with significantly higher movement latencies and higher error rates. We interpret these results in an action selection framework with two actions (move left or right) and competing 'action requests' offered by the target and distractor. As such, the results provide insights into action selection performance in humans and supply data for directly constraining future computational models therein.

The age-dependent absolute protein abundance of carboxylesterase (CES) 1 and CES2 in human liver was investigated and applied to predict infant pharmacokinetics (PK) of oseltamivir. The CES absolute protein abundance was determined by liquid chromatography-tandem mass spectrometry proteomics in human liver microsomal and cytosolic fractions prepared from tissue samples obtained from 136 pediatric donors and 35 adult donors. Two surrogate peptides per protein were selected for the quantification of CES1 and CES2 protein abundance. Purified CES1 and CES2 protein standards were used as calibrators, and the heavy labeled peptides were used as the internal standards. In hepatic microsomes, CES1 and CES2 abundance (in picomoles per milligram total protein) increased approximately 5-fold (315.2 vs. 1664.4) and approximately 3-fold (59.8 vs. 174.1) from neonates to adults, respectively. CES1 protein abundance in liver cytosol also showed age-dependent maturation. Oseltamivir carboxylase activity was correlated with protein abundance in pediatric and adult liver microsomes. The protein abundance data were then used to model in vivo PK of oseltamivir in infants using pediatric physiologically based PK modeling and incorporating the protein abundance-based ontogeny function into the existing pediatric Simcyp model. The predicted pediatric area under the curve, maximal plasma concentration, and time for maximal plasma concentration values were below 2.1-fold of the clinically observed values, respectively., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Although CYP2B6 catalyzes the biotransformation of many drugs used clinically for children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric, and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs), and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes such that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to postnatal samples and fetal HLMs did not appear to catalyze bupropion hydroxylation; however, fetal CYP2B6 protein levels were not significantly different from postnatal levels. Considerable interindividual variation in CYP2B6 mRNA expression, protein levels, and activity was observed in postnatal HLMs (mRNA, ∼40,000-fold; protein, ∼300-fold; activity, ∼600-fold). The extremely wide range of interindividual variability in CYP2B6 expression and activity was significantly associated with age (P < 0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 interindividual variability was not significantly associated with genetic variation in CYP2B6, nor was it associated with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Atomoxetine (ATX) is a second-line nonstimulant medication used to control symptoms of attention deficit hyperactivity disorder (ADHD). Inconsistent therapeutic efficacy has been reported with ATX, which may be related to variable CYP2D6-mediated drug clearance. We characterized ATX metabolism in a panel of human liver samples as a basis for a bottom-up PBPK model to aid in ATX exposure prediction and control. Km, Vmax, and Clint values in pooled human liver microsomes (HLMs) were 2.4 µM, 479 pmol/min/mg protein, and 202 µl/min/mg protein, respectively. Mean population values of kinetic parameters are not adequate to describe variability in a population, given that Km, Vmax, and Clint values from single-donor HLMs ranged from 0.93 to 79.2 µM, 20.0 to 1600 pmol/min/mg protein, and 0.3 to 936 µl/min/mg protein. All kinetic parameters were calculated from 4-hydroxyatomoxetine (4-OH-ATX) formation. CYP2E1 and CYP3A contributed to 4-OH-ATX formation in livers with CYP2D6 intermediate and poor metabolizer status. In HLMs with lower CYP2D6 activity levels, 2-hydroxymethylatomoxetine (2-CH2OH-ATX) formation became a more predominant pathway of metabolism, which appeared to be catalyzed by CYP2B6. ATX biotransformation at clinically relevant plasma concentrations was characterized in a panel of pediatric HLM (n = 116) samples by evaluating primary metabolites. Competing pathways of ATX metabolism [N-desmethylatomoxetine (NDM-ATX) and 2-CH2OH-ATX formation] had increasing importance in livers with lesser CYP2D6 activity, but, overall ATX clearance was still compromised. Modeling ATX exposure to individualize therapy would require comprehensive knowledge of factors that affect CYP2D6 activity as well as an understanding of competing pathways, particularly for individuals with lower CYP2D6 activity., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Trimethoprim (TMP) has been widely used since the 1960s, both alone and in combination with sulfamethoxazole. Unfortunately, information regarding the role that cytochrome P450 enzymes (P450s) play in the formation of TMP primary metabolites is scarce. Hence, we undertook in vitro studies to identify and more fully characterize the P450s that catalyze formation of six TMP primary metabolites: TMP 1-N-oxide (1-NO-TMP) and 3-N-oxide (3-NO-TMP), 3'- and 4'-desmethyl-TMP, a benzylic alcohol (Cα-OH-TMP), and an N-acetyl cysteine (NAC) adduct of TMP (Cα-NAC-TMP). Formation kinetics for each TMP metabolite in human liver microsomes (HLMs) were consistent with single-enzyme Michaelis-Menten kinetics, and Km values were markedly above (≥10-fold) the therapeutic concentrations of TMP (50 µM). The combined results from correlation studies between rates of metabolite formation and marker P450 activities in a panel of HLMs along with inhibition studies utilizing selective P450 inhibitors incubated with pooled HLMs suggested that 1-NO-TMP, Cα-NAC-TMP, and Cα-OH-TMP were predominantly formed by CYP3A4. In contrast, 3-NO-TMP was formed predominantly by CYP1A2 in HLMs and inhibited by α-naphthoflavone. 4'-Desmethyl-TMP, which is believed to be a reactive TMP metabolite precursor, was formed by several P450s, including CYP3A4, correlated with multiple P450 activities, but was inhibited primarily by ketoconazole (up to 50%), suggesting that CYP3A4 makes a major contribution to TMP 4'-demethylation. TMP 3'-demethylation was catalyzed by multiple P450s, including CYP2C9, correlated with CYP2C9 activity, and was inhibited by sulfaphenazole (up to 40%). Overall, CYP2C9 and CYP3A4 appear to be the most significant contributors to TMP primary metabolism., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Adult studies indicate a role for ghrelin in functional dyspepsia (FD) mediated through ghrelin's effect on gastric emptying (GE). This study examines the relationship between ghrelin, liquid GE, and pain in children with FD., Methods: Thirteen FD patients reporting symptoms consistent with post-prandial distress syndrome (PDS) and 17 healthy controls were enrolled. All participants received a liquid meal containing (13) C-sodium acetate. Pain severity, liquid GE utilizing exhaled (13) CO2 from the sodium acetate breath tests (ABT), plasma acyl ghrelin (AG), and des-acyl ghrelin concentrations were measured at specific intervals over 240 min following ingestion., Key Results: FD-PDS patients demonstrated lower mean baseline AG (14.8 ± 9.7 vs 27.2 ± 14.0 fmol/mL; p = 0.013), AG Cmax (24.6 ± 8.2 vs 40.5 ± 16.8 fmol/mL; p = 0.007), and AG flux (18.2 ± 7.8 vs 32.7 ± 17.3 fmol/mL; p = 0.015) than controls. The time to reach maximum exhaled (13) CO2 concentration (T max ) was longer in FD patients than controls (47.5 ± 18.5 vs 35.8 ± 11.8 min; p = 0.046). Significant relationships between ghrelin analyte ratios and ABT parameters were largely confined to control participants., Conclusions & Inferences: FD-PDS in children is associated with lower fasting and maximum AG concentrations, and dampened AG flux. These data suggest a possible role for altered ghrelin physiology in the pathogenesis of PDS., (© 2015 John Wiley & Sons Ltd.)

Members of the cytochrome P450 3A (CYP3A) subfamily of drug metabolizing enzymes exhibit developmental changes in expression in human liver characterized by a transition between CYP3A7 and CYP3A4 over the first few years of life. In contrast, the developmental expression of CYP3A5 is less well understood due to polymorphic expression of the enzyme in human tissues as a result of the prevalence of the CYP3A5*3 allele, which leads to alternative splicing. We further explored the expression of CYP3A5 and the impact of alternative splicing on the variability of CYP3A5 functional activity in a large bank of human prenatal liver samples (7 to 32 weeks of age postconception). The expression of normally spliced CYP3A5 mRNA in all human fetal liver samples varied 235-fold whereas CYP3A5 SV1 mRNA was only detected in fetal liver samples with at least one CYP3A5*3 allele. Formation of 1'-OH midazolam (MDZ) varied 79-fold, and the ratio of 1'-OH MDZ to 4-OH MDZ varied 8-fold and depended on the presence or absence of the CYP3A5*3 allele. Formation of 4-OH MDZ was significantly associated with 1'-OH MDZ (r(2) = 0.76, P < 0.0001) but varied (36-fold) independently of CYP3A5 genotype or expression. The substantial interindividual variability that remains even after stratification for CYP3A5 genotype suggests that factors such as environmental exposure and epigenetic alterations act in addition to genetic variation to contribute to the variability of CYP3A5 expression in human prenatal liver., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Aim: We sought to discover endogenous urinary biomarkers of human CYP2D6 activity., Patients & Methods: Healthy pediatric subjects (n = 189) were phenotyped using dextromethorphan and randomized for candidate biomarker selection and validation. Global urinary metabolomics was performed using liquid chromatography quadrupole time-of-flight mass spectrometry. Candidate biomarkers were tested in adults receiving fluoxetine, a CYP2D6 inhibitor., Results: A biomarker, M1 (m/z 444.3102) was correlated with CYP2D6 activity in both the pediatric training and validation sets. Poor metabolizers had undetectable levels of M1, whereas it was present in subjects with other phenotypes. In adult subjects, a 9.56-fold decrease in M1 abundance was observed during CYP2D6 inhibition., Conclusion: Identification and validation of M1 may provide a noninvasive means of CYP2D6 phenotyping.

The antimicrobial trimethoprim-sulfamethoxazole (TMP-SMX) is widely used for the treatment of skin and soft-tissue infections in the outpatient setting. Despite its therapeutic benefits, TMP-SMX has been associated with a number of adverse drug reactions, which have been primarily attributed to the formation of reactive metabolites from SMX. Recently, in vitro experiments have demonstrated that TMP may form reactive intermediates as well. However, evidence of TMP bioactivation in patients has not yet been demonstrated. In this study, we performed in vitro trapping experiments with N-acetyl-l-cysteine (NAC) to determine stable markers of reactive TMP intermediates, focusing on eight potential markers (NAC-TMP adducts), some of which were previously identified in vitro. We developed a specific and sensitive assay involving liquid chromatography followed by tandem mass spectrometry for measurement of these adducts in human liver microsomal samples and expanded the methodology toward the detection of these analytes in human urine. Urine samples from four patients receiving TMP-SMX treatment were analyzed, and all samples demonstrated the presence of six NAC-TMP adducts, which were also detected in vitro. These adducts are consistent with the formation of imino-quinone-methide and para-quinone-methide reactive intermediates in vivo. As a result, the TMP component of TMP-SMX should be considered as well when evaluating adverse drug reactions to TMP-SMX.

Aim: The aim of this study was to establish reference intervals in healthy children for two novel urinary biomarkers of acute kidney injury, kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL)., Materials & Methods: Urinary biomarkers were determined in samples from children in the UK (n = 120) and the USA (n = 171) using both Meso Scale Discovery (MSD) and Luminex-based analytical approaches., Results: 95% reference intervals for each biomarker in each cohort are presented and stratified by sex or ethnicity where necessary, and age-related variability is explored using quantile regression. We identified consistently higher NGAL concentrations in females than males (p < 0.0001), and lower KIM-1 concentrations in African-Americans than Caucasians (p = 0.02). KIM-1 demonstrated diurnal variation, with higher concentrations in the morning (p < 0.001)., Conclusion: This is the first report of reference intervals for KIM-1 and NGAL using two analytical methods in a healthy pediatric population in both UK and US-based populations.

Despite metronidazole's widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a "therapeutic concentration" of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo.

Young children are at increased risk for valproic acid (VPA) hepatotoxicity. Urinary organic acid profiles, as a surrogate of mitochondrial function, were obtained in children 1.9 to 17.3 years of age (n = 52) who were undergoing treatment with VPA for seizure disorders. Age-matched patients receiving treatment with carbamazepine (CBZ; n = 50) and healthy children not undergoing treatment (n = 22) served as controls. Age-related changes in organic acid profiles were observed in all three groups. Although the untreated and CBZ control groups were indistinguishable from each other with respect to the principal-component analysis (PCA) score plots of the subjects, a distinct boundary was apparent between the VPA and each of the control groups. Interindividual variability was observed in the VPA-induced alterations in endogenous pathways corresponding to branched-chain amino acid metabolism and oxidative stress. The data suggest that more detailed metabolomic analysis may provide novel insights into biological mechanisms and predictive biomarkers for children at highest risk for serious toxicity.

Background: CYP2D6 31 (4042G>A, R(440)H) is an allelic variant of the highly polymorphic cytochrome P450 2D6 enzyme that has been associated with reduced functional activity. The US Food and Drug Administration (FDA)-cleared AmpliChip CYP450 test detects the 4042G>A single nucleotide polymorphism (SNP) but an allele assignment could not be made in two Spanish and two Puerto Rican individuals heterozygous for 4042G>A, resulting in no-calls. We aimed to resolve the CYP2D6 31 no-calls, determine the allele haplotype, and corroborate that CYP2D6 31 is associated with a poor metabolizer phenotype., Methods: CYP2D6 genotyping was carried out using the AmpliChip CYP450 test and long-range polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) platforms. Allele haplotype was determined by cloning and sequence analysis. Allele frequencies were determined in five population samples., Results: A 6.6-kb long-range PCR product comprising the entire CYP2D6 gene and flanking regions was sequenced to determine the CYP2D6 31 haplotype. Identical sequences were obtained from both Puerto Ricans selected for sequence analysis. One Spanish individual with a CYP2D6 4/31 genotype was phenotyped as a poor metabolizer with the CYP2D6 probe drug dextromethorphan (urinary ratio DM/DX=0.71). The frequency of CYP2D6 31 was determined in 176 Spanish (0.57%), 50 Puerto Rican (2.0%), and 150 Hispanic (0.33%) people. CYP2D6 31 was absent in 237 North American Caucasians and 154 African Americans., Conclusions: CYP2D6 31 was associated with poor metabolism of dextromethorphan in vivo, which is consistent with a previous report classifying this allelic variant as nonfunctional. The discovery of CYP2D6 31 in Spanish people only (or of Spanish ancestry) suggests that it may contribute to CYP2D6 variability in individuals of Spanish ancestry.

The (13)C-acetate breath test represents a potential alternative to conventional scintigraphy to measure liquid gastric emptying (GE). The purpose of this study was to compare the (13)C-acetate breath test to gastric scintigraphy in children with functional dyspepsia. Simultaneous assessment of GE was performed in 28 children (9-17 years of age) using a liquid test meal that was double labeled with (13)C-acetate and (99 m)Technetium. (13)CO(2) versus time profiles were fit using traditional pharmacokinetic analyses. For each subject, GE half-life [Formula: see text] determined by scintigraphy was plotted against parameters determined from the (13)C-acetate breath test. Linear regression was used to explore the associations between the tests. Complete (13)CO(2) versus time profiles were available for 25 subjects. There was no association between the scintigraphy GE T½ and the(13)CO(2) half-exhalation time. However, significant associations were observed between the gastric half-emptying time as determined by scintigraphy and two of the breath test parameters: the enrichment of (13)CO(2) present in breath samples at 60 min (DOB(60)) (r = -0.52, p = 0.01) and the area under the curve from 0 to 60 min (AUC(0-60 min)) (r = -0.54; p < 0.01). The (13)C-acetate breath test has the potential to serve as a rapid, technically simple and inexpensive means to assess liquid GE in children with functional dyspepsia and possibly serve as a pharmacodynamic surrogate in studies of prokinetic drugs in children.

Carboxylesterases hydrolyze chemicals containing such functional groups as a carboxylic acid ester, amide and thioester. The liver contains the highest carboxylesterase activity and expresses two major carboxylesterases: HCE1 and HCE2. In this study, we analyzed 104 individual liver samples for the expression patterns of both carboxylesterases. These samples were divided into three age groups: adults (>or= 18 years of age), children (0 days-10 years) and fetuses (82-224 gestation days). In general, the adult group expressed significantly higher HCE1 and HCE2 than the child group, which expressed significantly higher than the fetal group. The age-related expression was confirmed by RT-qPCR and Western immunoblotting. To determine whether the expression patterns reflected the hydrolytic activity, liver microsomes were pooled from each group and tested for the hydrolysis of drugs such as oseltamivir and insecticides such as deltamethrin. Consistent with the expression patterns, adult microsomes were approximately 4 times as active as child microsomes and 10 times as active as fetal microsomes in hydrolyzing these chemicals. Within the same age group, particularly in the fetal and child groups, a large inter-individual variability was detected in mRNA (430-fold), protein (100-fold) and hydrolytic activity (127-fold). Carboxylesterases are recognized to play critical roles in drug metabolism and insecticide detoxication. The findings on the large variability among different age groups or even within the same age group have important pharmacological and toxicological implications, particularly in relation to pharmacokinetic alterations of ester drugs in children and vulnerability of fetuses and children to pyrethroid insecticides.

Background: Recombinant human growth hormone (r-hGH) is increasingly being used in children. Although growth hormone (GH) may alter the clearance of concomitantly administered medications, its effects on individual drug-metabolizing enzymes in children have not been characterized., Objective: The goal of this study was to assess the activities of cytochrome P450 (CYP) 1A2, N-acetyltransferase 2, xanthine oxidase, and CYP2D6 in children with isolated idiopathic GH deficiency before and 3 and 6 months after initiation of r-hGH treatment., Methods: This 6-month, prospective, longitudinal, open-label phenotyping study was conducted at 4 academic tertiary care centers within the Pediatric Pharmacology Research Unit network. Prepubertal or early pubertal children (4-14 years) with short stature and isolated idiopathic GH deficiency were enrolled. Patients were given 4 ounces of a cola beverage and 0.5 mg/kg of dextromethorphan (DM) before and 3 and 6 months after initiation of r-hGH treatment. Urine was collected for 8 hours after probe substrate administration, and enzyme activity was assessed using validatedcaffeine/metaboliteandDM/metabolitemolar ratios. Patients with a DM/dextrorphan molar ratio > or =0.3 were classified as poor metabolizers, and those with a ratio <0.3 were classified as extensive metabolizers. Anthropometric and biochemical responses were assessed at each visit. Blood was also obtained for determination of serum insulinlike growth factor-1 (IGF-1) levels and CYP2D6 genotype., Results: Fourteen patients (mean [SD] age, 11.5 [2.6] years [age range, 4.5-14.6 years]; 11 males, 3 females; 100% white; median height and weight, 131.8 cm and 29.2 kg, respectively) completed the 3 study visits. However, data from 2 patients were excluded from analysis due to procedural violations. In all patients, growth velocity and serum IGF-1 concentrations were significantly higher (P < 0.001) after r-hGH treatment (mean doses, 0.32 and 0.33 mg/kg per week at 3 and 6 months, respectively). However, molar ratio values did not significantly change after initiation of r-hGH., Conclusions: In this study population of children with isolated idiopathic GH deficiency, no significant differences in caffeine/metabolite and DM/metabolite molar ratios were observed after initiation of r-hGH treatment.

Conversion of the carbamazepine metabolite 3-hydroxycarbamazepine (3-OHCBZ) to the catechol 2,3-dihydroxycarbamazepine (2,3-diOHCBZ) followed by subsequent oxidation to a reactive o-quinone species has been proposed as a possible bioactivation pathway in the pathogenesis of carbamazepine-induced hypersensitivity. Initial in vitro phenotyping studies implicated CYP3A4 as a primary catalyst of 2,3-diOHCBZ formation: 2-hydroxylation of 3-OHCBZ correlated significantly (r(2) > or = 0.929, P < 0.001) with CYP3A4/5 activities in a panel of human liver microsomes (n = 14) and was markedly impaired by CYP3A inhibitors (>80%) but not by inhibitors of other cytochrome P450 enzymes (< or = 20%). However, in the presence of troleandomycin, the rate of 2,3-diOHCBZ formation correlated significantly with CYP2C19 activity (r(2) = 0.893, P < 0.001) in the panel of human liver microsomes. Studies with a panel of cDNA-expressed enzymes revealed that CYP2C19 and CYP3A4 were high (S50 = 30 microM) and low (S50 = 203 microM) affinity enzymes, respectively, for 2,3-diOHCBZ formation and suggested that CYP3A4, but not CYP2C19, might be inactivated by a metabolite formed from 3-OHCBZ. Subsequent experiments demonstrated that preincubation of 3-OHCBZ with human liver microsomes or recombinant CYP3A4 led to decreased CYP3A4 activity, which was both preincubation time- and concentration-dependent, but not inhibited by inclusion of glutathione or N-acetylcysteine. CYP3A4, CYP3A5, CYP3A7, CYP2C19, and CYP1A2 converted [14C]3-OHCBZ into protein-reactive metabolites, but CYP3A4 was the most catalytically active enzyme. The results of this study suggest that CYP3A4-dependent secondary oxidation of 3-OHCBZ represents a potential carbamazepine bioactivation pathway via formation of reactive metabolites capable of inactivating CYP3A4, potentially generating a neoantigen that may play a role in the etiology of carbamazepine-induced idiosyncratic toxicity.

Carbamazepine (CBZ) is a widely prescribed anticonvulsant whose use is often associated with idiosyncratic hypersensitivity. Sera of CBZ-hypersensitive patients often contain anti-CYP3A antibodies, including those to a CYP3A23 K-helix peptide that is also modified during peroxidative CYP3A4 heme-fragmentation. We explored the possibility that cytochromes P450 (P450s) such as CYP3A4 bioactivate CBZ to reactive metabolite(s) that irreversibly modify the P450 protein. Such CBZ-P450 adducts, if stable in vivo, could engender corresponding serum P450 autoantibodies. Incubation with CBZ not only failed to inactivate functionally reconstituted, purified recombinant CYP3A4 or CYP3A4 Supersomes in a time-dependent manner, but the inclusion of CBZ (0-1 mM) also afforded a concentration-dependent protection to CYP3A4 from inactivation by NADPH-induced oxidative uncoupling. Incubation of CYP3A4 Supersomes with (3)H-CBZ resulted in its irreversible binding to CYP3A4 protein with a stoichiometry of 1.58 +/- 0.15 pmol (3)H-CBZ bound/pmol CYP3A4. Inclusion of glutathione (1.5 mM) in the incubation reduced this level to 1.09. Similar binding (1.0 +/- 0.4 pmol (3)H-CBZ bound/pmol CYP3A4) was observed after (3)H-CBZ incubation with functionally reconstituted, purified recombinant CYP3A4(His)(6). The CBZ-modified CYP3A4 retained its functional activity albeit at a reduced level, but its testosterone 6beta-hydroxylase kinetics were altered from sigmoidal (a characteristic profile of substrate cooperativity) to near-hyperbolic (Michaelis-Menten) type, suggesting that CBZ may have modified CYP3A4 within its active site.

Inferring CYP2D6 phenotype from genotype is increasingly challenging, considering the growing number of alleles and their range of activity. This complexity poses a challenge in translational research where genotyping is being considered as a tool to personalize drug therapy. To simplify genotype interpretation and improve phenotype prediction, we evaluated the utility of an "activity score" (AS) system. Over 25 CYP2D6 allelic variants were genotyped in 672 subjects of primarily Caucasian and African-American heritage. The ability of genotype and AS to accurately predict phenotype using the CYP2D6 probe substrate dextromethorphan was evaluated using linear regression and clustering methods. Phenotype prediction, given as a probability for each AS group, was most accurate if ethnicity was considered; among subjects with genotypes containing a CYP2D6*2 allele, CYP2D6 activity was significantly slower in African Americans compared to Caucasians. The AS tool warrants further prospective evaluation for CYP2D6 substrates and in additional ethnic populations.

A 5-year-old African-American girl presented with a CYP2D6*4xN/*10 genotype that was discordant with her poor metabolizer phenotype determined with the probe drug dextromethorphan. Both phenotype and genotype were confirmed in repeat assessments, suggesting that the CYP2D6*10 allele carried a novel debilitating sequence variation(s). The rationale for this study was to resolve the discordance and to describe the novel non-functional allelic variant of CYP2D6 and its frequency in populations of different ethnic backgrounds.

The exponential increase in the number of drugs used to treat infant and childhood illnesses necessitates an understanding of the ontogeny of drug biotransformation for the development of safe and effective therapies. Healthy infants received an oral dose (0.3 mg/kg) of dextromethorphan (DM) at 0.5, 1, 2, 4, 6, and 12 months of age. DM and its major metabolites were measured in urine. CYP2D6 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism. Genotyping data indicated a strong correlation between CYP2D6 genotype and DM O-demethylation (beta=-0.638; 95% CI: -0.745, -0.532; P<0.001). CYP2D6 activity was detectable and concordant with genotype by 2 weeks of age, showed no relationship with gestational age, and did not change with post natal age up to 1 year. In contrast, DM N-demethylation developed significantly more slowly over the first year of life. Genotype and the temporal acquisition of drug biotransformation are critical determinants of a drug response in infants.

Orally administered caffeine and dextromethorphan (DM) were used as pharmacologic probes to determine the effect of infant diet on acquisition of cytochrome P-450 (CYP) enzyme activity during the first 6 mo of life. The caffeine elimination rate constant (ke) was determined from serum, and concentrations of caffeine, DM, and their respective metabolites were measured in urine by high-performance liquid chromatography (HPLC). Caffeine ke was low at 2 wk and displayed a significant positive linear correlation with age (p < 0.001); increasing faster in formula-fed than in breast-fed infants (p < 0.001). This occurred concomitantly with a significant increase in urinary 1,7-dimethylxanthine (17X) and 1-methylxanthine (1X) (p < 0.001), suggesting faster acquisition of CYP1A2 activity in formula-fed infants. The urinary molar ratio of (17X + 1X)/caffeine and age strongly predicted caffeine ke (r2 = 0.65; p < 0.001) irrespective of feeding type. CYP3A4 activity, assessed as the molar ratio of 3-hydroxymorphinan/dextrorphan showed a similar marked increase with postnatal age (p < 0.001) that was also greater in formula-fed than in breast-fed infants. Formula feeding appears to accelerate maturation of caffeine and DM metabolism by increasing the activity of CYP1A2 and CYP3A4, respectively. Dietary modification of CYP activity may modulate drug biotransformation and thus alter systemic exposure to xenobiotics from a very early age.

CYP2J2 metabolizes arachidonic acid to 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids (EETs), which play a critical role in the regulation of renal, pulmonary, cardiac, and vascular function. However, the contribution of CYP2J2 to EET formation in the liver remains poorly characterized. Likewise, information is sparse regarding the extent and variability of CYP2J2 expression during human development. This investigation was undertaken to characterize the variability of CYP2J2 expression in fetal liver, heart, kidney, lung, intestine, and brain and in postnatal liver samples. CYP2J2 mRNA expression was measured using quantitative polymerase chain reaction, and immunoreactive CYP2J2 was examined using two anti-CYP2J2 antibodies. CYP2J2 mRNA was ubiquitously expressed in pre- and postnatal samples. Fetal hepatic mRNA expression varied 127-fold (1351 +/- 717 transcripts/ng total RNA), but this variation was reduced to 8-fold after exclusion of four samples with extremely low levels of mRNA. Amounts of immunoreactive protein also varied substantially among samples without an apparent relationship with transcript number or genotype. Western blot analysis revealed a different protein pattern between prenatal and postnatal liver samples. DNA resequencing of selected subjects identified a single novel single-nucleotide polymorphism (CYP2J2*10), which was found in only one subject and therefore did not explain the large variability in CYP2J2 protein content. In vitro expression suggests that the protein product of CYP2J2*10 confers reduced enzymatic activity. Aberrant splicing produces three minor transcripts, which were present in all samples tested. Due to premature termination codons, none encodes functional protein. The mechanisms leading to variable amounts of immunoreactive protein and distinct pre- and postnatal CYP2J2 protein patterns warrant further investigation.

Fluticasone propionate (FTP) is a synthetic trifluorinated glucocorticoid with potent anti-inflammatory action that is commonly used in patients with asthma. After oral or intranasal administration, FTP undergoes rapid hepatic biotransformation; the principal metabolite formed is a 17beta-carboxylic acid derivative (M1). M1 formation has been attributed largely to cytochrome P450 3A4 (CYP3A4); however, there are no published data that confirm this assertion. Hence, in vitro studies were conducted to determine the role that human P450s play in the metabolism of FTP. Consistent with in vivo data, human liver microsomes catalyzed the formation of a single metabolite (M1) at substrate concentrations 0.95) with CYP3A4/5 activities in a panel of human liver microsomes (n = 14) and was markedly impaired by the CYP3A inhibitor ketoconazole (>94%) but not by inhibitors of other P450 enzymes (

Conversion of the carbamazepine metabolite, 2-hydroxycarbamazepine, to the potentially reactive species, carbamazepine iminoquinone (CBZ-IQ), has been proposed as a possible bioactivation pathway in the pathogenesis of carbamazepine-induced hypersensitivity. Generation of CBZ-IQ has been proposed to proceed through the intermediate, 2-hydroxyiminostilbene (2-OHIS); however, data suggested that 2-hydroxycarbamazepine is oxidized by cytochromes P450 (P450s) directly to CBZ-IQ, followed by NADPH-mediated reduction to 2-OHIS. In vitro studies were conducted to identify the P450s responsible for converting 2-hydroxycarbamazepine to 2-OHIS and to determine functional consequences of this bioactivation pathway. Formation of 2-OHIS in human liver microsomes (HLMs) was consistent with monophasic, Michaelis-Menten kinetics. The sample-to-sample variation in the rate of 2-OHIS formation correlated significantly (r2 > or = 0.706) with CYP3A4/5 and CYP2B6 activities in a panel of HLMs (n = 10). Studies with a panel of cDNA-expressed enzymes revealed that CYP3A4 preferentially catalyzed 2-OHIS formation; CYP3A4 formed 2-OHIS at a rate >10 times that of other enzymes capable of forming 2-OHIS (CYP1A1, CYP2C19, and CYP3A7). Inhibitors of CYP3A enzymes markedly impaired 2-OHIS formation in HLMs, whereas inhibitors of other P450s resulted in < or = 20% inhibition. Although CYP3A4 was primarily responsible for converting 2-hydroxycarbamazepine to 2-OHIS, neither 2-hydroxycarbamazepine, 2-OHIS, nor CBZ-IQ caused time-dependent inactivation of CYP3A activity. No thiol adducts were formed directly from 2-hydroxycarbamazepine. However, glutathione- and N-acetylcysteine-conjugates were formed with 2-OHIS or CBZ-IQ as substrates. Thus, CYP3A4-dependent secondary oxidation of 2-hydroxycarbamazepine represents a potential carbamazepine bioactivation pathway leading to the formation of thiol-reactive metabolites, intermediates that may play a role in the etiology of idiosyncratic toxicity attributed to carbamazepine.