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1.
J Biol Chem ; 300(5): 107278, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38599380

RESUMO

Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmax is the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.


Assuntos
Glucuronosiltransferase , Isoenzimas , Microssomos Hepáticos , Proteínas Recombinantes , Animais , Humanos , Ácidos Graxos/metabolismo , Ácidos Graxos/química , Glucuronosiltransferase/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/química , Isoenzimas/metabolismo , Isoenzimas/genética , Cinética , Microssomos Hepáticos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Magnetismo , Microssomos/química , Microssomos/metabolismo
2.
Pharm Res ; 40(12): 3025-3042, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37821766

RESUMO

OBJECTIVE: An in vitro relative activity factor (RAF) technique combined with mechanistic static modeling was examined to predict drug-drug interaction (DDI) magnitude and analyze contributions of different clearance pathways in complex DDIs involving transporter substrates. Atorvastatin and rifampicin were used as a model substrate and inhibitor pair. METHODS: In vitro studies were conducted with transfected HEK293 cells, hepatocytes and human liver microsomes. Prediction success was defined as predictions being within twofold of observations. RESULTS: The RAF method successfully translated atorvastatin uptake from transfected cells to hepatocytes, demonstrating its ability to quantify transporter contributions to uptake. Successful translation of atorvastatin's in vivo intrinsic hepatic clearance (CLint,h,in vivo) from hepatocytes to liver was only achieved through consideration of albumin facilitated uptake or through application of empirical scaling factors to transporter-mediated clearances. Transporter protein expression differences between hepatocytes and liver did not affect CLint,h,in vivo predictions. By integrating cis and trans inhibition of OATP1B1/OATP1B3, atorvastatin-rifampicin (single dose) DDI magnitude could be accurately predicted (predictions within 0.77-1.0 fold of observations). Simulations indicated that concurrent inhibition of both OATP1B1 and OATP1B3 caused approximately 80% of atorvastatin exposure increases (AUCR) in the presence of rifampicin. Inhibiting biliary elimination, hepatic metabolism, OATP2B1, NTCP, and basolateral efflux are predicted to have minimal to no effect on AUCR. CONCLUSIONS: This study demonstrates the effective application of a RAF-based translation method combined with mechanistic static modeling for transporter substrate DDI predictions and subsequent mechanistic interpretation.


Assuntos
Transportadores de Ânions Orgânicos , Rifampina , Humanos , Atorvastatina/metabolismo , Rifampina/farmacologia , Rifampina/metabolismo , Células HEK293 , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Interações Medicamentosas , Transportadores de Ânions Orgânicos/metabolismo
3.
Pharm Res ; 40(8): 1901-1913, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37280472

RESUMO

PURPOSE: After single oral dosing of the glycine reuptake transporter (GlyT1) inhibitor, iclepertin (BI 425809), a single major circulating metabolite, M530a, was identified. However, upon multiple dosing, a second major metabolite, M232, was observed with exposure levels ~ twofold higher than M530a. Studies were conducted to characterize the metabolic pathways and enzymes responsible for formation of both major human metabolites. METHODS: In vitro studies were conducted with human and recombinant enzyme sources and enzyme-selective inhibitors. The production of iclepertin metabolites was monitored by LC-MS/MS. RESULTS: Iclepertin undergoes rapid oxidation to a putative carbinolamide that spontaneously opens to an aldehyde, M528, which then undergoes reduction by carbonyl reductase to the primary alcohol, M530a. However, the carbinolamide can also undergo a much slower oxidation by CYP3A to form an unstable imide metabolite, M526, that is subsequently hydrolyzed by a plasma amidase to form M232. This difference in rate of metabolism of the carbinolamine explains why high levels of the M232 metabolite were not observed in vitro and in single dose studies in humans, but were observed in longer-term multiple dose studies. CONCLUSIONS: The long half-life iclepertin metabolite M232 is formed from a common carbinolamine intermediate, that is also a precursor of M530a. However, the formation of M232 occurs much more slowly, likely contributing to its extensive exposure in vivo. These results highlight the need to employ adequate clinical study sampling periods and rigorous characterization of unexpected metabolites, especially when such metabolites are categorized as major, thus requiring safety assessment.


Assuntos
Inibidores Enzimáticos , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida , Meia-Vida , Inibidores Enzimáticos/metabolismo , Redes e Vias Metabólicas , Microssomos Hepáticos/metabolismo
4.
J Appl Toxicol ; 42(10): 1570-1584, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35393688

RESUMO

Inhibition of sodium-glucose cotransporter-2 (SGLT2) has been shown to be a safe and efficacious approach to support managing Type 2 diabetes. In the 2-year carcinogenicity study with the SGLT2 inhibitor empagliflozin in CD-1 mice, an increased incidence of renal tubular adenomas and carcinomas was identified in the male high-dose group but was not observed in female mice. An integrated review of available nonclinical data was conducted to establish a mode-of-action hypothesis for male mouse-specific tumorigenesis. Five key events were identified through systematic analysis to form the proposed mode-of-action: (1) Background kidney pathology in CD-1 mice sensitizes the strain to (2) pharmacology-related diuretic effects associated with SGLT2 inhib ition. (3) In male mice, metabolic demand increases with the formation of a sex- and species-specific empagliflozin metabolite. These features converge to (4) deplete oxidative stress handling reserve, driving (5) constitutive cellular proliferation in male CD-1 mice. The proposed mode of action requires all five key events for empagliflozin to present a carcinogenicity risk in the CD-1 mouse. Considering that empagliflozin is not genotoxic in the standard battery of genotoxicity tests, and not all five key events are present in the context of female mice, rats, or humans, nor for other osmotic diuretics or other SGLT2 inhibitors, the observed male mouse renal tumors are not considered relevant to humans.


Assuntos
Carcinoma de Células Renais , Diabetes Mellitus Tipo 2 , Neoplasias Renais , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Antígenos CD1/metabolismo , Compostos Benzidrílicos/toxicidade , Carcinoma de Células Renais/complicações , Carcinoma de Células Renais/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/patologia , Feminino , Glucosídeos , Humanos , Hipoglicemiantes/toxicidade , Rim , Neoplasias Renais/induzido quimicamente , Neoplasias Renais/complicações , Neoplasias Renais/tratamento farmacológico , Masculino , Camundongos , Ratos , Transportador 2 de Glucose-Sódio/metabolismo , Transportador 2 de Glucose-Sódio/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/toxicidade
5.
Drug Metab Rev ; 52(3): 395-407, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32456484

RESUMO

The 12th International Society for the Study of Xenobiotics (ISSX) meeting, held in Portland, OR, USA from July 28 to 31, 2019, was attended by diverse members of the pharmaceutical sciences community. The ISSX New Investigators Group provides learning and professional growth opportunities for student and early career members of ISSX. To share meeting content with those who were unable to attend, the ISSX New Investigators herein elected to highlight the "Advances in the Study of Drug Metabolism" symposium, as it engaged attendees with diverse backgrounds. This session covered a wide range of current topics in drug metabolism research including predicting sites and routes of metabolism, metabolite identification, ligand docking, and medicinal and natural products chemistry, and highlighted approaches complemented by computational modeling. In silico tools have been increasingly applied in both academic and industrial settings, alongside traditional and evolving in vitro techniques, to strengthen and streamline pharmaceutical research. Approaches such as quantum mechanics simulations facilitate understanding of reaction energetics toward prediction of routes and sites of drug metabolism. Furthermore, in tandem with crystallographic and orthogonal wet lab techniques for structural validation of drug metabolizing enzymes, in silico models can aid understanding of substrate recognition by particular enzymes, identify metabolic soft spots and predict toxic metabolites for improved molecular design. Of note, integration of chemical synthesis and biosynthesis using natural products remains an important approach for identifying new chemical scaffolds in drug discovery. These subjects, compiled by the symposium organizers, presenters, and the ISSX New Investigators Group, are discussed in this review.


Assuntos
Biologia Computacional , Descoberta de Drogas , Xenobióticos , Congressos como Assunto , Aprendizado de Máquina , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Teoria Quântica
6.
Drug Metab Dispos ; 48(8): 690-697, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32503882

RESUMO

Long-term hepatocyte culture systems such as HepatoPac are well suited to evaluate the metabolic turnover of low clearance (CL) drugs because of their sustained metabolic capacity and longer-term viability. Erythromycin (ERY), a moderate, mechanism-based inhibitor of CYP3A, was evaluated as a tool in the HepatoPac model to assess contribution of CYP3A to the clearance of drug candidates. ERY inhibited CYP3A activity by 58% and 80% at 3 and 10 µM, respectively, for up to 72 hours. At 30 µM, ERY inhibited midazolam hydroxylation by >85% for the entire 144-hour duration of the incubation. Alprazolam CLint was inhibited 58% by 3 µM of ERY, 75% by 15 µM of ERY, 89% by 30 µM of ERY, and 94% by 60 µM of ERY. ERY (30 µM) did not markedly affect CLint of substrates for several other major cytochrome P450 isoforms evaluated and did not markedly inhibit uridine diphosphoglucuronosyl transferase (UGT) isoforms 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, or 2B15 as assessed using recombinant UGTs. ERY only mildly increased CYP3A4 gene expression by 2.1-fold (14% of rifampicin induction) at 120 µM, indicating that at effective concentrations for inhibition of CYP3A activity (30-60 µM), arylhydrocarbon receptor, constitutive androstane receptor, and pregnane-X-receptor activation are not likely to markedly increase levels of other drug-metabolizing enzymes or transporters. ERY at concentrations up to 60 µM was not toxic for up to 6 days of incubation. Use of ERY to selectively inhibit CYP3A in high-functioning, long-term hepatocyte models such as HepatoPac can be a valuable strategy to evaluate the contribution of CYP3A metabolism to the overall clearance of slowly metabolized drug candidates. SIGNIFICANCE STATEMENT: This work describes the use of erythromycin as a selective inhibitor of CYP3A to assess the contribution of CYP3A in the metabolism of compounds using long-term hepatocyte cultures.


Assuntos
Inibidores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/metabolismo , Eritromicina/farmacologia , Eliminação Hepatobiliar/efeitos dos fármacos , Adulto , Alprazolam/farmacocinética , Células Cultivadas , Técnicas de Cocultura/métodos , Indutores do Citocromo P-450 CYP3A/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Glucuronosiltransferase/metabolismo , Hepatócitos , Humanos , Masculino , Midazolam/farmacocinética , Pessoa de Meia-Idade , Cultura Primária de Células/métodos , Rifampina/farmacologia , Fatores de Tempo
7.
Drug Metab Dispos ; 48(8): 645-654, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32474441

RESUMO

Human liver microsomes (HLM) are a commonly used tool to study drug metabolism in vitro. Typical experiments conducted using suspensions of HLM can be challenging to separate from the incubation solution without lengthy ultracentrifugation steps. Magnetizable beads coated with silica (MGBS) were found to bind strongly to HLM, which could then be isolated and purified using a magnet. Binding of HLM to the MGBS (HLM-MGBS) was demonstrated to be mediated by strong interactions between microsomal phospholipids and MGBS, as artificially prepared phosphatidylcholine (PC) liposomes could be more efficiently captured by the MGBS. HLM-MGBS complexes retained functional cytochrome P450 and uridine-diphosphate-glucuronosyltransferase (UGT) activity as indicated by CYP2C8-mediated amodiaquine de-ethylation, CYP3A4-mediated midazolam 1'hydroxylation, UGT1A1-mediated glucuronidation of estradiol, UGT1A9-mediated glucuronidation of propofol, and UGT2B7-mediated glucuronidation of zidovudine. When comparing suspension HLM alone with HLM-MGBS complexes containing equivalent amounts of HLM, the intrinsic clearance (CLint) of CYP450 substrates was comparable; however, CLint of UGT1A1, UGT1A9, and UGT2B7 was increased in the HLM-MGBS system between 1.5- and 6-fold. HLM-MGBS used in an incubation could also be readily replaced with fresh HLM-MGBS to maintain the presence of active enzymes. Thus, HLM-MGBS demonstrate increased in vitro metabolic efficiency and manipulability, providing a new platform for determination of accurate metabolic parameters. SIGNIFICANCE STATEMENT: The following work describes the strong binding of HLM to magnetizable beads. In addition, the preservation of enzyme activity on the bound HLM provides a novel means to conduct preclinical metabolism studies.


Assuntos
Técnicas de Cultura de Células/métodos , Eliminação Hepatobiliar , Separação Celular/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios Enzimáticos , Glucuronosiltransferase/metabolismo , Humanos , Imãs , Microssomos Hepáticos/metabolismo
8.
Pharm Res ; 37(12): 250, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33237382

RESUMO

PURPOSE: To provide whole-body physiologically based pharmacokinetic (PBPK) models of the potent clinical organic anion transporter (OAT) inhibitor probenecid and the clinical OAT victim drug furosemide for their application in transporter-based drug-drug interaction (DDI) modeling. METHODS: PBPK models of probenecid and furosemide were developed in PK-Sim®. Drug-dependent parameters and plasma concentration-time profiles following intravenous and oral probenecid and furosemide administration were gathered from literature and used for model development. For model evaluation, plasma concentration-time profiles, areas under the plasma concentration-time curve (AUC) and peak plasma concentrations (Cmax) were predicted and compared to observed data. In addition, the models were applied to predict the outcome of clinical DDI studies. RESULTS: The developed models accurately describe the reported plasma concentrations of 27 clinical probenecid studies and of 42 studies using furosemide. Furthermore, application of these models to predict the probenecid-furosemide and probenecid-rifampicin DDIs demonstrates their good performance, with 6/7 of the predicted DDI AUC ratios and 4/5 of the predicted DDI Cmax ratios within 1.25-fold of the observed values, and all predicted DDI AUC and Cmax ratios within 2.0-fold. CONCLUSIONS: Whole-body PBPK models of probenecid and furosemide were built and evaluated, providing useful tools to support the investigation of transporter mediated DDIs.


Assuntos
Furosemida/farmacocinética , Modelos Biológicos , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Probenecid/farmacocinética , Administração Intravenosa , Administração Oral , Adulto , Biotransformação , Simulação por Computador , Vias de Eliminação de Fármacos , Interações Medicamentosas , Feminino , Furosemida/administração & dosagem , Furosemida/sangue , Humanos , Masculino , Transportadores de Ânions Orgânicos/metabolismo , Probenecid/administração & dosagem , Probenecid/sangue , Rifampina/farmacocinética
9.
Drug Metab Dispos ; 46(7): 953-963, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29666154

RESUMO

Quantification of the fraction transported (ft) by a particular transporter will facilitate more robust estimations of transporter interactions. Using pitavastatin as a model uptake transporter substrate, we investigated the utility of the relative activity factor (RAF) approach and mechanistic modeling to estimate ft in hepatocytes. The transporters evaluated were organic anion-transporting polypeptides OATP1B1 and OATP1B3 and sodium-taurocholate cotransporting polypeptide. Transporter-expressing human embryonic kidney 293 cells and human hepatocytes were used for determining RAF values, which were then incorporated into the mechanistic model to simulate hepatocyte uptake of pitavastatin over time. There was excellent agreement between simulated and observed hepatocyte uptake of pitavastatin, indicating the suitability of this approach for translation of uptake from individual transporter-expressing cells to more holistic in vitro models. Subsequently, ft values were determined. The largest contributor to hepatocyte uptake of pitavastatin was OATP1B1, which correlates with what is known about the in vivo disposition of pitavastatin. The ft values were then used for evaluating in vitro-in vivo correlations of hepatic uptake inhibition with OATP inhibitors rifampicin and cyclosporine. Predictions were compared with previously reported plasma exposure changes of pitavastatin with these inhibitors. Although hepatic uptake inhibition of pitavastatin was 2-3-fold underpredicted, incorporation of scaling factors (SFs) into RAF values significantly improved the predictive ability. We propose that calibration of hepatocytes with standard transporter substrates and inhibitors would allow for determination of system-specific SFs, which could subsequently be used for refining predictions of clinical DDI potential for new chemical entities that undergo active hepatic uptake.


Assuntos
Transporte Biológico/fisiologia , Hepatócitos/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Quinolinas/metabolismo , Adulto , Linhagem Celular , Ciclosporina/metabolismo , Interações Medicamentosas/fisiologia , Feminino , Células HEK293 , Hepatócitos/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Fígado/efeitos dos fármacos , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Rifampina/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Simportadores/metabolismo
10.
Drug Metab Dispos ; 46(6): 770-778, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29514826

RESUMO

BI 187004, an 11ß-hydroxysteroid dehydrogenase 1 inhibitor, was administered once daily for 14 days to eight patients with type 2 diabetes mellitus. N-methylation was identified as a major biotransformation pathway. In four patients treated with BI 187004, the plasma exposure of an N-methylbenzimidazole metabolite [N-methylbenzimidazole regioisomer 1 (M1)] was 7-fold higher than the remaining four patients, indicating a substantial degree of metabolic variation. To identify the methyltransferase enzymes responsible for N-methylation, BI 187004 was incubated with human liver microsomes (HLM), human kidney microsomes (HKM), and their respective cytosolic preparations in the presence and absence of isoform-selective chemical inhibitors. Additionally, BI 187004 was incubated with several human recombinant methyltransferases: catechol O-methyltransferase (rhCOMT), histamine N-methyltransferase (rhHNMT), nicotinamide N-methyltransferase (rhNNMT), glycine N-methyltransferase (rhGNMT), and thiopurine S-methyltransferase (rhTPMT). M1 was principally observed in HLM and HKM incubations, minimally formed in liver and kidney cytosol, and not formed during incubations with recombinant methyltransferase enzymes. In all microsomal and cytosolic incubations, the formation of M1 was inhibited only by 2,3-dichloro-α-methylbenzylamine (DCMB), an inhibitor of thiol S-methyltransferase (TMT), providing evidence that TMT catalyzed the formation of M1. Interestingly, the N-methylbenzimidazole regioisomer (M14) was only observed in vitro, predominantly during incubations with human kidney cytosol and rhHNMT. The formation of M14 was inhibited by amodiaquine (an HNMT inhibitor) and DCMB, providing additional evidence that both HNMT and TMT catalyzed M14 formation. Overall, using BI 187004 as a substrate, this study demonstrates a novel TMT-mediated N-methylation biotransformation and an HNMT-mediated regioselective N-methylation.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Metiltransferases/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biotransformação/fisiologia , Criança , Pré-Escolar , Citosol/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Metilação , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade , Proteínas Recombinantes/metabolismo , Compostos de Sulfidrila , Adulto Jovem
11.
Br J Clin Pharmacol ; 84(9): 1941-1949, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29665130

RESUMO

AIMS: Previous pharmacokinetic characterization of a transporter probe cocktail containing digoxin (P-gp), furosemide (OAT1, OAT3), metformin (OCT2, MATE1, MATE2-K) and rosuvastatin (OATP1B1, OATP1B3, BCRP) in healthy subjects showed increases in rosuvastatin systemic exposure compared to rosuvastatin alone. In this trial, the doses of metformin and furosemide as putative perpetrators were reduced to eliminate their drug-drug interaction (DDI) with rosuvastatin. METHODS: In a randomized, open-label, single-centre, five-treatment, five-period crossover trial, 30 healthy male subjects received as reference treatments separately 0.25 mg digoxin, 1 mg furosemide, 10 mg metformin and 10 mg rosuvastatin, and as test treatment all four drugs administered together as a cocktail. Primary pharmacokinetic endpoints were AUC0-tz (area under the plasma concentration-time curve from time zero to the last quantifiable concentration) and Cmax (maximum plasma concentration) of each probe drug. RESULTS: Geometric mean ratios and 90% confidence intervals of test (cocktail) to reference (single drug) for AUC0-tz were 96.4% (88.2-105.3%) for digoxin, 102.6% (93.8-112.3%) for furosemide, 97.5% (93.5-101.6%) for metformin and 105.0% (96.4-114.4%) for rosuvastatin, indicating lack of interaction. The same analysis for Cmax and for pharmacokinetic parameters of urinary excretion of all cocktail components also indicated no DDI. CONCLUSIONS: Digoxin (0.25 mg), furosemide (1 mg), metformin (10 mg) and rosuvastatin (10 mg) exhibit no mutual pharmacokinetic interactions and are well tolerated administered as a cocktail. The cocktail is thus optimized and has the potential to be used as a screening tool for clinical investigation of transporter-mediated DDI.


Assuntos
Desenvolvimento de Medicamentos/métodos , Interações Medicamentosas , Proteínas de Membrana Transportadoras/metabolismo , Adulto , Área Sob a Curva , Estudos Cross-Over , Digoxina/administração & dosagem , Digoxina/metabolismo , Digoxina/farmacocinética , Relação Dose-Resposta a Droga , Furosemida/administração & dosagem , Furosemida/metabolismo , Furosemida/farmacocinética , Voluntários Saudáveis , Humanos , Masculino , Metformina/administração & dosagem , Metformina/metabolismo , Metformina/farmacocinética , Pessoa de Meia-Idade , Eliminação Renal , Rosuvastatina Cálcica/administração & dosagem , Rosuvastatina Cálcica/metabolismo , Rosuvastatina Cálcica/farmacocinética , Adulto Jovem
12.
Drug Metab Dispos ; 43(4): 490-509, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25587128

RESUMO

Breast cancer resistance protein (BCRP; ABCG2) limits intestinal absorption of low-permeability substrate drugs and mediates biliary excretion of drugs and metabolites. Based on clinical evidence of BCRP-mediated drug-drug interactions (DDIs) and the c.421C>A functional polymorphism affecting drug efficacy and safety, both the US Food and Drug Administration and European Medicines Agency recommend preclinical evaluation and, when appropriate, clinical assessment of BCRP-mediated DDIs. Although many BCRP substrates and inhibitors have been identified in vitro, clinical translation has been confounded by overlap with other transporters and metabolic enzymes. Regulatory recommendations for BCRP-mediated clinical DDI studies are challenging, as consensus is lacking on the choice of the most robust and specific human BCRP substrates and inhibitors and optimal study design. This review proposes a path forward based on a comprehensive analysis of available data. Oral sulfasalazine (1000 mg, immediate-release tablet) is the best available clinical substrate for intestinal BCRP, oral rosuvastatin (20 mg) for both intestinal and hepatic BCRP, and intravenous rosuvastatin (4 mg) for hepatic BCRP. Oral curcumin (2000 mg) and lapatinib (250 mg) are the best available clinical BCRP inhibitors. To interrogate the worst-case clinical BCRP DDI scenario, study subjects harboring the BCRP c.421C/C reference genotype are recommended. In addition, if sulfasalazine is selected as the substrate, subjects having the rapid acetylator phenotype are recommended. In the case of rosuvastatin, subjects with the organic anion-transporting polypeptide 1B1 c.521T/T genotype are recommended, together with monitoring of rosuvastatin's cholesterol-lowering effect at baseline and DDI phase. A proof-of-concept clinical study is being planned by a collaborative consortium to evaluate the proposed BCRP DDI study design.


Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Interações Medicamentosas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Preparações Farmacêuticas/metabolismo , Farmacocinética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Ensaios Clínicos como Assunto , Resistência a Múltiplos Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Humanos , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Guias de Prática Clínica como Assunto , Projetos de Pesquisa , Especificidade por Substrato
13.
Chem Res Toxicol ; 28(1): 103-15, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25489797

RESUMO

Following oral administration of empagliflozin (1000 mg/kg/day) to male and female CD-1 mice for 2 years, renal tubular injury was identified in male mice. Renal injury was not detected in male mice (≤300 mg/kg/day), in female mice (1000 mg/kg/day), or in male or female Han Wistar rats (700 mg/kg/day). Using transfected HEK293 cells and Xenopus oocytes, empagliflozin was found to be a substrate of various mouse and rat organic anion transporters (oat/Oat) and organic anion transporting polypeptide (oatp/Oatp) transporters: mouse oat3, rat Oat3, mouse oatp1a1, and rat Oatp1a1. However, using isolated kidney slices from male and female mice and rats, no sex-based difference in the extent of uptake of empagliflozin occurred. Metabolism studies using hepatic and renal microsomes from male and female mice, rats, and humans revealed a hemiacetal metabolite of empagliflozin (M466/2), predominantly formed in male mouse kidney microsomes. Formation of M466/2 in male mouse kidney microsomes was 31-fold higher compared to that in female mouse kidney microsomes and was ∼29- and ∼20-fold higher compared to that in male and female mouse liver microsomes, respectively. M466/2 is unstable and degrades to form a phenol metabolite (M380/1) and 4-hydroxycrotonaldehyde (4-OH CTA). Formed 4-OH CTA was trapped by reduced GSH, and the structure of the GSH adduct was confirmed by mass spectrometry. Stoichiometric formation of M380/1 from M466/2 was observed (93-96% at 24 h); however, formation of 4-OH CTA was considerably lower (∼17.5% at 40 h), which is consistent with 4-OH CTA being a highly reactive species. These data represent a highly selective tissue-, species-, and sex-specific lesion in male CD-1 mice associated with a cytotoxic metabolite product, 4-OH CTA. In humans, glucuronidation of empagliflozin is the most prevalent metabolic pathway, and oxidation is a minor pathway. Thus, renal toxicity due to the formation of 4-OH CTA from empagliflozin is not expected in humans.


Assuntos
Aldeídos/metabolismo , Compostos Benzidrílicos/metabolismo , Citotoxinas/metabolismo , Glucosídeos/metabolismo , Hipoglicemiantes/metabolismo , Rim/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Microssomos/metabolismo , Oócitos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Ratos Wistar , Fatores Sexuais , Especificidade da Espécie , Xenopus laevis
14.
J Pharmacol Exp Ther ; 351(2): 403-12, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25204339

RESUMO

Faldaprevir, an investigational agent for hepatitis C virus treatment, is well tolerated but associated with rapidly reversible, dose-dependent, clinically benign, unconjugated hyperbilirubinemia. Multidisciplinary preclinical and clinical studies were used to characterize mechanisms underlying this hyperbilirubinemia. In vitro, faldaprevir inhibited key processes involved in bilirubin clearance: UDP glucuronosyltransferase (UGT) 1A1 (UGT1A1) (IC50 0.45 µM), which conjugates bilirubin, and hepatic uptake and efflux transporters, organic anion-transporting polypeptide (OATP) 1B1 (IC50 0.57 µM), OATP1B3 (IC50 0.18 µM), and multidrug resistance-associated protein (MRP) 2 (IC50 6.2 µM), which transport bilirubin and its conjugates. In rat and human hepatocytes, uptake and biliary excretion of [(3)H]bilirubin and/or its glucuronides decreased on coincubation with faldaprevir. In monkeys, faldaprevir (≥20 mg/kg per day) caused reversible unconjugated hyperbilirubinemia, without hemolysis or hepatotoxicity. In clinical studies, faldaprevir-mediated hyperbilirubinemia was predominantly unconjugated, and levels of unconjugated bilirubin correlated with the UGT1A1*28 genotype. The reversible and dose-dependent nature of the clinical hyperbilirubinemia was consistent with competitive inhibition of bilirubin clearance by faldaprevir, and was not associated with liver toxicity or other adverse events. Overall, the reversible, unconjugated hyperbilirubinemia associated with faldaprevir may predominantly result from inhibition of bilirubin conjugation by UGT1A1, with inhibition of hepatic uptake of bilirubin also potentially playing a role. Since OATP1B1/1B3 are known to be involved in hepatic uptake of circulating bilirubin glucuronides, inhibition of OATP1B1/1B3 and MRP2 may underlie isolated increases in conjugated bilirubin. As such, faldaprevir-mediated hyperbilirubinemia is not associated with any liver injury or toxicity, and is considered to result from decreased bilirubin elimination due to a drug-bilirubin interaction.


Assuntos
Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hiperbilirrubinemia/induzido quimicamente , Oligopeptídeos/efeitos adversos , Oligopeptídeos/uso terapêutico , Tiazóis/efeitos adversos , Tiazóis/uso terapêutico , Ácidos Aminoisobutíricos , Animais , Bilirrubina/metabolismo , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Método Duplo-Cego , Glucuronosiltransferase/genética , Hepatite C/genética , Hepatite C/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Hiperbilirrubinemia/genética , Hiperbilirrubinemia/metabolismo , Leucina/análogos & derivados , Fígado/efeitos dos fármacos , Fígado/virologia , Macaca mulatta , Estudos Multicêntricos como Assunto , Proteína 2 Associada à Farmacorresistência Múltipla , Oligopeptídeos/farmacologia , Prolina/análogos & derivados , Quinolinas , Ensaios Clínicos Controlados Aleatórios como Assunto , Ratos , Tiazóis/farmacologia
15.
Drug Metab Dispos ; 42(3): 394-406, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24366904

RESUMO

An increased appreciation of the importance of transporter and enzyme interplay in drug clearance and a desire to delineate these mechanisms necessitates the utilization of models that contain a full complement of enzymes and transporters at physiologically relevant activities. Additionally, the development of drugs with longer half-lives requires in vitro systems with extended incubation times that allow characterization of metabolic pathways for low-clearance drugs. A recently developed coculture hepatocyte model, HepatoPac, has been applied to meet these challenges. Faldaprevir is a drug in late-stage development for the treatment of hepatitis C. Faldaprevir is a low-clearance drug with the somewhat unique characteristic of being slowly metabolized, producing two abundant hydroxylated metabolites (M2a and M2b) in feces (∼40% of the dose) without exhibiting significant levels of circulating metabolites in humans. The human HepatoPac model was investigated to characterize the metabolism and transport of faldaprevir. In human HepatoPac cultures, M2a and M2b were the predominant metabolites formed, with extents of formation comparable to in vivo. Direct glucuronidation of faldaprevir was shown to be a minor metabolic pathway. HepatoPac studies also demonstrated that faldaprevir is concentrated in liver with active uptake by multiple transporters (including OATP1B1 and Na(+)-dependent transporters). Overall, human HepatoPac cultures provided valuable insights into the metabolism and disposition of faldaprevir in humans and demonstrated the importance of enzyme and transporter interplay in the clearance of the drug.


Assuntos
Antivirais/metabolismo , Hepatócitos/efeitos dos fármacos , Fígado/metabolismo , Oligopeptídeos/metabolismo , Tiazóis/metabolismo , Ácidos Aminoisobutíricos , Transporte Biológico , Biotransformação , Células Cultivadas , Técnicas de Cocultura , Criopreservação , Meios de Cultura , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Feminino , Fibroblastos/citologia , Glucuronídeos/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Cinética , Leucina/análogos & derivados , Transportador 1 de Ânion Orgânico Específico do Fígado , Taxa de Depuração Metabólica , Estrutura Molecular , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Prolina/análogos & derivados , Quinolinas , Sódio/metabolismo
16.
Drug Metab Dispos ; 42(3): 384-93, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24346834

RESUMO

Faldaprevir is a hepatitis C virus protease inhibitor that effectively reduces viral load in patients. Since faldaprevir exhibits slow metabolism in vitro and low clearance in vivo, metabolism was expected to be a minor clearance pathway. The human [(14)C] absorption, distribution, metabolism, and excretion study revealed that two monohydroxylated metabolites (M2a and M2b) were the most abundant excretory metabolites in feces, constituting 41% of the total administered dose. To deconvolute the formation and disposition of M2a and M2b in humans and determine why the minor change in structure [the addition of 16 atomic mass units (amu)] produced chemical entities that were excreted and were not present in the circulation, multiple in vitro test systems were used. The results from these in vitro studies clarified the formation and clearance of M2a and M2b. Faldaprevir is metabolized primarily in the liver by CYP3A4/5 to form M2a and M2b, which are also substrates of efflux transporters (P-glycoprotein and breast cancer resistance protein). The role of transporters is considered important for M2a and M2b as they demonstrate low permeability. It is proposed that both metabolites are efficiently excreted via bile into feces and do not enter the systemic circulation to an appreciable extent. If these metabolites permeate to blood, they can be readily taken up into hepatocytes from the circulation by uptake transporters (likely organic anion transporting polypeptides). These results highlight the critical role of drug-metabolizing enzymes and multiple transporters in the process of the formation and clearance of faldaprevir metabolites. Faldaprevir metabolism also provides an interesting case study for metabolites that are exclusively excreted in feces but are of clinical relevance.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antivirais , Sistema Enzimático do Citocromo P-450/metabolismo , Fezes/enzimologia , Oligopeptídeos , Tiazóis , Ácidos Aminoisobutíricos , Antivirais/sangue , Antivirais/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Fezes/química , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Absorção Intestinal , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/enzimologia , Cinética , Leucina/análogos & derivados , Masculino , Taxa de Depuração Metabólica , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Microssomos/metabolismo , Oligopeptídeos/sangue , Oligopeptídeos/metabolismo , Prolina/análogos & derivados , Ligação Proteica , Quinolinas , Tiazóis/sangue , Tiazóis/metabolismo
17.
J Pharm Sci ; 113(7): 1987-1995, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38615815

RESUMO

Accurate measurement of non-specific binding of a drug candidate to human liver microsomes (HLM) can be critical for the accurate determination of key enzyme kinetic parameters such as Michaelis-Menton (Km), reversible inhibition (Ki), or inactivation (KI) constants. Several methods have been developed to determine non-specific binding of small molecules to HLM, such as rapid equilibrium dialysis (RED), ultrafiltration (UF), HLM bound to magnetizable beads (HLM-beads), ultracentrifugation (UC), the linear extrapolation stability assay (LESA), and the Transil™ system. Despite various differences in methodology between these methods, it is generally presumed that similar free fraction values (fu,mic) should be generated. To evaluate this hypothesis, a test set of 9 compounds were selected, representing low (high fu,mic value) and significant (low fu,mic value) HLM binding, respectively, across HLM concentrations tested in this manuscript. The fu,mic values were determined using a single compound concentration (1.0 µM) and three HLM concentrations (0.025, 0.50, and 1.0 mg/mL). When the HLM non-specific binding event is not extensive resulting in high fu,mic values, all methods generated similar fu,mic values. However, fu,mic values varied markedly across assay formats when high binding to HLM occurred, where fu,mic values differed by up to 33-fold depending on the method used. Potential causes for such discrepancies across the various methods employed, practical implications related to conduct the different assays, and implications to clinical drug-drug interaction (DDI) predictions are discussed.


Assuntos
Microssomos Hepáticos , Ultrafiltração , Humanos , Microssomos Hepáticos/metabolismo , Ultrafiltração/métodos , Ligação Proteica , Cinética , Ultracentrifugação/métodos , Preparações Farmacêuticas/metabolismo , Preparações Farmacêuticas/química , Diálise/métodos
18.
Drug Metab Dispos ; 41(7): 1347-66, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23620485

RESUMO

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Digoxina/farmacocinética , Medição de Risco , Animais , Transporte Biológico , Células CACO-2 , Cães , Interações Medicamentosas , Humanos , Concentração Inibidora 50 , Células LLC-PK1 , Análise de Componente Principal , Suínos
19.
Drug Metab Dispos ; 41(7): 1367-74, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23620486

RESUMO

In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when the maximum concentration of inhibitor at steady state divided by IC50 ([I1]/IC50) is ≥0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC50 ([I2]/IC50) is ≥10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC50 values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC50 values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I1]/IC50 ≥ 0.03 and [I2]/IC50 ≥ 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC50 values, a theoretical 95% confidence interval calculation was developed for single laboratory IC50 values, translating into a range of [I1]/IC50 and [I2]/IC50 values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC50 values.


Assuntos
Digoxina/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Árvores de Decisões , Interações Medicamentosas , Humanos , Curva ROC , Estados Unidos , United States Food and Drug Administration
20.
Drug Metab Dispos ; 39(11): 2013-6, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21832002

RESUMO

Gemcitabine (dFdC) is a chemotherapeutic nucleoside analog that undergoes uptake via equilibrative nucleoside transporters (hENT) followed by sequential phosphorylation to the active triphosphate moiety (dFdCTP). Its deaminated metabolite, 2',2'-difluorodeoxyuridine (dFdU), competes with the parent compound for cellular entry via hENTs, but over time dFdU increases the net intracellular accumulation of dFdC by a currently unknown mechanism. In this study, we investigated whether dFdU affects intracellular phosphorylation of gemcitabine by modulating the activity of deoxycytidine kinase (dCK). We report here that coincubation of dFdU with dFdC significantly increases intracellular levels of dFdCTP. dFdCTP was not identified as a substrate for hENTs, suggesting that dFdU affects the formation rather than elimination of the triphosphate. To further characterize the disposition of dFdC in the presence of dFdU, the net intracellular radioactivity of [5-(3)H]dFdC and corresponding metabolic profile were evaluated in HeLa cells transfected with dCK-targeting small interfering RNA. Intracellular radioactivity significantly decreased in cells with compromised intracellular phosphorylation, which was mainly due to a loss in dFdCTP. Although dFdU increased the net intracellular radioactivity of [5-(3)H]dFdC at 24 h in control cells, this increase was abolished in the absence of dCK activity, strongly suggesting that the interaction between dFdU and dFdC occurs via modulation of both transport and metabolism. In conclusion, we have demonstrated that the intracellular distribution of dFdC is dependent on both transport and metabolic processes, and that by affecting the rate at which dFdC enters the cell, the presence of dFdU may be altering the metabolic fate of the parent compound (dFdC).


Assuntos
Desoxicitidina Quinase/metabolismo , Desoxicitidina/análogos & derivados , Floxuridina/análogos & derivados , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Transporte Biológico , Desaminação , Desoxicitidina/metabolismo , Desoxicitidina/farmacocinética , Floxuridina/metabolismo , Floxuridina/farmacocinética , Células HeLa , Humanos , Inativação Metabólica , Fosforilação , Polifosfatos/metabolismo , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas , Gencitabina
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