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A reliable translation of in vitro and preclinical data on drug absorption, distribution, metabolism, and excretion (ADME) to humans is important for safe and effective drug development. Precision medicine that is expected to provide the right clinical dose for the right patient at the right time requires a comprehensive understanding of population factors affecting drug disposition and response. Characterization of drug-metabolizing enzymes and transporters for the protein abundance and their interindividual as well as differential tissue and cross-species variabilities is important for translational ADME and precision medicine. This review first provides a brief overview of quantitative proteomics principles including liquid chromatography-tandem mass spectrometry tools, data acquisition approaches, proteomics sample preparation techniques, and quality controls for ensuring rigor and reproducibility in protein quantification data. Then, potential applications of quantitative proteomics in the translation of in vitro and preclinical data as well as prediction of interindividual variability are discussed in detail with tabulated examples. The applications of quantitative proteomics data in physiologically based pharmacokinetic modeling for ADME prediction are discussed with representative case examples. Finally, various considerations for reliable quantitative proteomics analysis for translational ADME and precision medicine and the future directions are discussed. SIGNIFICANCE STATEMENT: Quantitative proteomics analysis of drug-metabolizing enzymes and transporters in humans and preclinical species provides key physiological information that assists in the translation of in vitro and preclinical data to humans. This review provides the principles and applications of quantitative proteomics in characterizing in vitro, ex vivo, and preclinical models for translational research and interindividual variability prediction. Integration of these data into physiologically based pharmacokinetic modeling is proving to be critical for safe, effective, timely, and cost-effective drug development.
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Medicina de Precisión , Proteómica , Humanos , Proteínas de Transporte de Membrana/metabolismo , Proteómica/métodos , Reproducibilidad de los Resultados , Investigación Biomédica TraslacionalRESUMEN
Tubular secretion is a primary mechanism along with glomerular filtration for renal elimination of drugs and toxicants into urine. Organic cation transporters (OCTs) and multidrug and toxic extrusion (MATE) transporters facilitate the active secretion of cationic substrates, including drugs such as metformin and endogenous cations. We hypothesized that administration of cimetidine, an Oct/Mate inhibitor, will result in increased plasma levels and decreased renal clearance of metformin and endogenous Oct/Mate substrates in rats. A paired rat pharmacokinetic study was carried out in which metformin (5 mg/kg, intravenous) was administered as an exogenous substrate of Oct/Mate transporters to six Sprague-Dawley rats with and without cimetidine (100 mg/kg, intraperitoneal). When co-administered with cimetidine, metformin area under the curve increased significantly by 3.2-fold, and its renal clearance reduced significantly by 73%. Untargeted metabolomics was performed to investigate the effect of cimetidine on endogenous metabolome in the blood and urine samples. Over 8,000 features (metabolites) were detected in the blood, which were shortlisted using optimized criteria, i.e., a significant increase (P value < 0.05) in metabolite peak intensity in the cimetidine-treated group, reproducible retention time, and quality of chromatogram peak. The metabolite hits were classified into three groups that can potentially distinguish inhibition of i) extra-renal uptake transport or catabolism, ii) renal Octs, and iii) renal efflux transporters or metabolite formation. The metabolomics approach identified novel putative endogenous substrates of cationic transporters that could be tested as potential biomarkers to predict Oct/Mate transporter mediated drug-drug interactions in the preclinical stages. SIGNIFICANCE STATEMENT: Endogenous substrates of renal transporters in animal models could be used as potential biomarkers to predict renal drug-drug interactions in early drug development. Here we demonstrated that cimetidine, an inhibitor of organic cation transporters (Oct/Mate), could alter the pharmacokinetics of metformin and endogenous cationic substrates in rats. Several putative endogenous metabolites of Oct/Mate transporters were identified using metabolomics approach, which could be tested as potential transporter biomarkers to predict renal drug-drug interaction of Oct/Mate substrates.
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Metformina , Ratas , Animales , Metformina/farmacocinética , Cimetidina/farmacología , Proteínas de Transporte de Catión Orgánico/metabolismo , Ratas Sprague-Dawley , Interacciones Farmacológicas , Preparaciones Farmacéuticas/metabolismo , Riñón/metabolismo , Biomarcadores/metabolismo , Cationes/metabolismoRESUMEN
Over the past 20 years, quantitative proteomics has contributed a wealth of protein expression data, which are currently used for a variety of systems pharmacology applications, as a complement or a surrogate for activity of the corresponding proteins. A symposium at the 25th North American ISSX meeting, in Boston, in September 2023, was held to explore current and emerging applications of quantitative proteomics in translational pharmacology and strategies for improved integration into model-informed drug development based on practical experience of each of the presenters. A summary of the talks and discussions is presented in this perspective alongside future outlooks that were outlined for future meetings. Significance Statement This perspective explores current and emerging applications of quantitative proteomics in translational pharmacology and precision medicine, and outlines outlooks for improved integration into model-informed drug development.
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Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are critical for scaling in vitro and animal data to humans for accurate prediction and interpretation of drug clearance and toxicity. Targeted DMET proteomics which relies on synthetic stable isotope-labeled surrogate peptides as calibrators, is routinely used for the quantification of selected proteins; however, the technique is limited to the quantification of a small number of proteins. Although the global proteomics-based total protein approach (TPA) is emerging as a better alternative for large-scale protein quantification, the conventional TPA doesn't consider differential sequence coverage by identifying unique peptides across proteins. Here, we optimized the TPA approach by correcting protein abundance data by the sequence coverage (SC-TPA), which was applied to quantify 54 DMETs for characterization of i) differential tissue DMET abundance in the human liver, kidney, and intestine, and ii) interindividual variability of DMET proteins in individual intestinal samples (n=13). UGT2B7, MGST1, MGST2, MGST3, CES2, and MRP2 were expressed in all three tissues, whereas, as expected CYP3A4, CYP3A5, CYP2C9, CYP4F2, UGT1A1, UGT2B17, CES1, FMO5, MRP3, and P-gp were present in the liver and intestine. The top three DMET proteins in individual tissues were: CES1>CYP2E1>UGT2B7 (liver), CES2>UGT2B17>CYP3A4 (intestine), and MGST1>UGT1A6>MGST2 (kidney). CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2 showed high interindividual variability in the intestine. These data are relevant for enhancing in vitro to in vivo extrapolation (IVIVE) of drug absorption and disposition and can be used to enhance the accuracy of physiologically based pharmacokinetic (PBPK) prediction of systemic and tissue concentration of drugs. Significance Statement We quantified the abundance and compositions of drug-metabolizing enzymes and transporters (DMETs) in pooled human liver, intestine, and kidney microsomes using an optimized sequence coverage-informed total protein approach. The quantification of DMETs revealed quantitative differences in their levels in the liver, intestine, and kidney. Further, the analysis of individual intestine samples confirmed high variability in the levels of CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2. These data are applicable for the prediction of first-pass metabolism and tissue-specific drug clearance.
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Despite the increasing importance of aldehyde oxidase (AO) in the drug metabolism of clinical candidates, ontogeny data for AO are limited. The objective of our study was to characterize the age-dependent AO content and activity in the human liver cytosolic fraction (HLC) and human hepatocytes (HH). HLC (n = 121 donors) and HH (n = 50 donors) were analyzed for (1) AO protein content by quantitative proteomics and (2) enzyme activity using carbazeran as a probe substrate. AO activity showed high technical variability and poor correlation with the content in HLC samples, whereas hepatocyte samples showed a strong correlation between the content and activity. Similarly, AO content and activity showed no significant age-dependent differences in HLC samples, whereas the average AO content and activity in hepatocytes increased significantly (â¼20-40-fold) from the neonatal levels (0-28 days). Based on the hepatocyte data, the age at which 50% of the adult AO content is reached (age50) was 3.15 years (0.32-13.97 years, 95% CI). Metabolite profiling of carbazeran revealed age-dependent metabolic switching and the role of non-AO mechanisms (glucuronidation and desmethylation) in carbazeran elimination. The content-activity correlation in hepatocytes improved significantly (R2 = 0.95; p < 0.0001) in samples showing <10% contribution of glucuronidation toward the overall metabolism, confirming that AO-mediated oxidation and glucuronidation are the key routes of carbazeran metabolism. Considering the confounding effect of glucuronidation on AO activity, AO content-based ontogeny data are a more direct reflection of developmental changes in protein expression. The comprehensive ontogeny data of AO in HH samples are more reliable than HLC data, which are important for developing robust physiologically based pharmacokinetic models for predicting AO-mediated metabolism in children.
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Aldehído Oxidasa , Hepatocitos , Hígado , Humanos , Aldehído Oxidasa/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Niño , Lactante , Adulto , Preescolar , Adolescente , Recién Nacido , Masculino , Adulto Joven , Femenino , Persona de Mediana Edad , Citosol/metabolismo , Proteómica/métodosRESUMEN
PURPOSE: Predicting the quantitative fraction of glucuronidation (fgluc) by individual UDP-glucuronosyltransferase enzymes (UGTs) is challenging due to the lack of selective inhibitors and inconsistent activity of recombinant UGT systems (rUGTs). Our study compares the relative expression versus activity factors (REF versus RAF) to predict fgluc based on rUGT data to human liver and intestinal microsomes (HLM and HIM). METHODS: REF scalars were derived from a previous in-house proteomics study for eleven UGT enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17), whereas RAF was calculated by measuring activities in rUGTs to microsomes of selective UGT probe substrates. Protein-normalized activity factor (pnAF) values were generated after correcting activity of individual UGTs to their corresponding protein abundance. The utility of REF and RAF in predicting fgluc was assessed for three UGT substrates-diclofenac, vorinostat, and raltegravir. RESULTS: The REF values ranged from 0.02 to 1.75, RAF based on activity obtained in rUGTs to HLM/HIM were from 0.1 to 274. pnAF values were ~ 5 to 80-fold, except for UGT2B4 and UGT2B15, where pnAF was ~ 180 and > 1000, respectively. The results revealed confounding effect of differential specific activities (per pmol) of rUGTs in fgluc prediction. CONCLUSION: The data suggest that the activity of UGT enzymes was significantly lower when compared to their activity in microsomes at the same absolute protein amount (pmol). Collectively, results of this study demonstrate poor and variable specific activity of different rUGTs (per pmol protein), as determined by pnAF values, which should be considered in fgluc scaling.
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Uridine 5'-diphospho-glulcuronosyltransferase 2B17 (UGT2B17) is important in the metabolism of steroids and orally administered drugs due to its high interindividual variability. However, the structural basis governing the substrate selectivity or inhibition of UGT2B17 remains poorly understood. This study investigated 76 FDA-approved drugs and 20 steroids known to undergo glucuronidation for their metabolism by UGT2B17. Specifically, we assessed the substrate selectivity for UGT2B17 over other UGT enzymes using recombinant human UGT2B17 (rUGT2B17), human intestinal microsomes, and human liver microsomes. The quantitative contribution of intestinal UGT2B17 in the glucuronidation of these compounds was characterized using intestinal microsomes isolated from UGT2B17 expressors and nonexpressors. In addition, a structure-based pharmacophore model for UGT2B17 substrates was built and validated using the studied pool of substrates and nonsubstrates. The results show that UGT2B17 could metabolize 23 out of 96 compounds from various chemical classes, including alcohols and carboxylic acids, particularly in the intestine. Interestingly, amines were less susceptible to UGT2B17 metabolism, though they could inhibit the enzyme. Three main pharmacophoric features of UGT2B17 substrates include (1) the presence of an accessible -OH or -COOH group near His35 residue, (2) a hydrophobic functional group at â¼4.5-5 Å from feature 1, and (3) an aromatic ring â¼5-7 Å from feature 2. Most of the studied compounds inhibited UGT2B17 activity irrespective of their substrate potential, indicating the possibility of multiple mechanisms. These data suggest that UGT2B17 is promiscuous in substrate selectivity and inhibition and has a high potential to produce significant variability in the absorption and disposition of orally administered drugs.
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Glucuronosiltransferasa , Esteroides , Humanos , Glucuronosiltransferasa/metabolismo , Uridina , Antígenos de Histocompatibilidad Menor/metabolismoRESUMEN
Cysteine-rich angiogenic factor 61 (CCN1/Cyr61) is a matricellular protein that is induced and secreted in response to growth factors. Our previous work showed that 18:1-lysophosphatidic acid (LPA), which activates the G protein-coupled receptor LPAR1, induces CCN1 between 2-4 h in PC-3 human prostate cancer cells in a manner than enhances cell-substrate adhesion. While the time course of induction suggests that CCN1 contributes to intermediate events in LPA action, the roles of CCN1 in LPA-mediated signal transduction have not been fully elucidated. This study utilized a comprehensive global proteomics approach to identify proteins up- or down-regulated in response to treatment of PC-3 cells with LPA for three hours, during the time of peak CCN1 levels. In addition, the effects of siRNA-mediated CCN1 knockdown on LPA responses were analyzed. The results show that, in addition to CCN1, LPA increased the levels of multiple proteins. Proteins up-regulated by LPA included metastasis-associated in colon cancer protein 1 (MACC1) and thrombospondin-1 (TSP1/THBS1); both MACC1 and TSP1 regulated cancer cell adhesion and motility. LPA down-regulated thioredoxin interacting protein (TXNIP). CCN1 knockdown suppressed the LPA-induced up-regulation of 30 proteins; these included MACC1 and TSP1, as confirmed by immunoblotting. Gene ontology and STRING analyses revealed multiple pathways impacted by LPA and CCN1. These results indicate that CCN1 contributes to LPA signaling cascades that occur during the intermediate phase after the initial stimulus. The study provides a rationale for the development of interventions to disrupt the LPA-CCN1 axis.
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Proteína 61 Rica en Cisteína , Neoplasias de la Próstata , Proteómica , Humanos , Masculino , Lisofosfolípidos/metabolismo , Células PC-3 , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Proteína 61 Rica en Cisteína/genética , Proteína 61 Rica en Cisteína/metabolismoRESUMEN
Neuroblastoma (NB) is a pediatric cancer with low survival rates in high-risk patients. 131I-mIBG has emerged as a promising therapy for high-risk NB and kills tumor cells by radiation. Consequently, 131I-mIBG tumor uptake and retention are major determinants for its therapeutic efficacy. mIBG enters NB cells through the norepinephrine transporter (NET), and accumulates in mitochondria through unknown mechanisms. Here we evaluated the expression of monoamine and organic cation transporters in high-risk NB tumors and explored their relationship with MYCN amplification and patient survival. We found that NB mainly expresses NET, the plasma membrane monoamine transporter (PMAT), and the vesicular membrane monoamine transporter 1/2 (VMAT1/2), and that the expression of these transporters is significantly reduced in MYCN-amplified tumor samples. PMAT expression is the highest and correlates with overall survival in high-risk NB patients without MYCN amplification. Immunostaining showed that PMAT resides intracellularly in NB cells and co-localizes with mitochondria. Using cells expressing PMAT, mIBG was identified as a PMAT substrate. In mitochondria isolated from NB cell lines, mIBG uptake was reduced by â¼50% by a PMAT inhibitor. Together, our data suggest that PMAT is a previously unrecognized transporter highly expressed in NB and could impact intracellular transport and therapeutic response to 131I-mIBG. SIGNIFICANCE STATEMENT: This study identified that plasma membrane monoamine transporter (PMAT) is a novel transporter highly expressed in neuroblastoma and its expression level is associated with overall survival rate in high-risk patients without MYCN amplification. PMAT is expressed intracellularly in neuroblastoma cells, transports meta-iodobenzylguanidine (mIBG) and thus could impact tumor retention and response to 131I-mIBG therapy. These findings have important clinical implications as PMAT could represent a novel molecular marker to help inform disease prognosis and predict response to 131I-mIBG therapy.
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3-Yodobencilguanidina , Neuroblastoma , Niño , Humanos , 3-Yodobencilguanidina/farmacología , Proteína Proto-Oncogénica N-Myc/metabolismo , Proteínas de Transporte de Membrana , Membrana Celular/metabolismoRESUMEN
Characterization of accurate compositions and total abundance of homologous drug-metabolizing enzymes, such as UDP glucuronosyltransferases (UGTs), is important for predicting the fractional contribution of individual isoforms involved in the metabolism of a drug for applications in physiologically based pharmacokinetic (PBPK) modeling. Conventional targeted proteomics utilizes surrogate peptides, which often results in high technical and interlaboratory variability due to peptide-specific digestion leading to data inconsistencies. To address this problem, we developed a novel conserved-plus-surrogate peptide (CPSP) approach for determining the accurate compositions and total or cumulative abundance of homologous UGTs in commercially available pooled human liver microsomes (HLM), human intestinal microsomes (HIM), human kidney microsomes (HKM), and human liver S9 (HLS9) fraction. The relative percent composition of UGT1A and UGT2B isoforms in the human liver was 35:5:36:11:13 for UGT1A1:1A3:1A4:1A6:1A9 and 20:32:22:21:5 for UGT2B4:2B7:2B10:2B15:2B17. The human kidney and intestine also showed unique compositions of UGT1As and UGT2Bs. The reproducibility of the approach was validated by assessing correlations of UGT compositions between HLM and HLS9 (R2> 0.91). The analysis of the conserved peptides also provided the abundance for individual UGT isoforms included in this investigation as well as the total abundance (pmol/mg protein) of UGT1As and UGT2Bs across tissues, i.e., 268 and 342 (HLM), 21 and 92 (HIM), and 138 and 99 (HKM), respectively. The CPSP approach could be used for applications in the in-vitro-to-in-vivo extrapolation of drug metabolism and PBPK modeling. SIGNIFICANCE STATEMENT: We quantified the absolute compositions and total abundance of UDP glucuronosyltransferases (UGTs) in pooled human liver, intestine, and kidney microsomes using a novel conserved-plus-surrogate peptide (CPSP) approach. The CPSP approach addresses the surrogate peptide-specific variability in the determination of the absolute composition of UGTs. The data presented in this manuscript are applicable for the estimation of the fraction metabolized by individual UGTs towards better in vitro-to-in vivo extrapolation of UGT-mediated drug metabolism.
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Glucuronosiltransferasa , Microsomas Hepáticos , Humanos , Reproducibilidad de los Resultados , Microsomas Hepáticos/metabolismo , Glucuronosiltransferasa/metabolismo , Isoformas de Proteínas/metabolismo , Péptidos/metabolismo , Uridina Difosfato/metabolismoRESUMEN
Drug-metabolizing enzymes and transporters (DMETs) are key regulators of the pharmacokinetics, efficacy, and toxicity of therapeutics. Over the past two decades, significant advancements in in vitro methodologies, targeted proteomics, in vitro to in vivo extrapolation methods, and integrated computational approaches such as physiologically based pharmacokinetic modeling have unequivocally contributed to improving our ability to quantitatively predict the role of DMETs in absorption, distribution, metabolism, and excretion and drug-drug interactions. However, the paucity of data regarding alterations in DMET activity in specific populations such as pregnant individuals, lactation, pediatrics, geriatrics, organ impairment, and disease states such as, cancer, kidney, and liver diseases and inflammation has restricted our ability to realize the full potential of these recent advancements. We envision that a series of carefully curated articles in a special supplementary issue of Drug Metabolism and Disposition will summarize the latest progress in in silico, in vitro, and in vivo approaches to characterize alteration in DMET activity and quantitatively predict drug disposition in specific populations. In addition, the supplementary issue will underscore the current scientific knowledge gaps that present formidable barriers to fully understand the clinical implications of altered DMET activity in specific populations and highlight opportunities for multistakeholder collaboration to advance our collective understanding of this rapidly emerging area. SIGNIFICANCE STATEMENT: This commentary highlights current knowledge and identifies gaps and key challenges in understanding the role of drug-metabolizing enzymes and transporters (DMETs) in drug disposition in specific populations. With this commentary for the special issue in Drug Metabolism and Disposition, the authors intend to increase interest and invite potential contributors whose research is focused or has aided in expanding the understanding around the role and impact of DMETs in drug disposition in specific populations.
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Hepatopatías , Proteínas de Transporte de Membrana , Embarazo , Femenino , Humanos , Niño , Proteínas de Transporte de Membrana/metabolismo , Interacciones Farmacológicas , Inflamación , Tasa de Depuración MetabólicaRESUMEN
Testosterone exhibits high variability in pharmacokinetics and glucuronidation after oral administration. Although testosterone metabolism has been studied for decades, the impact of UGT2B17 gene deletion and the role of gut bacterial ß-glucuronidases on its disposition are not well characterized. We first performed an exploratory study to investigate the effect of UGT2B17 gene deletion on the global liver proteome, which revealed significant increases in proteins from multiple biological pathways. The most upregulated liver proteins were aldoketoreductases [AKR1D1, AKR1C4, AKR7A3, AKR1A1, and 7-dehydrocholesterol reductase (DHCR7)] and alcohol or aldehyde dehydrogenases (ADH6, ADH1C, ALDH1A1, ALDH9A1, and ALDH5A). In vitro assays revealed that AKR1D1 and AKR1C4 inactivate testosterone to 5ß-dihydrotestosterone (5ß-DHT) and 3α,5ß-tetrahydrotestosterone (3α,5ß-THT), respectively. These metabolites also appeared in human hepatocytes treated with testosterone and in human serum collected after oral testosterone dosing in men. Our data also suggest that 5ß-DHT and 3α, 5ß-THT are then eliminated through glucuronidation by UGT2B7 in UGT2B17 deletion individuals. Second, we evaluated the potential reactivation of testosterone glucuronide (TG) after its secretion into the intestinal lumen. Incubation of TG with purified gut microbial ß-glucuronidase enzymes and with human fecal extracts confirmed testosterone reactivation into testosterone by gut bacterial enzymes. Both testosterone metabolic switching and variable testosterone activation by gut microbial enzymes are important mechanisms for explaining the disposition of orally administered testosterone and appear essential to unraveling the molecular mechanisms underlying UGT2B17-associated pathophysiological conditions. SIGNIFICANCE STATEMENT: This study investigated the association of UGT2B17 gene deletion and gut bacterial ß-glucuronidases with testosterone disposition in vitro. The experiments revealed upregulation of AKR1D1 and AKR1C4 in UGT2B17 deletion individuals, and the role of these enzymes to inactivate testosterone to 5ß-dihydrotestosterone and 3α, 5ß-tetrahydrotestosterone, respectively. Key gut bacterial species responsible for testosterone glucuronide activation were identified. These data are important for explaining the disposition of exogenously administered testosterone and appear essential to unraveling the molecular mechanisms underlying UGT2B17-associated pathophysiological conditions.
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Dihidrotestosterona , Glucuronidasa , Masculino , Humanos , Dihidrotestosterona/metabolismo , Testosterona/metabolismo , Hígado/metabolismo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismoRESUMEN
We investigated the effect of variability and instability in aldehyde oxidase (AO) content and activity on the scaling of in vitro metabolism data. AO content and activity in human liver cytosol (HLC) and five recombinant human AO preparations (rAO) were determined using targeted proteomics and carbazeran oxidation assay, respectively. AO content was highly variable as indicated by the relative expression factor (REF; i.e., HLC to rAO content) ranging from 0.001 to 1.7 across different in vitro systems. The activity of AO in HLC degrades at a 10-fold higher rate in the presence of the substrate as compared with the activity performed after preincubation without substrate. To scale the metabolic activity from rAO to HLC, a protein-normalized activity factor (pnAF) was proposed wherein the activity was corrected by AO content, which revealed up to sixfold higher AO activity in HLC versus rAO systems. A similar value of pnAF was observed for another substrate, ripasudil. Physiologically based pharmacokinetic (PBPK) modeling revealed a significant additional clearance (CL; 66%), which allowed for the successful prediction of in vivo CL of four other substrates, i.e., O-benzyl guanine, BIBX1382, zaleplon, and zoniporide. For carbazeran, the metabolite identification study showed that the direct glucuronidation may be contributing to around 12% elimination. Taken together, this study identified differential protein content, instability of in vitro activity, role of additional AO clearance, and unaccounted metabolic pathways as plausible reasons for the underprediction of AO-mediated drug metabolism. Consideration of these factors and integration of REF and pnAF in PBPK models will allow better prediction of AO metabolism. SIGNIFICANCE STATEMENT: This study elucidated the plausible reasons for the underprediction of aldehyde oxidase (AO)-mediated drug metabolism and provided recommendations to address them. It demonstrated that integrating protein content and activity differences and accounting for the loss of AO activity, as well as consideration of extrahepatic clearance and additional pathways, would improve the in vitro to in vivo extrapolation of AO-mediated drug metabolism using physiologically based pharmacokinetic modeling.
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Aldehído Oxidasa , Carbamatos , Humanos , Aldehído Oxidasa/metabolismo , Carbamatos/metabolismo , Cinética , Tasa de Depuración Metabólica , Hígado/metabolismoRESUMEN
The placenta is a fetal organ that performs critical functions to maintain pregnancy and support fetal development, including metabolism and transport of xenobiotics and steroids between the maternal-fetal unit. In vitro placenta models are used to study xenobiotic and steroid disposition, but how well these models recapitulate the human placenta is not well understood. We first characterized the abundance of proteins involved in xenobiotic and steroid disposition in human placental tissue. In pooled human placenta, the following xenobiotic and steroid disposition proteins were detected (highest to lowest), 1) enzymes: glutathione S-transferase P, carbonyl reductase 1, aldo-keto reductase 1B1, hydroxysteroid dehydrogenases (HSD3B1 and HSD11B1), aromatase, epoxide hydrolase 1 (EPHX1) and steryl-sulfatase, and 2) transporters: monocarboxylate transporters (MCT1 and 4), organic anion transporting polypeptide 2B1, organic anion transporter 4, and breast cancer resistance protein (BCRP). Then, the tissue proteomics data were compared with four placental cell lines (BeWo, JEG-3, JAR, and HTR-8/SVneo). The differential global proteomics analysis revealed that the tissue and cell lines shared 1420 cytosolic and 1186 membrane proteins. Although extravillous trophoblast and cytotrophoblast marker proteins were detected in all cell lines, only BeWo and JEG-3 cells expressed the syncytiotrophoblast marker, chorionic somatomammotropin hormone 1. BeWo and JEG-3 cells expressed most target proteins including aromatase, HSDs, EPHX1, MCT1, and BCRP. JEG-3 cells treated with commonly detected phthalates in human biofluids showed dysregulation of steroid pathways. The data presented here show that BeWo and JEG-3 cells are closer to the placental tissue for studying xenobiotic and steroid disposition. SIGNIFICANCE STATEMENT: This is the first study to compare proteomics data of human placental tissue and cell lines (BeWo, JAR, JEG-3, and HTR-8/SVneo). The placental cell line and tissue proteomes are vastly different, but BeWo and JEG-3 cells showed greater resemblance to the tissue in the expression of xenobiotic and steroid disposition proteins. These data will assist researchers to select an optimum cell model for mechanistic investigations on xenobiotic and steroid disposition in the placenta.
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Aromatasa , Placenta , Embarazo , Humanos , Femenino , Placenta/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Línea Celular Tumoral , Aromatasa/metabolismo , Xenobióticos/metabolismo , Proteómica , Proteínas de Neoplasias/metabolismo , Esteroides/metabolismoRESUMEN
Rats are extensively used as a preclinical model for assessing drug pharmacokinetics (PK) and tissue distribution; however, successful translation of the rat data requires information on the differences in drug metabolism and transport mechanisms between rats and humans. To partly fill this knowledge gap, we quantified clinically relevant drug-metabolizing enzymes and transporters (DMETs) in the liver and different intestinal segments of Sprague-Dawley rats. The levels of DMET proteins in rats were quantified using the global proteomics-based total protein approach (TPA) and targeted proteomics. The abundance of the major DMET proteins was largely comparable using quantitative global and targeted proteomics. However, global proteomics-based TPA was able to detect and quantify a comprehensive list of 66 DMET proteins in the liver and 37 DMET proteins in the intestinal segments of SD rats without the need for peptide standards. Cytochrome P450 (Cyp) and UDP-glycosyltransferase (Ugt) enzymes were mainly detected in the liver with the abundance ranging from 8 to 6502 and 74 to 2558 pmol/g tissue. P-gp abundance was higher in the intestine (124.1 pmol/g) as compared to that in the liver (26.6 pmol/g) using the targeted analysis. Breast cancer resistance protein (Bcrp) was most abundant in the intestinal segments, whereas organic anion transporting polypeptides (Oatp) 1a1, 1a4, 1b2, and 2a1 and multidrug resistance proteins (Mrp) 2 and 6 were predominantly detected in the liver. To demonstrate the utility of these data, we modeled digoxin PK by integrating protein abundance of P-gp and Cyp3a2 into a physiologically based PK (PBPK) model constructed using PK-Sim software. The model was able to reliably predict the systemic as well as tissue concentrations of digoxin in rats. These findings suggest that proteomics-informed PBPK models in preclinical species can allow mechanistic PK predictions in animal models including tissue drug concentrations.
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Proteínas de Transporte de Membrana , Proteínas de Neoplasias , Humanos , Ratas , Animales , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Ratas Sprague-Dawley , Proteínas de Neoplasias/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Hígado/metabolismo , Intestinos , Digoxina/metabolismoRESUMEN
Microcystin-leucine arginine (MCLR) is one of the most common and toxic microcystin variants, a class of cyanotoxins produced by cyanobacteria. A major molecular mechanism for MCLR-elicited liver toxicity involves the dysregulation of protein phosphorylation through protein phosphatase (PP) inhibition and mitogen-activated protein kinase (MAPK) modulation. In this study, specific pharmacological MAPK inhibitors were used in HepaRG cells to examine the pathways associated with MCLR cytotoxicity. SB203580 (SB), a p38 inhibitor, rescued HepaRG cell viability, whereas treatment with SP600125 (JNK inhibitor), MK2206 (AKT inhibitor), or N-acetylcysteine (reactive oxygen species scavenger) did not. Phosphoproteomic analysis revealed that phosphosites-which were altered by the addition of SB compared to MCLR treatment alone-included proteins involved in RNA processing, cytoskeletal stability, DNA damage response, protein degradation, and cell death. A closer analysis of specific proteins in some of these pathways indicated that SB reversed the MCLR-mediated phosphorylation of the necroptosis-associated proteins, the mixed lineage kinase domain-like protein (MLKL), receptor-interacting serine/threonine kinase 1 (RIP1), DNA damage response proteins, ataxia telangiectasia and Rad3-related kinase (ATR), and checkpoint kinase 1 (CHK1). Overall, these data implicate p38/MK2, DNA damage, and necroptosis in MCLR-mediated hepatotoxicity, and suggest these pathways may be targets for prevention prior to, or treatment after, MCLR toxicity.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Proteínas Quinasas Activadas por Mitógenos , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Microcistinas/toxicidad , Fosforilación , Fosfoproteínas Fosfatasas/metabolismo , Citoesqueleto/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Mitochondrial amidoxime-reducing component (mARC) enzymes are molybdenum-containing proteins that metabolize a number of endobiotics and xenobiotics. The interindividual variability and differential tissue abundance of mARC1 and mARC2 were quantified using targeted proteomics in three types of tissue fractions: 1) pediatric liver tissue homogenates, 2) total membrane fraction of the paired liver and kidney samples from pediatric and adult donors, and 3) pooled S9 fractions of the liver, intestine, kidney, lung, and heart. The absolute levels of mARC1 and mARC2 in the pediatric liver homogenate were 40.08 ± 4.26 and 24.58 ± 4.02 pmol/mg homogenate protein, respectively, and were independent of age and sex. In the total membrane fraction of the paired liver and kidney samples, the abundance of hepatic mARC1 and mARC2 was comparable, whereas mARC2 abundance in the kidney was approximately 9-fold higher in comparison with mARC1. The analysis of the third set of samples (i.e., S9 fraction) revealed that mARC1 abundance in the kidney, intestine, and lung was 5- to 13-fold lower than the liver S9 abundance, whereas mARC2 abundance was approximately 3- and 16-fold lower in the intestine and lung than the liver S9, respectively. In contrast, the kidney mARC2 abundance in the S9 fraction was approximately 2.5-fold higher as compared with the hepatic mARC2 abundance. The abundance of mARC enzymes in the heart was below the limit of quantification (â¼0.6 pmol/mg protein). The mARC enzyme abundance data presented here can be used to develop physiologically based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates. SIGNIFICANCE STATEMENT: A precise targeted quantitative proteomics method was developed and applied to quantify newly discovered drug-metabolizing enzymes, mARC1 and mARC2, in pediatric and adult tissue samples. The data suggest that mARC enzymes are ubiquitously expressed in an isoform-specific manner in the human liver, kidney, intestine, and lung, and the enzyme abundance is not associated with age and sex. These data are important for developing physiologically based pharmacokinetic models for the prediction of in vivo pharmacokinetics of mARC substrates.
Asunto(s)
Proteínas Mitocondriales , Oximas , Adulto , Niño , Humanos , Hígado/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Oximas/metabolismoRESUMEN
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
The use of animal pharmacokinetic models as surrogates for humans relies on the assumption that the drug disposition mechanisms are similar between preclinical species and humans. However, significant cross-species differences exist in the tissue distribution and protein abundance of drug-metabolizing enzymes (DMEs) and transporters. We quantified non-cytochrome P450 (non-CYP) DMEs across commonly used preclinical species (cynomolgus and rhesus monkeys, beagle dog, Sprague Dawley and Wistar Han rats, and CD1 mouse) and compared these data with previously obtained human data. Aldehyde oxidase was abundant in humans and monkeys while poorly expressed in rodents, and not expressed in dogs. Carboxylesterase (CES) 1 abundance was highest in the liver while CES2 was primarily expressed in the intestine in all species with notable species differences. For example, hepatic CES1 was 3× higher in humans than in monkeys, but hepatic CES2 was 3-5× higher in monkeys than in humans. Hepatic UDP-glucuronosyltransferase (UGT) 1A2 abundance was â¼4× higher in dogs compared with rats, whereas UGT1A3 abundance was 3-5× higher in dog livers than its ortholog in human and monkey livers. UGT1A6 abundance was 5-6× higher in human livers compared with monkey and dog livers. Hepatic sulfotransferase 1B1 abundance was 5-7× higher in rats compared with the rest of the species. These quantitative non-CYP proteomics data can be used to explain unique toxicological profiles across species and can be integrated into physiologically based pharmacokinetic models for the mechanistic explanation of pharmacokinetics and tissue distribution of xenobiotics in animal species. SIGNIFICANCE STATEMENT: We characterized the quantitative differences in non-cytochrome P450 (non-CYP) drug-metabolizing enzymes across commonly used preclinical species (cynomolgus and rhesus monkeys, beagle dogs, Sprague Dawley and Wistar Han rats, and CD1 mice) and compared these data with previously obtained human data. Unique differences in non-CYP enzymes across species were observed, which can be used to explain significant pharmacokinetic and toxicokinetic differences between experimental animals and humans.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Proteómica , Animales , Animales de Laboratorio/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Humanos , Hígado/metabolismo , Ratones , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Especificidad de la EspecieRESUMEN
Dimethandrolone undecanoate (DMAU), an oral investigational male hormonal contraceptive, is a prodrug that is rapidly converted to its active metabolite, dimethandrolone (DMA). Poor and variable oral bioavailability of DMA after DMAU dosing is a critical challenge to develop it as an oral drug. The objective of our study was to elucidate the mechanisms of variable pharmacokinetics of DMA. We first identified DMA metabolites formed in vitro and in vivo in human hepatocyte incubation and serum samples following oral DMAU administration in men, respectively. The metabolite identification study revealed two metabolites, DMA-glucuronide (DMA-G; major) and the androstenedione analog of DMA (minor), in the hepatocyte incubations. After oral DMAU administration, only DMA-G was detected in serum, which was >100-fold compared with DMA levels, supporting glucuronidation as the major elimination mechanism for DMA. Next, 13 clinically relevant UDP-glucuronosyltransferase (UGT) enzymes were tested for their involvement in DMA-G formation, which revealed a major role of UDP-glucuronosyltransferase 2B17 (UGT2B17) isoform with a smaller contribution of UGT1A9 in DMA-G formation. These data were confirmed by dramatically higher DMA glucuronidation rates (>200- and sevenfold) in the high versus the null UGT2B17-expressing human intestinal and liver microsomes, respectively. Since human UGT2B17 is a highly variable enzyme with a 20%-80% gene deletion frequency, the in vitro data suggest a major role of UGT2B17 polymorphism on the first-pass metabolism of DMA. Further, considering DMA is a selective and sensitive UGT2B17 substrate, it could be used as a clinical probe of UGT2B17 activity. SIGNIFICANCE STATEMENT: Dimethandrolone (DMA) is an active metabolite of dimethandrolone undecanoate (DMAU), an investigational male hormonal contraceptive. Previous studies have indicated poor and inconsistent bioavailability of DMAU following oral administration. This study found that UDP-glucuronosyltransferase 2B17-mediated high intestinal first-pass metabolism is the key mechanism of variable DMA bioavailability.