RESUMO
Per- and polyfluoroalkyl substances (PFAS) encompass a diverse group of synthetic fluorinated chemicals known to elicit adverse health effects in animals and humans. However, only a few studies investigated the mechanisms underlying clearance of PFAS. Herein, the relevance of human renal transporters and permeability to clearance and bioaccumulation for 14 PFAS containing three to eleven perfluorinated carbon atoms (ηpfc = 3-11) and several functional head-groups was investigated. Apparent permeabilities and interactions with human transporters were measured using in vitro cell-based assays, including the MDCK-LE cell line, and HEK293 stable transfected cell lines expressing organic anion transporter (OAT) 1-4 and organic cation transporter (OCT) 2. The results generated align with the Extended Clearance Classification System (ECCS), affirming that permeability, molecular weight, and ionization serve as robust predictors of clearance and renal transporter engagement. Notably, PFAS with low permeability (ECCS 3A and 3B) exhibited substantial substrate activity for OAT1 and OAT3, indicative of active renal secretion. Furthermore, we highlight the potential contribution of OAT4-mediated reabsorption to the renal clearance of PFAS with short ηpfc, such as perfluorohexane sulfonate (PFHxS). Our data advance our mechanistic understanding of renal clearance of PFAS in humans, provide useful input parameters for toxicokinetic models, and have broad implications for toxicological evaluation and regulatory considerations.
Assuntos
Fluorocarbonos , Rim , Transportadores de Ânions Orgânicos , Humanos , Fluorocarbonos/metabolismo , Células HEK293 , Rim/metabolismo , Animais , Transportadores de Ânions Orgânicos/metabolismo , Células Madin Darby de Rim Canino , Cães , Permeabilidade , Poluentes Ambientais/metabolismo , Transporte BiológicoRESUMO
The International Consortium for Innovation and Quality in Pharmaceutical Development Transporter Working Group had a rare opportunity to analyze a crosspharma collation of in vitro data and assay methods for the evaluation of drug transporter substrate and inhibitor potential. Experiments were generally performed in accordance with regulatory guidelines. Discrepancies, such as not considering the impact of preincubation for inhibition and free or measured in vitro drug concentrations, may be due to the retrospective nature of the dataset and analysis. Lipophilicity was a frequent indicator of crosstransport inhibition (P-gp, BCRP, OATP1B, and OCT1), with high molecular weight (MW ≥500 Da) also common for OATP1B and BCRP inhibitors. A high level of overlap in in vitro inhibition across transporters was identified for BCRP, OATP1B1, and MATE1, suggesting that prediction of DDIs for these transporters will be common. In contrast, inhibition of OAT1 did not coincide with inhibition of any other transporter. Neutrals, bases, and compounds with intermediate-high lipophilicity tended to be P-gp and/or BCRP substrates, whereas compounds with MW <500 Da tended to be OAT3 substrates. Interestingly, the majority of in vitro inhibitors were not reported to be followed up with a clinical study by the submitting company, whereas those compounds identified as substrates generally were. Approaches to metabolite testing were generally found to be similar to parent testing, with metabolites generally being equally or less potent than parent compounds. However, examples where metabolites inhibited transporters in vitro were identified, supporting the regulatory requirement for in vitro testing of metabolites to enable integrated clinical DDI risk assessment. SIGNIFICANCE STATEMENT: A diverse dataset showed that transporter inhibition often correlated with lipophilicity and molecular weight (>500 Da). Overlapping transporter inhibition was identified, particularly that inhibition of BCRP, OATP1B1, and MATE1 was frequent if the compound inhibited other transporters. In contrast, inhibition of OAT1 did not correlate with the other drug transporters tested.
Assuntos
Indústria Farmacêutica , Proteínas de Membrana Transportadoras , Humanos , Indústria Farmacêutica/métodos , Proteínas de Membrana Transportadoras/metabolismo , Desenvolvimento de Medicamentos/métodos , Interações Medicamentosas/fisiologia , Preparações Farmacêuticas/metabolismo , Transporte Biológico/fisiologia , Inquéritos e Questionários , AnimaisRESUMO
Drug interactions involving the inhibition of hepatic organic anion transporting polypeptides (OATPs) 1B1 and OATP1B3 are considered important. Therefore, we sought to study various sulfated bile acids (BA-S) as potential clinical OATP1B1/3 biomarkers. It was determined that BA-S [e.g., glycochenodeoxycholic acid 3-O-sulfate (GCDCA-S) and glycodeoxycholic acid 3-O-sulfate (GDCA-S)] are substrates of OATP1B1, OATP1B3, and sodium-dependent taurocholic acid cotransporting polypeptide (NTCP) transfected into human embryonic kidney 293 cells, with minimal uptake evident for other solute carriers (SLCs) like OATP2B1, organic anion transporter 2, and organic cation transporter 1. It was also shown that BA-S uptake by plated human hepatocytes (PHH) was inhibited (≥96%) by a pan-SLC inhibitor (rifamycin SV), and there was greater inhibition (≥77% versus ≤12%) with rifampicin (OATP1B1/3-selective inhibitor) than a hepatitis B virus myristoylated-preS1 peptide (NTCP-selective inhibitor). Estrone 3-sulfate was also used as an OATP1B1-selective inhibitor. In this instance, greater inhibition was observed with GDCA-S (76%) than GCDCA-S (52%). The study was expanded to encompass the measurement of GCDCA-S and GDCA-S in plasma of SLCO1B1 genotyped subjects. The geometric mean GDCA-S concentration was 2.6-fold (90% confidence interval 1.6, 4.3; P = 2.1 × 10-4) and 1.3-fold (1.1, 1.7; P = 0.001) higher in individuals homozygous and heterozygous for the SLCO1B1 c.521T > C loss-of-function allele, respectively. For GCDCA-S, no significant difference was noted [1.2-fold (0.8, 1.7; P = 0.384) and 0.9-fold (0.8, 1.1; P = 0.190), respectively]. This supported the in vitro data indicating that GDCA-S is a more OATP1B1-selective substrate (versus GCDCA-S). It is concluded that GCDCA-S and GDCA-S are viable plasma-based OATP1B1/3 biomarkers, but they are both less OATP1B1-selective when compared to their corresponding 3-O-glucuronides (GCDCA-3G and GDCA-3G). Additional studies are needed to determine their utility versus more established biomarkers, such as coproporphyrin I, for assessing inhibitors with different OATP1B1 (versus OATP1B3) inhibition signatures.
Assuntos
Transportadores de Ânions Orgânicos , Humanos , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Sulfatos , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Ácidos e Sais Biliares , Transporte Biológico/fisiologia , Biomarcadores/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismoRESUMO
P-glycoprotein (P-gp) may limit oral drug absorption of substrate drugs due to intestinal efflux. Therefore, regulatory agencies require investigation of new chemical entities as possible inhibitors of P-gp in vitro. Unfortunately, inter-laboratory and inter-assay variability have hindered the translatability of in vitro P-gp inhibition data to predict clinical drug interaction risk. The current study was designed to evaluate the impact of potential IC50 discrepancies between two commonly utilized assays, i.e., bi-directional Madin-Darby Canine Kidney-MDR1 cell-based and MDR1 membrane vesicle-based assays. When comparing vesicle- to cell-based IC50 values (n = 28 inhibitors), non-P-gp substrates presented good correlation between assay formats, whereas IC50s of P-gp substrates were similar or lower in the vesicle assays. The IC50s obtained with a cell line expressing relatively low P-gp aligned more closely to those obtained from the vesicle assay, but passive permeability of the inhibitors did not appear to influence the correlation of IC50s, suggesting that efflux activity reduces intracellular inhibitor concentrations. IC50s obtained between two independent laboratories using the same assay type showed good correlation. Using the G-value (i.e., ratio of estimated gut concentration-to-inhibition potency) >10 cutoff recommended by regulatory agencies resulted in minimal differences in predictive performance, suggesting this cutoff is appropriate for either assay format.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Animais , Cães , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Interações Medicamentosas , Transporte Biológico , Linhagem CelularRESUMO
Hepatic impairment (HI) is known to modulate drug disposition and may lead to elevated plasma exposure. The aim of this study was to quantitate the in vivo OATP1B-mediated hepatic uptake activity in populations with varying degrees of HI. First, we measured baseline levels of plasma coproporphyrin-I, an endogenous OATP1B biomarker, in an open-label, parallel cohort study in adult subjects with normal liver function and mild, moderate, and severe HI (n = 24, 6/cohort). The geometric mean plasma concentrations of coproporphyrin-I were 1.66-fold, 2.81-fold (P < 0.05), and 7.78-fold (P < 0.0001) higher in mild, moderate, and severe impairment than those healthy controls. Second, we developed a dataset of 21 OATP1B substrate drugs with HI data extracted from literature. Median disease-to-healthy plasma area under the curve (AUC) ratios for substrate drugs were ~ 1.4, 3.0, and 6.4 for mild, moderate, and severe HI, respectively. Additionally, significant linear relationship was noted between AUC ratios of substrate drugs without and with co-administration of rifampin, a prototypic OATP1B inhibitor, and AUC ratios in moderate (P < 0.01) and severe (P < 0.001) HI. Third, a physiologically-based pharmacokinetic model analysis was conducted with 10 substrate drugs following estimation of relative OATP1B functional activity in virtual disease population models using coproporphyrin-I data and verification of substrate models with rifampin drug-drug interaction data. This approach adequately predicted plasma AUC change particularly in moderate (9 of 10 within 2-fold) and severe (5 of 5 within 2-fold) HI. Collective findings indicate progressive reduction, by as much as 90-92%, in OATP1B activity in the HI population.
Assuntos
Hepatopatias , Rifampina , Adulto , Humanos , Coproporfirinas , Preparações Farmacêuticas , Estudos de Coortes , Biomarcadores , Interações Medicamentosas , Área Sob a CurvaRESUMO
Vero cells are widely used for antiviral tests and virology research for SARS-CoV-2 as well as viruses from various other families. However, Vero cells generally express high levels of multi-drug resistance 1 (MDR1) or Pgp protein, the efflux transporter of foreign substances including many antiviral compounds, affecting the antiviral activity as well as interpretation of data. To address this, a Pgp gene knockout VeroE6 cell line (VeroE6-Pgp-KO) was generated using CRISPR-CAS9 technology. These cells no longer expressed the Pgp protein as indicated by flow cytometry analysis following staining with a Pgp-specific monoclonal antibody. They also showed significantly reduced efflux transporter activity in the calcein acetoxymethyl ester (calcein AM) assay. The VeroE6-Pgp-KO cells and the parental VeroE6 cells were each infected with SARS-CoV-2 to test antiviral activities of remdesivir and nirmatrelvir, two known Pgp substrates, in the presence or absence of a Pgp inhibitor. The compounds showed antiviral activities in VeroE6-Pgp-KO cells similar to that observed in the presence of the Pgp inhibitor. Thus, the newly established VeroE6-Pgp-KO cell line adds a new in vitro virus infection system for SARS-CoV-2 and possibly other viruses to test antiviral therapies without a need to control the Pgp activity. Removal of the Pgp inhibitor for antiviral assays will lead to less data variation and prevent failed assays.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Humanos , Chlorocebus aethiops , Animais , SARS-CoV-2/genética , Antivirais/farmacologia , Técnicas de Inativação de Genes , Células Vero , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Linhagem CelularRESUMO
Quantitative prediction of drug-drug interactions (DDIs) involving organic anion transporting polypeptide (OATP)1B1/1B3 inhibition is limited by uncertainty in the translatability of experimentally determined in vitro inhibition potency (half-maximal inhibitory concentration (IC50 )). This study used an OATP1B endogenous biomarker-informed physiologically-based pharmacokinetic (PBPK) modeling approach to predict the effect of inhibitor drugs on the pharmacokinetics (PKs) of OATP1B substrates. Initial static analysis with about 42 inhibitor drugs, using in vitro IC50 values and unbound liver inlet concentrations (Iin,max,u ), suggested in vivo OATP1B inhibition risk for drugs with R-value (1+ Iin,max,u /IC50 ) above 1.5. A full-PBPK model accounting for transporter-mediated hepatic disposition was developed for coproporphyrin I (CP-I), an endogenous OATP1B biomarker. For several inhibitors (cyclosporine, diltiazem, fenebrutinib, GDC-0810, itraconazole, probenecid, and rifampicin at 3 different doses), PBPK models were developed and verified against available CP-I plasma exposure data to obtain in vivo OATP1B inhibition potency-which tend to be lower than the experimentally measured in vitro IC50 by about 2-fold (probenecid and rifampicin) to 37-fold (GDC-0810). Models verified with CP-I data are subsequently used to predict DDIs with OATP1B probe drugs, rosuvastatin and pitavastatin. The predicted and observed area under the plasma concentration-time curve ratios are within 20% error in 55% cases, and within 30% error in 89% cases. Collectively, this comprehensive study illustrates the adequacy and utility of endogenous biomarker-informed PBPK modeling in mechanistic understanding and quantitative predictions of OATP1B-mediated DDIs in drug development.
Assuntos
Atorvastatina/farmacocinética , Coproporfirinas/sangue , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Fígado/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Modelos Biológicos , Rosuvastatina Cálcica/farmacocinética , Biomarcadores/sangue , Simulação por Computador , Interações Medicamentosas , Células HEK293 , Humanos , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Medição de Risco , Fatores de RiscoRESUMO
Quantitative assessment of drug-drug interactions (DDIs) involving breast cancer resistance protein (BCRP) inhibition is challenged by overlapping substrate/inhibitor specificity. This study used physiologically-based pharmacokinetic (PBPK) modeling to delineate the effects of inhibitor drugs on BCRP- and organic anion transporting polypeptide (OATP)1B-mediated disposition of rosuvastatin, which is a recommended BCRP clinical probe. Initial static model analysis using in vitro inhibition data suggested BCRP/OATP1B DDI risk while considering regulatory cutoff criteria for a majority of inhibitors assessed (25 of 27), which increased rosuvastatin plasma exposure to varying degree (~ 0-600%). However, rosuvastatin area under plasma concentration-time curve (AUC) was minimally impacted by BCRP inhibitors with calculated G-value (= gut concentration/inhibition potency) below 100. A comprehensive PBPK model accounting for intestinal (OATP2B1 and BCRP), hepatic (OATP1B, BCRP, and MRP4), and renal (OAT3) transport mechanisms was developed for rosuvastatin. Adopting in vitro inhibition data, rosuvastatin plasma AUC changes were predicted within 25% error for 9 of 12 inhibitors evaluated via PBPK modeling. This study illustrates the adequacy and utility of a mechanistic model-informed approach in quantitatively assessing BCRP-mediated DDIs.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Rosuvastatina Cálcica/farmacocinética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Adolescente , Adulto , Idoso , Área Sob a Curva , Interações Medicamentosas , Feminino , Células HEK293 , Humanos , Intestinos/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Adulto JovemRESUMO
The aim of this study was to investigate the sensitivity and specificity of endogenous glycochenodeoxycholate and glycodeoxycholate 3-O-glucuronides (GCDCA-3G and GDCA-3G) as substrates for organic anion transporting polypeptide 1B1 (OATP1B1) in humans. We measured fasting levels of plasma GCDCA-3G and GDCA-3G using liquid chromatography-tandem mass spectrometry in 356 healthy volunteers. The mean plasma levels of both compounds were ~ 50% lower in women than in men (P = 2.25 × 10-18 and P = 4.73 × 10-9 ). In a microarray-based genome-wide association study, the SLCO1B1 rs4149056 (c.521T>C, p.Val174Ala) variation showed the strongest association with the plasma GCDCA-3G (P = 3.09 × 10-30 ) and GDCA-3G (P = 1.60 × 10-17 ) concentrations. The mean plasma concentration of GCDCA-3G was 9.2-fold (P = 8.77 × 10-31 ) and that of GDCA-3G was 6.4-fold (P = 2.45x10-13 ) higher in individuals with the SLCO1B1 c.521C/C genotype than in those with the c.521T/T genotype. No other variants showed independent genome-wide significant associations with GCDCA-3G or GDCA-3G. GCDCA-3G was highly efficacious in detecting the SLCO1B1 c.521C/C genotype with an area under the receiver operating characteristic curve of 0.996 (P < 0.0001). The sensitivity (98-99%) and specificity (100%) peaked at a cutoff value of 180 ng/mL for men and 90 ng/mL for women. In a haplotype-based analysis, SLCO1B1*5 and *15 were associated with reduced, and SLCO1B1*1B, *14, and *35 with increased OATP1B1 function. In vitro, both GCDCA-3G and GDCA-3G showed at least 6 times higher uptake by OATP1B1 than OATP1B3 or OATP2B1. These data indicate that the hepatic uptake of GCDCA-3G and GDCA-3G is predominantly mediated by OATP1B1. GCDCA-3G, in particular, is a highly sensitive and specific OATP1B1 biomarker in humans.
Assuntos
Glucuronídeos/metabolismo , Ácido Glicoquenodesoxicólico/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Fígado/metabolismo , Adulto , Biomarcadores/metabolismo , Cromatografia Líquida , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Glucuronídeos/sangue , Ácido Glicoquenodesoxicólico/sangue , Células HEK293 , Voluntários Saudáveis , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado/deficiência , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Masculino , Desintoxicação Metabólica Fase II , Análise de Sequência com Séries de Oligonucleotídeos , Variantes Farmacogenômicos , Fenótipo , Polimorfismo de Nucleotídeo Único , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
There is a growing interest in using endogenous compounds as drug transporter biomarkers to facilitate drug-drug interaction (DDI) risk assessment in early phase I clinical trials. Compared to other drug transporters, however, no valid biomarker for hepatic organic cation transporter (OCT) 1 has been described to date. The present work represents the first report of an endogenous compound, isobutyryl-l-carnitine (IBC), as a potential clinical OCT1 biomarker for DDI assessment. A hydrophilic interaction chromatography (HILIC)-mass spectrometry/high resolution mass spectrometry (MS/HRMS) assay with a simple sample preparation method was developed. The assay is capable of simultaneously quantifying multiple endogenous compounds, including IBC, thiamine, N1-methylnicotinamide (1-NMN), creatinine, carnitine, and metformin, which is a probe for OCT1 and OCT2 and MATE1 and MATE2K (multidrug and toxin extrusion proteins) in clinical studies. The HRMS assay was fit-for-purpose validated in human plasma and demonstrated good linearity, accuracy, and precision for all analytes. It was further applied to two phase I clinical trials to evaluate potential biomarkers for OCT1 and additional cation transporters (renal OCT2, MATE1, and MATE2K). The clinical data demonstrated that plasma IBC changes correlated well with in vitro data and supported its use as a liver OCT1 biomarker. The described HILIC-MS/HRMS assay can be used as a "biomarker cocktail" to simultaneously assess clinical DDI risk for the inhibition of OCT1/2 and MATEs in clinical studies with new drug candidates.
Assuntos
Biomarcadores/química , Carnitina/análogos & derivados , Inibidores Enzimáticos/farmacocinética , Transportador 1 de Cátions Orgânicos/metabolismo , Carnitina/química , Ensaios Clínicos Fase I como Assunto , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Humanos , Metformina/farmacocinética , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/antagonistas & inibidores , Transportador 1 de Cátions Orgânicos/química , Transportador 2 de Cátion Orgânico/metabolismoRESUMO
Nicotinic acid (NA) and nicotinamide (NAM) are biosynthetic precursors of nicotinamide adenine dinucleotide (NAD+) - a physiologically important coenzyme that maintains the redox state of cells. Mechanisms driving their entry into cells are not well understood. Here we evaluated the hepatic uptake mechanism(s) of NA and NAM using transporter-transfected cell systems and primary human hepatocytes. NA showed robust organic anion transporter (OAT)2-mediated transport with an uptake ratio (i.e., ratio of accumulation in transfect cells to wild-type cells) of 9.7 ± 0.3, and a Michaelis-Menten constant (Km) of 13.5 ± 3.3 µM. However, no transport was apparent via other major hepatic uptake and renal secretory transporters, including OAT1/3/4, organic anion transporting polypeptide (OATP)1B1/1B3/2B1, sodium-taurocholate co-transporting polypeptide, organ cation transporter 1/2/3. OAT2-specific transport of NA was inhibited by ketoprofen and indomethacin (known OAT2 inhibitors) in a concentration-dependent manner. Similarly, NA uptake into primary human hepatocytes showed pH- and concentration-dependence and was subject to inhibition by specific OAT2 inhibitors. Unlike NA, NAM was not transported by the hepatic and renal solute carriers upon assessment in transfected cells, although its uptake into human hepatocytes was significantly inhibited by excess unlabelled NAM and a pan-SLC inhibitor (rifamycin SV 1 mM). In conclusion, these studies demonstrate, for the first time, a specific transport mechanism for NA uptake in the human liver and suggest that OAT2 (SLC22A7) has a critical role in its physiological and pharmacological functions.
Assuntos
Hepatócitos/metabolismo , Fígado/metabolismo , Niacina/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células HEK293 , Hepatócitos/efeitos dos fármacos , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Rifamicinas/farmacologiaRESUMO
The hepatic risk matrix (HRM) was developed and used to differentiate lead clinical and back-up drug candidates against competitor/marketed drugs within the same pharmaceutical class for their potential to cause human drug-induced liver injury (DILI). The hybrid HRM scoring system blends physicochemical properties (Rule of Two Model: dose and lipophilicity or Partition Model: dose, ionization state, lipophilicity, and fractional carbon bond saturation) with common toxicity mechanisms (cytotoxicity, mitochondrial dysfunction, and bile salt export pump (BSEP) inhibition) that promote DILI. HRM scores are based on bracketed safety margins (<1, 1-10, 10-100, and >100× clinical Cmax,total). On the basis of well-established clinical safety experience of marketed/withdrawn drug candidates, the background analysis consists of 200 drugs from the Liver Toxicity Knowledge Base annotated as Most-DILI- (79), Less-DILI- (56), No-DILI- (47), and Ambiguous-DILI-concern (18) drugs. Scores were generated for over 21 internal and 7 external drug candidates discontinued for unacceptable incidence/magnitude of liver transaminase elevations during clinical trials or withdrawn for liver injury severity. Both hybrid scoring systems identified 70-80% Most-DILI-concern drugs, but more importantly, stratified successful/unsuccessful drug candidates for liver safety (incidence/severity of transaminase elevations and approved drug labels). Incorporating other mechanisms (reactive metabolite and cytotoxic metabolite generation and hepatic efflux transport inhibition, other than BSEP) to the HRM had minimal beneficial impact in DILI prediction/stratification. As is, the hybrid scoring system was positioned for portfolio assessments to contrast DILI risk potential of small molecule drug candidates in early clinical development. This stratified approach for DILI prediction aided decisions regarding drug candidate progression, follow-up mechanistic work, back-up selection, clinical dose selection, and due diligence assessments in favor of compounds with less implied clinical hepatotoxicity risk.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Sobrevivência Celular , Desenvolvimento de Medicamentos/métodos , Células Hep G2 , Humanos , Mitocôndrias Hepáticas/efeitos dos fármacos , Ratos , Medição de Risco/métodosRESUMO
Physiologically-based pharmacokinetic (PBPK) modeling is a powerful tool to quantitatively describe drug disposition profiles in vivo, thereby providing an alternative to predict drug-drug interactions (DDIs) that have not been tested clinically. This study aimed to predict effects of rifampin-mediated intestinal P-glycoprotein (Pgp) induction on pharmacokinetics of Pgp substrates via PBPK modeling. First, we selected four Pgp substrates (digoxin, talinolol, quinidine, and dabigatran etexilate) to derive in vitro to in vivo scaling factors for intestinal Pgp kinetics. Assuming unbound Michaelis-Menten constant (Km ) to be intrinsic, we focused on the scaling factors for maximal efflux rate (Jmax ) to adequately recover clinically observed results. Next, we predicted rifampin-mediated fold increases in intestinal Pgp abundances to reasonably recover clinically observed DDI results. The modeling results suggested that threefold to fourfold increases in intestinal Pgp abundances could sufficiently reproduce the DDI results of these Pgp substrates with rifampin. Hence, the obtained fold increases can potentially be applicable to DDI prediction with other Pgp substrates.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Mucosa Intestinal/metabolismo , Rifampina/farmacocinética , Administração Oral , Dabigatrana/farmacologia , Digoxina/administração & dosagem , Digoxina/farmacologia , Interações Medicamentosas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Voluntários Saudáveis , Humanos , Masculino , Modelos Biológicos , Propanolaminas/administração & dosagem , Propanolaminas/farmacologia , Quinidina/administração & dosagem , Quinidina/farmacologia , Rifampina/administração & dosagemRESUMO
Hepatic uptake transporters [solute carriers (SLCs)], including organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, sodium-dependent taurocholate cotransporting polypeptide (NTCP), and organic anion (OAT2) and organic cation (OCT1) transporters, play a key role in determining the systemic and liver exposure of chemically diverse drugs. Here, we established a phenotyping approach to quantify the contribution of the six SLCs, and passive diffusion, to the overall uptake using plated human hepatocytes (PHHs). First, selective inhibitor conditions were identified by screening about 20 inhibitors across the six SLCs using single-transfected human embryonic kidney 293 cells. Data implied rifamycin SV (20 µM) inhibits three OATPs, while rifampicin (5 µM) inhibits OATP1B1/1B3 only. Further, hepatitis B virus myristoylated-preS1 peptide (0.1 µM), quinidine (100 µM), and ketoprofen (100-300 µM) are relatively selective against NTCP, OCT1, and OAT2, respectively. Second, using these inhibitory conditions, the fraction transported (ft ) by the individual SLCs was characterized for 20 substrates with PHH. Generally, extended clearance classification system class 1A/3A (e.g., warfarin) and 1B/3B compounds (e.g., statins) showed predominant OAT2 and OATP1B1/1B3 contribution, respectively. OCT1-mediated uptake was prominent for class 2/4 compounds (e.g., metformin). Third, in vitro ft values were corrected using quantitative proteomics data to obtain "scaled ft " Fourth, in vitro-in vivo extrapolation of the scaled OATP1B1/1B3 ft was assessed, leveraging statin clinical drug-drug interaction data with rifampicin as the perpetrator. Finally, we outlined a novel stepwise strategy to implement phenotypic characterization of SLC-mediated hepatic uptake for new molecular entities and drugs in a drug discovery and development setting.
Assuntos
Hepatócitos/metabolismo , Fígado/citologia , Fígado/metabolismo , Preparações Farmacêuticas/metabolismo , Fenótipo , Proteínas Carreadoras de Solutos/metabolismo , Transporte Biológico/efeitos dos fármacos , Interações Medicamentosas , Células HEK293 , Hepatócitos/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fígado/efeitos dos fármacos , Rifampina/metabolismo , Rifampina/farmacologiaRESUMO
We aim to establish an in vivo preclinical model to enable simultaneous assessment of inhibition potential of an investigational drug on clinically relevant drug transporters, organic anion-transporting polypeptide (OATP)1B, breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), and organic anion transporter (OAT)3. Pharmacokinetics of substrate cocktail consisting of pitavastatin (OATP1B substrate), rosuvastatin (OATP1B/BCRP/OAT3), sulfasalazine (BCRP), and talinolol (P-gp) were obtained in cynomolgus monkey-alone or in combination with transporter inhibitors. Single-dose rifampicin (30 mg/kg) significantly (P < 0.01) increased the plasma exposure of all four drugs, with a marked effect on pitavastatin and rosuvastatin [area under the plasma concentration-time curve (AUC) ratio â¼21-39]. Elacridar, BCRP/P-gp inhibitor, increased the AUC of sulfasalazine, talinolol, as well as rosuvastatin and pitavastatin. An OAT1/3 inhibitor (probenecid) significantly (P < 0.05) impacted the renal clearance of rosuvastatin (â¼8-fold). In vitro, rifampicin (10 µM) inhibited uptake of pitavastatin, rosuvastatin, and sulfasalazine by monkey and human primary hepatocytes. Transport studies using membrane vesicles suggested that all probe substrates, except talinolol, are transported by cynoBCRP, whereas talinolol is a cynoP-gp substrate. Elacridar and rifampicin inhibited both cynoBCRP and cynoP-gp in vitro, indicating potential for in vivo intestinal efflux inhibition. In conclusion, a probe substrate cocktail was validated to simultaneously evaluate perpetrator impact on multiple clinically relevant transporters using the cynomolgus monkey. The results support the use of the cynomolgus monkey as a model that could enable drug-drug interaction risk assessment, before advancing a new molecular entity into clinical development, as well as providing mechanistic insights on transporter-mediated interactions.
Assuntos
Transporte Biológico/fisiologia , Interações Medicamentosas/fisiologia , Preparações Farmacêuticas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células HEK293 , Hepatócitos/metabolismo , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Macaca fascicularis , Masculino , Taxa de Depuração Metabólica/fisiologia , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismoRESUMO
Bosutinib is an orally available Src/Abl tyrosine kinase inhibitor indicated for the treatment of patients with Ph+ chronic myelogenous leukemia at a clinically recommended dose of 500 mg once daily. Clinical results indicated that increases in bosutinib oral exposures were supraproportional at the lower doses (50-200 mg) and approximately dose-proportional at the higher doses (200-600 mg). Bosutinib is a substrate of CYP3A4 and P-glycoprotein and exhibits pH-dependent solubility with moderate intestinal permeability. These findings led us to investigate the factors influencing the underlying pharmacokinetic mechanisms of bosutinib with physiologically based pharmacokinetic (PBPK) models. Our primary objectives were to: 1) refine the previously developed bosutinib PBPK model on the basis of the latest oral bioavailability data and 2) verify the refined PBPK model with P-glycoprotein kinetics on the basis of the bosutinib drug-drug interaction (DDI) results with ketoconazole and rifampin. Additionally, the verified PBPK model was applied to predict bosutinib DDIs with dual CYP3A/P-glycoprotein inhibitors. The results indicated that 1) the refined PBPK model adequately described the observed plasma concentration-time profiles of bosutinib and 2) the verified PBPK model reasonably predicted the effects of ketoconazole and rifampin on bosutinib exposures by accounting for intestinal P-glycoprotein inhibition/induction. These results suggested that bosutinib DDI mechanism could involve not only CYP3A4-mediated metabolism but also P-glycoprotein-mediated efflux on absorption. In summary, P-glycoprotein kinetics could constitute an element in the PBPK models critical to understanding the pharmacokinetic mechanism of dual CYP3A/P-glycoprotein substrates, such as bosutinib, that exhibit nonlinear pharmacokinetics owing largely to a saturation of intestinal P-glycoprotein-mediated efflux.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Compostos de Anilina/farmacocinética , Interações Medicamentosas/fisiologia , Mucosa Intestinal/metabolismo , Nitrilas/farmacocinética , Quinolinas/farmacocinética , Administração Oral , Disponibilidade Biológica , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacocinética , Feminino , Humanos , Cetoconazol/farmacocinética , Masculino , Rifampina/farmacocinéticaRESUMO
Tolbutamide is primarily metabolized by CYP2C9, and, thus, is frequently applied as a clinical probe substrate for CYP2C9 activity. However, there is a marked discrepancy in the in vitro-in vivo extrapolation of its metabolic clearance, implying a potential for additional clearance mechanisms. The goal of this study was to evaluate the role of hepatic uptake transport in the pharmacokinetics of tolbutamide and to identify the molecular mechanism thereof. Transport studies using singly transfected cells expressing six major hepatic uptake transporters showed that tolbutamide is a substrate to organic anion transporter 2 (OAT2) alone with transporter affinity [Michaelis-Menten constant (Km)] of 19.5 ± 4.3 µM. Additionally, OAT2-specific transport was inhibited by ketoprofen (an OAT2 inhibitor) and 1 mM rifamycin SV (pan inhibitor), but not by cyclosporine and rifampicin (OAT polypeptides/Na+-taurocholate cotransporting polypeptide inhibitors). Uptake studies in primary human hepatocytes confirmed the predominant role of OAT2 in the active uptake with significant inhibition by rifamycin SV and ketoprofen, but not by the other inhibitors. Concentration-dependent uptake was noted in human hepatocytes with active transport characterized by Km and Vmax values of 39.3 ± 6.6 µM and 426 ± 30 pmol/min per milligram protein, respectively. Bottom-up physiologically based pharmacokinetic modeling was employed to verify the proposed role of OAT2-mediated hepatic uptake. In contrast to the rapid equilibrium (CYP2C9-only) model, the permeability-limited (OAT2-CYP2C9 interplay) model better described the plasma concentration-time profiles of tolbutamide. Additionally, the latter well described tolbutamide pharmacokinetics in carriers of CYP2C9 genetic variants and quantitatively rationalized its known drug-drug interactions. Our results provide first-line evidence for the role of OAT2-mediated hepatic uptake in the pharmacokinetics of tolbutamide, and imply the need for additional clinical studies in this direction.
Assuntos
Citocromo P-450 CYP2C9/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Tolbutamida/metabolismo , Transporte Biológico , Células HEK293 , Hepatócitos/metabolismo , Humanos , Distribuição Tecidual , Tolbutamida/farmacocinética , Tolbutamida/farmacologiaRESUMO
1. Penciclovir, ganciclovir, creatinine, para-aminohippuric acid (PAH), ketoprofen, estrone 3-O-sulfate (E3S), dehydroepiandrosterone 3-O-sulfate (DHEAS) and cyclic guanosine monophosphate (cGMP) were screened as substrates of human liver organic anion transporters OAT2 and OAT7. 2. For OAT7, high uptake ratios (versus mock transfected HEK293 cells) of 29.6 and 15.3 were obtained with E3S and DHEAS. Less robust uptake ratios (≤3.6) were evident with the other substrates. OAT2 (transcript variant 1, OAT2-tv1) presented high uptake ratios of 30, 13, â¼35, â¼25, 8.5 and 9 with cGMP, PAH, penciclovir, ganciclovir, creatinine and E3S, respectively. No uptake was observed with DHEAS. 3. Although not a substrate of either transporter, ketoprofen did inhibit transfected OAT2-tv1 (IC50 of 17, 22, 23, 24, 35 and 586 µM; creatinine, ganciclovir, penciclovir, cGMP, E3S and prostaglandin F2α, respectively) and penciclovir uptake (IC50 = 27 µM; >90% inhibition) by plated human hepatocytes (PHH). 4. It is concluded that penciclovir and ketoprofen may serve as useful tools for the assessment of OAT2 activity in PHH. However, measurement of OAT7 activity therein will prove more challenging, as high uptake rates are evident with E3S and DHEAS only and both sulfoconjugates are known to be substrates of organic anion transporting polypeptides.
Assuntos
Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Aciclovir/análogos & derivados , Aciclovir/farmacologia , Adulto , Estrona/análogos & derivados , Estrona/metabolismo , Feminino , Guanina , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cetoprofeno/farmacologia , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Peptídeos/metabolismo , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato/efeitos dos fármacos , TransfecçãoRESUMO
Transporter-mediated hepatic uptake is proven to be the rate-determining step in the systemic clearance of several drugs. Therefore, accurate measurement of active and passive uptake clearances in vitro is critical to facilitate pharmacokinetics and drug-drug interaction predictions. Here, we evaluated the plated human hepatocytes (PHH) and studied the effect of incubation temperature and inhibitor concentration on uptake measurements, in order to reliably estimate hepatic uptake components. Uptake rates measured using PHH, at 37°C without and with rifamycin SV, were comparable with those obtained from suspension hepatocytes and sandwich-cultured hepatocytes for a set of 10-13 compounds. Apparent permeability across monolayers of low-efflux Madin-Darby canine kidney cells was measured at 4, 10, and 37°C. Of the 23 compounds evaluated, 13 compounds showed >2-fold reduction in passive permeability at 4°C compared to 37°C, inferring that low-temperature incubations may underestimate passive uptake. Inhibition studies using transporter-transfected cells suggested that â¼20 µM rifamycin SV completely inhibited organic anion-transporting polypeptides (OATPs), while no significant inhibition was noted for other hepatic uptake transporters. On the basis of inhibition profiles, the contribution of active versus passive and OATP versus non-OATP transport to the PHH uptake was discerned for various endogenous substrates and statins. With the exception of fluvastatin, the statins studied were predominantly transported by OATPs in PHH and the non-OATP transporters, such as Na+-taurocholate co-transporting polypeptide, played a minimal role. In conclusion, PHH is useful for uptake measurements, and rifamycin SV employed at different concentrations can reliably estimate active and passive uptake and characterize OATP-dependent active uptake.