RESUMEN
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 (r2 = 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.
RESUMEN
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 R2 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.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450 , Inhibidores de la Bomba de Protones , Niño , Humanos , Adolescente , Inhibidores de la Bomba de Protones/farmacocinética , Haplotipos , Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP2C19/genética , GenotipoRESUMEN
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).
Asunto(s)
Hígado , Microsomas Hepáticos , Adulto , Humanos , Niño , Microsomas Hepáticos/metabolismo , Hígado/metabolismo , Proteínas/metabolismo , Tasa de Depuración Metabólica , Citosol/metabolismo , Modelos BiológicosRESUMEN
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.
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Citocromo P-450 CYP2D6 , Adolescente , Niño , Humanos , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano , Dextrorfano , Estudios Longitudinales , FenotipoRESUMEN
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 CO2 (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 (Ti ) 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 DOBT30 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.
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Pruebas Respiratorias , Inhibidores de la Bomba de Protones , 2-Piridinilmetilsulfinilbencimidazoles/farmacocinética , Adulto , Pruebas Respiratorias/métodos , Niño , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Genotipo , Humanos , Pantoprazol , Inhibidores de la Bomba de Protones/farmacocinéticaRESUMEN
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.
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Citocromo P-450 CYP2D6/genética , Polimorfismo de Nucleótido Simple/genética , Adolescente , Alelos , Clorhidrato de Atomoxetina/uso terapéutico , Niño , Dextrometorfano/uso terapéutico , Genotipo , Haplotipos/genética , Humanos , Pruebas de Farmacogenómica/métodos , FenotipoRESUMEN
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 µM). Naltrexone biotransformation was determined by ultraperformance mass spectrometry quantification of the primary metabolite, 6-beta-naltrexol (6ßN), and 6ßN 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 6ßN 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 6ßN 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.
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Naltrexona , Antagonistas de Narcóticos , Adolescente , Adulto , Anciano , Biotransformación , Niño , Preescolar , Citosol/metabolismo , Femenino , Humanos , Lactante , Recién Nacido , Tasa de Depuración Metabólica , Persona de Mediana Edad , Naltrexona/farmacocinética , Antagonistas de Narcóticos/farmacocinética , Adulto JovenRESUMEN
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.
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Microsomas Hepáticos/metabolismo , Farmacocinética , Proteínas/metabolismo , Adolescente , Adulto , Envejecimiento/metabolismo , Niño , Preescolar , Sistema Enzimático del Citocromo P-450 , Femenino , Humanos , Lactante , Masculino , Modelos Biológicos , NADPH-Ferrihemoproteína Reductasa , Proteómica , Adulto JovenRESUMEN
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.
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Amoxicilina/sangre , Amoxicilina/farmacocinética , Antibacterianos/sangre , Antibacterianos/farmacocinética , Sepsis/tratamiento farmacológico , Administración Oral , Amoxicilina/administración & dosificación , Antibacterianos/administración & dosificación , Quimioterapia Combinada , Femenino , Gentamicinas/administración & dosificación , Humanos , Lactante , Recién Nacido , Inyecciones Intramusculares , Masculino , Pruebas de Sensibilidad Microbiana , Streptococcus pneumoniae/efectos de los fármacos , Factores de TiempoRESUMEN
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 (fm) of individual drug-metabolizing enzymes has better potential in prediction of drug-drug interactions and the effect of genetic polymorphisms in the pediatric population.
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Acetaminofén/farmacocinética , Variación Biológica Poblacional/fisiología , Citosol/metabolismo , Hígado/metabolismo , Sulfotransferasas/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Área Bajo la Curva , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Interacciones Farmacológicas/fisiología , Femenino , Humanos , Lactante , Recién Nacido , Hígado/citología , Masculino , Persona de Mediana Edad , Modelos Biológicos , Proteómica , Factores Sexuales , Sulfatos/metabolismo , Sulfotransferasas/análisis , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
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.
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Citocromo P-450 CYP2A6/genética , Metronidazol/farmacocinética , Nicotina/farmacocinética , Pruebas de Farmacogenómica/métodos , Adolescente , Adulto , Estudios Cruzados , Citocromo P-450 CYP2A6/metabolismo , Femenino , Voluntarios Sanos , Humanos , Masculino , Metronidazol/administración & dosificación , Persona de Mediana Edad , Nicotina/administración & dosificación , Chicles de Nicotina , Polimorfismo Genético , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
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 â¼8, 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.
Asunto(s)
Genotipo , Glucuronosiltransferasa/metabolismo , Microsomas Hepáticos/enzimología , Adolescente , Factores de Edad , Analgésicos Opioides/farmacología , Antimetabolitos/farmacología , Niño , Preescolar , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Lactante , Recién Nacido , Microsomas Hepáticos/efectos de los fármacos , Morfina/farmacología , Adulto Joven , Zidovudina/farmacologíaRESUMEN
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⯵L) 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⯵m, 2.1â¯×â¯100â¯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 0.25â¯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⯵M; 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 500â¯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.
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Cromatografía Líquida de Alta Presión/métodos , Metronidazol/análogos & derivados , Metronidazol/sangre , Metronidazol/farmacocinética , Espectrometría de Masas en Tándem/métodos , Adolescente , Adulto , Estabilidad de Medicamentos , Humanos , Límite de Detección , Modelos Lineales , Metronidazol/química , Persona de Mediana Edad , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
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 (â¼10-fold) with â¼2.6-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.
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Andrógenos/metabolismo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Hígado/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Variaciones en el Número de Copia de ADN/genética , Femenino , Genotipo , Humanos , Inactivación Metabólica/genética , Lactante , Recién Nacido , Masculino , Microsomas Hepáticos/metabolismo , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Testosterona/metabolismo , Adulto JovenRESUMEN
CYP2B6*6 and CYP2B6*18 are the most clinically important variants causing reduced CYP2B6 protein expression and activity. However, these variants do not account for all variability in CYP2B6 activity. Emerging evidence has shown that genetic variants in the 3'UTR may explain variable drug response by altering microRNA regulation. Five 3'UTR variants were associated with significantly altered efavirenz AUC0-48 (8-OH-EFV/EFV) ratios in healthy human volunteers. The rs70950385 (AG>CA) variant, predicted to create a microRNA binding site for miR-1275, was associated with a 33% decreased CYP2B6 activity among normal metabolizers (AG/AG vs. CA/CA (P < 0.05)). In vitro luciferase assays were used to confirm that the CA on the variant allele created a microRNA binding site causing an 11.3% decrease in activity compared to the AG allele when treated with miR-1275 (P = 0.0035). Our results show that a 3'UTR variant contributes to variability in CYP2B6 activity.
Asunto(s)
Regiones no Traducidas 3'/genética , Benzoxazinas/farmacocinética , Inductores del Citocromo P-450 CYP2B6/farmacocinética , Citocromo P-450 CYP2B6/genética , MicroARNs/metabolismo , Adolescente , Adulto , Alquinos , Alelos , Área Bajo la Curva , Benzoxazinas/metabolismo , Sitios de Unión , Simulación por Computador , Ciclopropanos , Citocromo P-450 CYP2B6/metabolismo , Inductores del Citocromo P-450 CYP2B6/metabolismo , Femenino , Voluntarios Sanos , Células Hep G2 , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Variantes Farmacogenómicas , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
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.
Asunto(s)
Hígado/enzimología , Oxigenasas/genética , Oxigenasas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Envejecimiento/metabolismo , Niño , Preescolar , Estudios de Cohortes , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Caracteres Sexuales , Adulto JovenRESUMEN
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 â¼40% 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.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa/metabolismo , Hígado/enzimología , Adolescente , Factores de Edad , Niño , Preescolar , Humanos , Lactante , Recién Nacido , Isoenzimas , Adulto JovenAsunto(s)
Antiinfecciosos/metabolismo , Antiinfecciosos/farmacocinética , Sistema Enzimático del Citocromo P-450/genética , Variación Genética , Metronidazol/metabolismo , Metronidazol/farmacocinética , Estudios de Cohortes , Sistema Enzimático del Citocromo P-450/metabolismo , Femenino , Humanos , Recién Nacido , MasculinoRESUMEN
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.
Asunto(s)
Envejecimiento/metabolismo , Carboxilesterasa/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Hígado/enzimología , Modelos Biológicos , Oseltamivir/farmacocinética , Adulto , Cromatografía Liquida , Citosol/efectos de los fármacos , Citosol/enzimología , Humanos , Técnicas In Vitro , Lactante , Hígado/efectos de los fármacos , Hígado/crecimiento & desarrollo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Oseltamivir/sangre , Proteómica , Espectrometría de Masas en TándemRESUMEN
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.