RESUMEN
Madin-Darby canine kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate transepithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits, and as such, laboratory-to-laboratory variability in drug permeability assessments is observed. Consequently, in vitro-in vivo extrapolation (IVIVE) approaches using permeability and/or transporter activity data require calibration. A comprehensive proteomic quantification of 11 filter-grown parental or mock-transfected MDCK monolayers from 8 different pharmaceutical laboratories using the total protein approach (TPA) is provided. The TPA enables estimations of key morphometric parameters such as monolayer cellularity and volume. Overall, metabolic liability to xenobiotics is likely to be limited for MDCK cells due to the low expression of required enzymes. SLC16A1 (MCT1) was the highest abundant SLC transporter linked to xenobiotic activity, while ABCC4 (MRP4) was the highest abundant ABC transporter. Our data supports existing findings that claudin-2 levels may be linked to tight junction modulation, thus impacting trans-epithelial resistance. This unique database provides data on more than 8000 protein copy numbers and concentrations, thus allowing an in-depth appraisal of the control monolayers used in each laboratory.
Asunto(s)
Proteoma , Proteómica , Animales , Perros , Células de Riñón Canino Madin Darby , Proteoma/metabolismo , Uniones Estrechas/metabolismo , Riñón/metabolismo , Proteínas Portadoras/metabolismoRESUMEN
Translation of information on drug exposure and effect is facilitated by in silico models that enable extrapolation of in vitro measurements to in vivo clinical outcomes. These models integrate drug-specific data with information describing physiological processes and pathological changes, including alterations to proteins involved in drug absorption, distribution and elimination. Over the past 15 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. In this review, we explore current and emerging applications of targeted and global (untargeted) proteomics in translational pharmacology as well as strategies for improved integration into model-based drug development.
Asunto(s)
Farmacología en Red , Proteómica , Modelos Biológicos , ProteínasRESUMEN
The intestinal epithelium represents a natural barrier against harmful xenobiotics, while facilitating the uptake of nutrients and other substances. Understanding the interaction of chemicals with constituents of the intestinal epithelium and their fate in the body requires quantitative measurement of relevant proteins in in vitro systems and intestinal epithelium. Recent studies have highlighted the mismatch between messenger RNA (mRNA) and protein abundance for several drug-metabolizing enzymes and transporters in the highly dynamic environment of the intestinal epithelium; mRNA abundances cannot therefore be used as a proxy for protein abundances in the gut, necessitating direct measurements. The objective was to determine the expression of a wide range proteins pertinent to metabolism and disposition of chemicals and nutrients in the intestinal epithelium. Ileum and jejunum biopsy specimens were obtained from 16 patients undergoing gastrointestinal elective surgery. Mucosal fractions were prepared and analyzed using targeted and global proteomic approaches. A total of 29 enzymes, 32 transporters, 6 tight junction proteins, 2 adhesion proteins, 1 alkaline phosphatase, 1 thioredoxin, 5 markers, and 1 regulatory protein were quantified-60 for the first time. The global proteomic method identified a further 5,222 proteins, which are retained as an open database for interested parties to explore. This study significantly expands our knowledge of a wide array of proteins important for xenobiotic handling in the intestinal epithelium. Quantitative systems biology models will benefit from the novel systems data generated in the present study and the translation path offered for in vitro to in vivo translation.
Asunto(s)
Íleon/metabolismo , Mucosa Intestinal/metabolismo , Yeyuno/metabolismo , Proteínas/metabolismo , Xenobióticos/farmacocinética , Fosfatasa Alcalina/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Enzimas/metabolismo , Humanos , Modelos Biológicos , Oxigenasas/metabolismo , Proteómica , Tiorredoxinas/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Transferasas/metabolismoRESUMEN
The levels of drug-metabolizing enzymes (DMEs) and transporter proteins in the human intestine are pertinent to determine oral drug bioavailability. Despite the paucity of reports on such measurements, it is well recognized that these values are essential for translating in vitro data on drug metabolism and transport to predict drug disposition in gut wall. In the current study, clinically relevant DMEs [cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT)] and drug transporters were quantified in total mucosal protein preparations from the human jejunum (n = 4) and ileum (n = 12) using quantification concatemer-based targeted proteomics. In contrast to previous reports, UGT2B15 and organic anion-transporting polypeptide 1 (OATP1A2) were quantifiable in all our samples. Overall, no significant disparities in protein expression were observed between jejunum and ileum. Relative mRNA expression for drug transporters did not correlate with the abundance of their cognate protein, except for P-glycoprotein 1 (P-gp) and organic solute transporter subunit alpha (OST-α), highlighting the limitations of RNA as a surrogate for protein expression in dynamic tissues with high turnover. Intercorrelations were found within P450 [2C9-2C19 (P = 0.002, R 2 = 0.63), 2C9-2J2 (P = 0.004, R 2 = 0.40), 2D6-2J2 (P = 0.002, R 2 = 0.50)] and UGT [1A1-2B7 (P = 0.02, R 2 = 0.87)] family of enzymes. There were also correlations between P-gp and several other proteins [OST-α (P < 0.0001, R 2 = 0.77), UGT1A6 (P = 0.009, R 2 = 0.38), and CYP3A4 (P = 0.007, R 2 = 0.30)]. Incorporating such correlations into building virtual populations is crucial for obtaining plausible characteristics of simulated individuals. SIGNIFICANCE STATEMENT: A number of drug transporters were quantified for the first time in this study. Several intercorrelations of protein abundance were reported. mRNA expression levels proved to be a poor reflection of differences between individuals regarding the level of protein expression in gut. The reported abundance of drug-metabolizing enzymes and transporters and their intercorrelations will contribute to better predictions of oral drug bioavailability and drug-drug interactions by linking in vitro observations to potential outcomes through physiologically based pharmacokinetic models.
Asunto(s)
Sistema Enzimático del Citocromo P-450/análisis , Glucuronosiltransferasa/análisis , Yeyuno/enzimología , Transportadores de Anión Orgánico/análisis , Administración Oral , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Disponibilidad Biológica , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Femenino , Humanos , Yeyuno/cirugía , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Modelos Biológicos , Transportadores de Anión Orgánico/metabolismo , Proteómica/métodosRESUMEN
The aim of this study was to derive region-specific transporter expression data suitable for in vitro-to-in vivo extrapolation (IVIVE) within a physiologically based pharmacokinetic (PBPK) modeling framework. A meta-analysis was performed whereby literary sources reporting region-specific transporter expression obtained via absolute and relative quantification approaches were considered in healthy adult Caucasian individuals. Furthermore, intestinal total membrane protein yield was calculated to enable mechanistic IVIVE via absolute transporter abundances. Where required, authors were contacted for additional information. A refined database was constructed where samples were excluded based on quantification in, non-Caucasian subjects, disease tissue, subjects <18 years old, duplicated samples, non-total membrane matrix, pooled matrices, or cDNA. Demographic data were collected where available. The weighted and geometric mean, coefficient of variation, and between-study homogeneity was calculated in each of eight gut segments (duodenum, two jejunum, four ileum, and colon) for 16 transporters. Expression data were normalized to that in the proximal jejunum. From a total of 47 articles, the final database consisted of 2238 measurements for 16 transporters. The solute carrier peptide transporter 1 (PepT1) showed the highest jejunal abundance, while multidrug resistance-associated protein (MRP) 2 was the highest abundance ATP-binding cassette transporter. Transporters displaying significant region-specific expression included the ileal bile acid transporter, which showed 18-fold greater terminal ileum expression compared with the proximal jejunum, while MRP3, organic cation transporter type 1 (OCTN1), and OCT1 showed >2-fold higher expression in other regions compared with the proximal jejunum. This is the first systematic analysis incorporating absolute quantification methodology to determine region-specific intestinal transporter expression. It is expected to be beneficial for mechanistic transporter IVIVE in healthy adult Caucasians. SIGNIFICANCE STATEMENT: Given the burgeoning reports of absolute transporter abundances in the human intestine, the incorporation of such information into mechanistic IVIVE-PBPK models could offer a distinct advantage in facilitating the robust assessment of the impact of gut transporters on drug disposition. The systematic and formal assessment via a literature meta-analysis described herein, enables assignment of the regional-specific expression, absolute transporter abundances, interindividual variability, and other associated scaling factors to healthy Caucasian populations within PBPK models. The resulting values are available to incorporate into PBPK models, and offer a verifiable account describing intestinal transporter expression within PBPK models for persons wishing to utilize them. Furthermore, these data facilitate the development of appropriate IVIVE scaling strategies using absolute transporter abundances.
Asunto(s)
Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Yeyuno/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Adulto , Humanos , Proteínas de Transporte de Membrana/análisis , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteómica , Población BlancaRESUMEN
The use of in vitro-in vivo extrapolation (IVIVE) techniques, mechanistically incorporated within physiologically based pharmacokinetic (PBPK) models, can harness in vitro drug data and enhance understanding of in vivo pharmacokinetics. This study's objective was to develop a user-friendly rat (250 g, male Sprague-Dawley) IVIVE-linked PBPK model. A 13-compartment PBPK model including mechanistic absorption models was developed, with required system data (anatomical, physiological, and relevant IVIVE scaling factors) collated from literature and analyzed. Overall, 178 system parameter values for the model are provided. This study also highlights gaps in available system data required for strain-specific rat PBPK model development. The model's functionality and performance were assessed using previous literature-sourced in vitro properties for diazepam, metoprolol, and midazolam. The results of simulations were compared against observed pharmacokinetic rat data. Predicted and observed concentration profiles in 10 tissues for diazepam after a single intravenous (i.v.) dose making use of either observed i.v. clearance (CLiv) or in vitro hepatocyte intrinsic clearance (CLint) for simulations generally led to good predictions in various tissue compartments. Overall, all i.v. plasma concentration profiles were successfully predicted. However, there were challenges in predicting oral plasma concentration profiles for metoprolol and midazolam, and the potential reasons and according solutions are discussed.
Asunto(s)
Adyuvantes Anestésicos/farmacocinética , Antiarrítmicos/farmacocinética , Anticonvulsivantes/farmacocinética , Diazepam/farmacocinética , Metoprolol/farmacocinética , Midazolam/farmacocinética , Adyuvantes Anestésicos/administración & dosificación , Adyuvantes Anestésicos/sangre , Adyuvantes Anestésicos/metabolismo , Administración Intravenosa , Animales , Antiarrítmicos/administración & dosificación , Antiarrítmicos/sangre , Antiarrítmicos/metabolismo , Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/sangre , Anticonvulsivantes/metabolismo , Simulación por Computador , Diazepam/administración & dosificación , Diazepam/sangre , Diazepam/metabolismo , Hepatocitos/metabolismo , Masculino , Tasa de Depuración Metabólica , Metoprolol/administración & dosificación , Metoprolol/sangre , Metoprolol/metabolismo , Midazolam/administración & dosificación , Midazolam/sangre , Midazolam/metabolismo , Modelos Biológicos , Ratas Sprague-DawleyRESUMEN
This study aimed to derive quantitative abundance values for key hepatic transporters suitable for in vitro-in vivo extrapolation within a physiologically based pharmacokinetic modeling framework. A meta-analysis was performed whereby data on abundance measurements, sample preparation methods, and donor demography were collated from the literature. To define values for a healthy Caucasian population, a subdatabase was created whereby exclusion criteria were applied to remove samples from non-Caucasian individuals, those with underlying disease, or those with subcellular fractions other than crude membrane. Where a clinically relevant active genotype was known, only samples from individuals with an extensive transporter phenotype were included. Authors were contacted directly when additional information was required. After removing duplicated samples, the weighted mean, geometric mean, standard deviation, coefficient of variation, and between-study homogeneity of transporter abundances were determined. From the complete database containing 24 transporters, suitable abundance data were available for 11 hepatic transporters from nine studies after exclusion criteria were applied. Organic anion transporting polypeptides OATP1B1 and OATP1B3 showed the highest population abundance in healthy adult Caucasians. For several transporters, the variability in abundance was reduced significantly once the exclusion criteria were applied. The highest variability was observed for OATP1B3 > OATP1B1 > multidrug resistance protein 2 > multidrug resistance gene 1. No relationship was found between transporter expression and donor age. To our knowledge, this study provides the first in-depth analysis of current quantitative abundance data for a wide range of hepatic transporters, with the aim of using these data for in vitro-in vivo extrapolation, and highlights the significance of investigating the background of tissue(s) used in quantitative transporter proteomic studies. Similar studies are now warranted for other ethnicities.
Asunto(s)
Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Hígado/metabolismo , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/metabolismo , Población Blanca , HumanosRESUMEN
Transporter mRNA and protein expression data are used to extrapolate in vitro transporter kinetics to in vivo drug disposition predictions. Breast cancer resistance protein (BCRP) possesses broad substrate specificity; therefore, understanding BCRP expression-activity relationships are necessary for the translation to in vivo. Bidirectional transport of estrone-3-sulfate (E-3-S), a BCRP probe, was evaluated with respect to relative BCRP mRNA expression and absolute protein abundance in 10- and 29-day cultured Caco-2 cells. BCRP mRNA expression was quantified by real-time PCR against a housekeeper gene, Cyclophilin A. The BCRP protein abundance in total membrane fractions was quantified by targeted proteomics, and [(3)H]-E-3-S bidirectional transport was determined in the presence or absence of Ko143, a potent BCRP inhibitor. BCRP mRNA expression was 1.5-fold higher in 29- versus 10-day cultured cells (n = 3), whereas a 2.4-fold lower (p < 0.001) BCRP protein abundance was observed in 29- versus 10-day cultured cells (1.28 ± 0.33 and 3.06 ± 0.22 fmol/µg protein, n = 6, respectively). This correlated to a 2.45-fold lower (p < 0.01) efflux ratio for E-3-S in 29- versus 10-day cultured cells (8.97 ± 2.51 and 3.32 ± 0.66, n = 6, respectively). Caco-2 cell BCRP protein abundance, but not mRNA levels, correlates with BCRP activity, suggesting that extrapolation strategies incorporating BCRP protein abundance-activity relationships may be more successful.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Estrona/análogos & derivados , Proteínas de Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transporte Biológico , Células CACO-2 , Estrona/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/metabolismo , ARN Mensajero/genéticaRESUMEN
Relative expression factors (REFs) are used to scale in vitro transporter kinetic data via in vitro-in vivo extrapolation linked to physiologically based pharmacokinetic (IVIVE-PBPK) models to clinical observations. Primarily two techniques to quantify transporter protein expression are available, immunoblotting and liquid chromatography-tandem mass spectrometry. Literature-collated REFs ranged from 0.4 to 5.1 and 1.1 to 90 for intestinal P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), respectively. The impact of using human jejunum-Caco-2 REFs for P-gp (REFiP-gp) and BCRP (REFiBCRP), generated from the same samples and using different proteomic methodologies from independent laboratories, on PBPK outcomes was assessed. A 5-fold decrease in REFiP-gp for a single oral dose of digoxin resulted in a 1.19- and 1.31-fold higher plasma area under the curve and Cmax, respectively. All generated REFiP-gp values led to simulated digoxin Cmax values within observed ranges; however, combining kinetic data generated from a different laboratory with the 5-fold lower REFiP-gp could not recover a digoxin-rifampicin drug-drug interaction, emphasizing the necessity to obtain transporter-specific kinetic estimates and REFs from the same in vitro system. For a theoretical BCRP compound, with absorption taking place primarily in the jejunum, a decrease in the REFiBCRP from 2.22 (University of Manchester) to 1.11 (Bertin Pharma) promoted proximal intestinal absorption while delaying tmax 1.44-fold. Laboratory-specific differences in REF may lead to different IVIVE-PBPK outcomes. To understand the mechanisms underlying projected pharmacokinetic liabilities, it is important to assess the potential impact of bias on the generation of REFs on an interindividual basis within a target population.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico/fisiología , Yeyuno/metabolismo , Proteínas de Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Células CACO-2 , Línea Celular Tumoral , Digoxina/metabolismo , Interacciones Farmacológicas/fisiología , Humanos , Absorción Intestinal/fisiología , Cinética , Proteómica/métodosRESUMEN
Over the last 5 years the quantification of transporter-protein absolute abundances has dramatically increased in parallel to the expanded use of in vitro-in vivo extrapolation (IVIVE) and physiologically based pharmacokinetics (PBPK)-linked models, for decision-making in pharmaceutical company drug development pipelines and regulatory submissions. Although several research groups have developed laboratory-specific proteomic workflows, it is unclear if the large range of reported variability is founded on true interindividual variability or experimental variability resulting from sample preparation or the proteomic methodology used. To assess the potential for methodological bias on end-point abundance quantification, two independent laboratories, the University of Manchester (UoM) and Bertin Pharma (BPh), employing different proteomic workflows, quantified the absolute abundances of Na/K-ATPase, P-gp, and breast cancer resistance protein (BCRP) in the same set of biologic samples from human intestinal and Caco-2 cell membranes. Across all samples, P-gp abundances were significantly correlated (P = 0.04, Rs = 0.72) with a 2.4-fold higher abundance (P = 0.001) generated at UoM compared with BPh. There was a systematically higher BCRP abundance in Caco-2 cell samples quantified by BPh compared with UoM, but not in human intestinal samples. Consequently, a similar intestinal relative expression factor (REF), derived from distal jejunum and Caco-2 monolayer samples, between laboratories was found for P-gp. However, a 2-fold higher intestinal REF was generated by UoM (2.22) versus BPh (1.11). We demonstrate that differences in absolute protein abundance are evident between laboratories and they probably result from laboratory-specific methodologies relating to peptide choice.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Neoplasias de la Mama/metabolismo , Yeyuno/metabolismo , Proteínas de Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Adulto , Anciano , Células CACO-2 , Línea Celular Tumoral , Femenino , Humanos , Absorción Intestinal/fisiología , Masculino , Proteómica/métodos , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
In drug development, considerable efforts are made to extrapolate from in vitro and preclinical findings to predict human drug disposition by using in vitro-in vivo extrapolation (IVIVE) approaches. Use of IVIVE strategies linked with physiologically based pharmacokinetic (PBPK) modeling is widespread, and regulatory agencies are accepting and occasionally requesting model analysis to support licensing submissions. Recently, there has been a drive to improve PBPK models by characterizing the absolute abundance of enzymes, transporters, and receptors within mammalian tissues and in vitro experimental systems using quantitative targeted absolute proteomics (QTAP). The absolute abundance of proteins relevant to processes governing drug disposition provided by QTAP will enable IVIVE-PBPK to incorporate terms for the abundance of enzymes and transporters in target populations. However, most studies that report absolute abundances of enzymes and transporter proteins do so in enriched membrane fractions so as to increase the abundance per sample, and thus the assay's sensitivity, rather than measuring the expected lower abundance in the more biologically meaningful whole cells or tissues. This communication discusses the balance between protein enrichment and potential loss during the preparation of membrane fractions from whole cells or tissues. Accounting for losses with recovery factors throughout the fractionation procedure provides a means to correct for procedural losses, thereby enabling the scaling of protein abundance from subcellular fractions to whole-cell or organ abundances. PBPK models based on corrected abundances will more closely resemble biological systems and facilitate development of more meaningful IVIVE scaling factors, producing more accurate quantitative predictions of drug disposition.
Asunto(s)
Centrifugación , Proteínas de Transporte de Membrana/análisis , Proteómica/métodos , Animales , Fraccionamiento Celular , Humanos , Proteínas de Transporte de Membrana/aislamiento & purificación , Modelos BiológicosRESUMEN
QconCAT is a tool for quantitative proteomics, consisting of an artificial protein, expressed from an artificial gene, made up of a concatenated string of proteotypic peptides selected from the proteins under study. Isotopically labeled QconCAT (usually containing (13)C6-arginine and (13)C6-lysine) provides a standard for each proteotypic peptide included in its sequence. In practice, some QconCAT proteins fail to express at sufficient levels for the purpose of quantitative analysis. Two complementary methods are presented to express recalcitrant QconCAT proteins intended to quantify human hepatic enzymes and transporters.
Asunto(s)
Regulación de la Expresión Génica , Inactivación Metabólica/genética , Hígado/química , Proteómica , Proteínas Recombinantes de Fusión/genética , Células CACO-2 , Isótopos de Carbono , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Sintéticos , Ingeniería Genética , Vectores Genéticos/metabolismo , Humanos , Hígado/enzimología , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Péptidos/metabolismo , Polilisina/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Inhibition of P-glycoprotein (PGP) resulting from the co-administration of substrate drugs represents a potential source of drug-drug interactions. Although in vitro screens can readily identify such interactions, the accuracy with which they mimic interactions in tissues or their value in predicting interactions in vivo is unresolved. This was addressed for the model PGP substrate digoxin by comparing the modulation of its permeability across Caco-2 cells and ex vivo human and rodent intestine by drugs for which pharmacokinetic data on interactions with digoxin in man is available. All five compounds (talinolol, omeprazole, verapamil, quinidine, cyclosporin) dose-dependently increased absorptive (A-B) digoxin permeability with maximal increases of 2.2-4.5-fold across Caco-2. Quantitatively similar increases were observed in ex vivo human and mouse intestine and studies in mdr1a(-/-) intestine confirmed that these interactions are mediated solely by PGP. In vitro changes in digoxin permeability were qualitative indicators of the increase in digoxin C(max) for these compounds in man, although accounting for the luminal drug concentrations expected for a given oral dose was a critical consideration. Based on a limited dataset these data suggest that Caco-2 accurately mimics intestinal digoxin interactions and may be useful in predicting the threshold dose at which interactions become clinically significant. Further studies across a wider range of drugs are needed to determine the broader applicability of in vitro data for quantitative prediction of clinical drug interactions.