RESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease inhibitor PF-07321332 (nirmatrelvir), in combination with ritonavir (Paxlovid), was recently granted emergency use authorization by multiple regulatory agencies for the treatment of coronavirus disease 2019 (COVID-19) in adults and pediatric patients. Disposition studies on nirmatrelvir in animals and in human reagents, which were used to support clinical studies, are described herein. Plasma clearance was moderate in rats (27.2 ml/min per kg) and monkeys (17.1 ml/min per kg), resulting in half-lives of 5.1 and 0.8 hours, respectively. The corresponding oral bioavailability was moderate in rats (34%-50%) and low in monkeys (8.5%), primarily due to oxidative metabolism along the gastrointestinal tract in this species. Nirmatrelvir demonstrated moderate plasma protein binding in rats, monkeys, and humans with mean unbound fractions ranging from 0.310 to 0.478. The metabolism of nirmatrelvir was qualitatively similar in liver microsomes and hepatocytes from rats, monkeys, and humans; prominent metabolites arose via cytochrome P450 (CYP450)-mediated oxidations on the P1 pyrrolidinone ring, P2 6,6-dimethyl-3-azabicyclo[3.1.0]hexane, and the tertiary-butyl group at the P3 position. Reaction phenotyping studies in human liver microsomes revealed that CYP3A4 was primarily responsible (fraction metabolized = 0.99) for the oxidative metabolism of nirmatrelvir. Minor clearance mechanisms involving renal and biliary excretion of unchanged nirmatrelvir were also noted in animals and in sandwich-cultured human hepatocytes. Nirmatrelvir was a reversible and time-dependent inhibitor as well as inducer of CYP3A activity in vitro. First-in-human pharmacokinetic studies have demonstrated a considerable boost in the oral systemic exposure of nirmatrelvir upon coadministration with the CYP3A4 inhibitor ritonavir, consistent with the predominant role of CYP3A4 in nirmatrelvir metabolism. SIGNIFICANCE STATEMENT: The manuscript describes the preclinical disposition, metabolism, and drug-drug interaction potential of PF-07321332 (nirmatrelvir), an orally active peptidomimetic-based inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease, which has been granted emergency use authorization by multiple regulatory agencies around the globe for the treatment of coronavirus disease 2019 (COVID-19) in COVID-19-positive adults and pediatric patients who are at high risk for progression to severe COVID-19, including hospitalization or death.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Administração Oral , Animais , Criança , Citocromo P-450 CYP3A/metabolismo , Haplorrinos , Humanos , Lactamas , Leucina , Microssomos Hepáticos/metabolismo , Nitrilas , Peptídeo Hidrolases/metabolismo , Prolina , Ratos , Ritonavir/metabolismoRESUMO
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
The aim of the present study was to quantitatively evaluate the drug-drug interactions (DDIs) of maraviroc (MVC) with various perpetrator drugs, including telaprevir (TVR), using an in vitro data-informed physiologically based pharmacokinetic (PBPK) model. MVC showed significant active uptake and biliary excretion in sandwich-cultured human hepatocytes, and biphasic organic anion transporting polypeptide (OATP)1B1-mediated uptake kinetics in transfected cells (high-affinity K m â¼5 µM). No measureable active uptake was noted in OATP1B3- and OATP2B1-transfceted cells. TVR inhibited OATP1B1-mediated MVC transport in vitro, and also exhibited CYP3A time-dependent inhibition in human hepatocytes (inactivation constant, K I = 2.24 µM, and maximum inactivation rate constant, k inact = 0.0112 minute-1). The inactivation efficiency (k inact/K I) was approximately 34-fold lower in human hepatocytes compared with liver microsomes. A PBPK model accounting for interactions involving CYP3A, P-glycoprotein (P-gp), and OATP1B1 was developed based on in vitro mechanistic data. MVC DDIs with ketoconazole (inhibition of CYP3A and P-gp), ritonavir (inhibition of CYP3A and P-gp), efavirenz (induction of CYP3A), rifampicin (induction of CYP3A and P-gp; inhibition of OATP1B1), and TVR (inhibition of CYP3A, P-gp, and OATP1B1) were well described by the PBPK model with optimized transporter K i values implying that OATP1B1-mediated uptake along with CYP3A metabolism determines the hepatic clearance of MVC, and P-gp-mediated efflux limits its intestinal absorption. In summary, MVC disposition involves intestinal P-gp/CYP3A and hepatic OATP1B1/CYP3A interplay, and TVR simultaneously inhibits these multiple mechanisms leading to a strong DDI-about 9.5-fold increase in MVC oral exposure.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas/fisiologia , Hepatócitos/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Fígado/metabolismo , Maraviroc/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Células HEK293 , Humanos , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Microssomos Hepáticos/metabolismoRESUMO
High-permeability-low-molecular-weight acids/zwitterions [i.e., extended clearance classification system class 1A (ECCS 1A) drugs] are considered to be cleared by metabolism with a minimal role of membrane transporters in their hepatic clearance. However, a marked disconnect in the in vitro-in vivo (IVIV) translation of hepatic clearance is often noted for these drugs. Metabolic rates measured using human liver microsomes and primary hepatocytes tend to underpredict. Here, we evaluated the role of organic anion transporter 2 (OAT2)-mediated hepatic uptake in the clearance of ECCS 1A drugs. For a set of 25 ECCS 1A drugs, in vitro transport activity was assessed using transporter-transfected cells and primary human hepatocytes. All but two drugs showed substrate affinity to OAT2, whereas four (bromfenac, entacapone, fluorescein, and nateglinide) also showed OATP1B1 activity in transfected cells. Most of these drugs (21 of 25) showed active uptake by plated human hepatocytes, with rifamycin SV (pan-transporter inhibitor) reducing the uptake by about 25%-95%. Metabolic turnover was estimated for 19 drugs after a few showed no measurable substrate depletion in liver microsomal incubations. IVIV extrapolation using in vitro data was evaluated to project human hepatic clearance of OAT2-alone substrates considering 1) uptake transport only, 2) metabolism only, and 3) transporter-enzyme interplay (extended clearance model). The transporter-enzyme interplay approach achieved improved prediction accuracy (average fold error = 1.9 and bias = 0.93) compared with the other two approaches. In conclusion, this study provides functional evidence for the role of OAT2-mediated hepatic uptake in determining the pharmacokinetics of several clinically important ECCS 1A drugs.
Assuntos
Fígado/efeitos dos fármacos , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Permeabilidade/efeitos dos fármacos , Preparações Farmacêuticas/administração & dosagem , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Peso MolecularRESUMO
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
(R)-4-((4-(((4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol (TBPT), a serotonin-4 receptor partial agonist, is metabolized to two metabolites: an N-dealkylation product [(R)-3-(piperidin-4-ylmethoxy)-4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazole (M1)] and a cyclized oxazolidine structure [7-(((4-(((R)-tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)octahydro-3H (M2)]. After administration of TBPT to humans the exposure to M1 was low and the exposure to M2 was high, relative to the parent drug, despite this being the opposite in vitro. In this study, projection of the plasma metabolite/parent (M/P) ratios for M1 and M2 was attempted using in vitro metabolism, binding, and permeability data in static and dynamic physiologically based pharmacokinetic (PBPK) models. In the static model, the fraction of parent clearance yielding the metabolite (which also required taking into account secondary metabolites of M1 and M2), the clearance of the metabolites and parent, and an estimate of the availability of the metabolites from the liver were combined to yield estimated parent/metabolite ratios of 0.32 and 23 for M1 and M2, respectively. PBPK modeling that used in vitro and physicochemical data input yielded estimates of 0.26 and 20, respectively. The actual values were 0.12 for M1/TBPT and 58 for M2/TBPT. Thus, the ratio for M1 was overpredicted, albeit at values less than unity. The ratio for M2/TBPT was underpredicted, and the high ratio of 58 may exceed a limiting ceiling of the approach. Nevertheless, when considered in the context of determining whether a potential circulating metabolite may be quantitatively important prior to administration of a drug for the first time to humans, the approaches succeeded in highlighting the importance of M2 (M/P ratio >> 1) relative to M1, despite M1 being much greater than M2 in vitro.
Assuntos
Furanos/sangue , Furanos/farmacocinética , Inativação Metabólica/fisiologia , Oxazóis/sangue , Oxazóis/farmacocinética , Agonistas do Receptor de Serotonina/sangue , Agonistas do Receptor de Serotonina/farmacocinética , Adulto , Ciclização/fisiologia , Remoção de Radical Alquila/fisiologia , Feminino , Hepatócitos/metabolismo , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Adulto JovemRESUMO
Predicting human pharmacokinetics of novel compounds is a critical step in drug discovery and clinical study design but continues to be a challenging task for hepatic transporter substrates, particularly in predicting their liver exposures. In this study, using bosentan as an example, we prospectively predicted systemic exposure and the (pseudo) steady-state unbound liver-to-unbound plasma ratio (Kpuu) in healthy subjects using 1) a mechanistic approach solely based on in vitro hepatocyte assays and 2) an approach based on hepatic process rates from monkey in vivo data but Michaelis-Menten constants from in vitro data. Both methods reasonably match the observed human systemic time course data, but the second method leads to better prediction accuracy. In addition, the second method can predict a human Kpuu value that is close to the value deduced using clinical data. We also generated rat and monkey liver Kpuu values in terminal studies. However, these directly measured animal values are different from the deduced human value.
Assuntos
Fígado/metabolismo , Sulfonamidas/farmacocinética , Animais , Bosentana , Descoberta de Drogas/métodos , Haplorrinos , Voluntários Saudáveis , Hepatócitos/metabolismo , Humanos , Modelos Biológicos , Ratos , Sulfonamidas/sangueRESUMO
Targeted protein quantification using liquid chromatography-tandem mass spectrometry with stable isotope-labeled standards is recognized as the gold standard of practice for protein quantification. Such assays, however, can only cover a limited number of proteins, and developing targeted methods for larger numbers of proteins requires substantial investment. Alternatively, large-scale global proteomic experiments along with computational methods such as the "total protein approach" (TPA) have the potential to provide extensive protein quantification. In this study, we compared the TPA-based quantitation of seven major hepatic uptake transporters in four human liver tissue samples using global proteomic data obtained from two multiplexed tandem mass tag experiments (performed in two independent laboratories) to the quantitative data from targeted proteomic assays. The TPA-based quantitation of these hepatic transporters [sodium-taurocholate cotransporting polypeptide (NTCP/SLC10A1), organic anion transporter 2 (OAT2/SLC22A7), OAT7/SLC22A9, organic anion-transporting polypeptide 1B1 (OATP1B1/SLCO1B1), OATP1B3/SLCO1B3, OATP2B1/SLCO2B1, and organic cation transporter (OCT1/SLC22A1)] showed good-to-excellent correlations (Pearson r = 0.74-1.00) to the targeted data. In addition, the values were similar to those measured by targeted proteomics with 71% and 86% of the data sets falling within 3-fold of the targeted data. A comparison of the TPA-based quantifications of enzyme abundances to available literature data showed that the majority of the enzyme quantifications fell within the reference data intervals. In conclusion, these results demonstrate the capability of multiplexed global proteomic experiments to detect differences in protein expression between samples and provide reasonable estimations of protein expression levels.
Assuntos
Transporte Biológico/fisiologia , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Preparações Farmacêuticas/metabolismo , Cromatografia Líquida/métodos , Hepatócitos/metabolismo , Humanos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Understanding liver exposure of hepatic transporter substrates in clinical studies is often critical, as it typically governs pharmacodynamics, drug-drug interactions, and toxicity for certain drugs. However, this is a challenging task since there is currently no easy method to directly measure drug concentration in the human liver. Using bosentan as an example, we demonstrate a new approach to estimate liver exposure based on observed systemic pharmacokinetics from clinical studies using physiologically based pharmacokinetic modeling. The prediction was verified to be both accurate and precise using sensitivity analysis. For bosentan, the predicted pseudo steady-state unbound liver-to-unbound systemic plasma concentration ratio was 34.9 (95% confidence interval: 4.2, 50). Drug-drug interaction (i.e., CYP3A and CYP2B6 induction) and inhibition of hepatic transporters (i.e., bile salt export pump, multidrug resistance-associated proteins, and sodium-taurocholate cotransporting polypeptide) were predicted based on the estimated unbound liver tissue or plasma concentrations. With further validation and refinement, we conclude that this approach may serve to predict human liver exposure and complement other methods involving tissue biopsy and imaging.
Assuntos
Fígado/metabolismo , Sulfonamidas/sangue , Sulfonamidas/farmacocinética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bosentana , Interações Medicamentosas/fisiologia , Voluntários Saudáveis , Hepatócitos/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismoRESUMO
Organic anion transporter (OAT) 2 and OAT7 were recently shown to be involved in the hepatic uptake of drugs; however, there is limited understanding of the population variability in the expression of these transporters in liver. There is also a need to derive relative expression-based scaling factors (REFs) that can be used to bridge in vitro functional data to the in vivo drug disposition. To this end, we quantified OAT2 and OAT7 surrogate peptide abundance in a large number of human liver tissue samples ( n = 52), as well as several single-donor cryopreserved human hepatocyte lots ( n = 30) by a novel, validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The average surrogate peptide expression of OAT2 and OAT7 in the liver samples was 1.52 ± 0.57 and 4.63 ± 1.58 fmol/µg membrane protein, respectively. While we noted statistically significant differences ( p < 0.05) in hepatocyte and liver tissue abundances for both OAT2 and OAT7, the differences were relatively small (1.8- and 1.5-fold difference in median values, respectively). Large interindividual variability was noted in the hepatic expression of OAT2 (16-fold in liver tissue and 23-fold in hepatocytes). OAT7, on the other hand, showed less interindividual variability (4-fold) in the livers, but high variability for the hepatocyte lots (27-fold). A significant positive correlation in OAT2 and OAT7 expression was observed, but expression levels were neither associated with age nor sex. In conclusion, our data suggest marked interindividual variability in the hepatic expression of OAT2/7, which may contribute to the pharmacokinetic variability of their substrates. Because both transporters were less abundant in hepatocytes than livers, a REF-based approach is recommended when scaling in vitro hepatocyte transport data to predict hepatic drug clearance and liver exposure of OAT2/7 substrates.
Assuntos
Hepatócitos/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteômica/métodos , Adulto , Fatores Etários , Idoso , Variação Biológica da População , Criança , Cromatografia Líquida , Feminino , Células HEK293 , Humanos , Fígado/citologia , Masculino , Pessoa de Meia-Idade , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Cultura Primária de Células , Fatores Sexuais , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
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
Targeting of the human ribosome is an unprecedented therapeutic modality with a genome-wide selectivity challenge. A liver-targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesteraseâ 1. The synthesis of select tetrazole prodrugs was crucial. A cell-free inâ vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. Inâ vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography 18 F-isotopologue validated our liver-targeting approach.
Assuntos
Fígado/efeitos dos fármacos , Inibidores de PCSK9 , Pró-Proteína Convertase 9/biossíntese , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/enzimologia , Fígado/metabolismo , Estrutura Molecular , Pró-Proteína Convertase 9/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Methods to predict the pharmacokinetics of drugs in humans from in vitro data have been established, but corresponding methods to predict exposure to circulating metabolites are unproven. The objective of this study was to use in vitro methods combined with static and dynamic physiologically based pharmacokinetic (PBPK) models to predict metabolite exposures, using midazolam and its major metabolites as a test system. Intrinsic clearances (CLint) of formation of individual metabolites were determined using human liver microsomes. Metabolic CLintof hydroxymidazolam metabolites via oxidation and glucuronidation were also determined. Passive diffusion intrinsic clearances of hydroxymidazolam metabolites were determined using sandwich cultured human hepatocytes and the combination of this term along with the metabolic CLint, and liver blood flow was used to estimate the fraction of the metabolite that can enter the systemic circulation after formation in the liver. The metabolite/parent drug area under the plasma concentration-time curve ratio (AUCm/AUCp) was predicted using a static model relating the fraction of midazolam clearance to each metabolite, the clearance rates of midazolam and hydroxymidazolam metabolites, and the availability of the metabolites. Additionally, the human disposition of midazolam metabolites was simulated using a SimCYP PBPK model. Both approaches yielded AUCm/AUCpratios that were in agreement with the in vivo ratios. This study shows that in vivo midazolam metabolite exposure can be predicted from in vitro data and PBPK modeling. This study emphasized the importance of metabolite systemic availability from its tissue of formation, which remains a challenge to quantitative prediction.
Assuntos
Midazolam/metabolismo , Hepatócitos/metabolismo , Humanos , Cinética , Fígado/metabolismo , Taxa de Depuração Metabólica/fisiologia , Microssomos Hepáticos/metabolismo , Modelos BiológicosRESUMO
Gemfibrozil has been suggested as a sensitive cytochrome P450 2C8 (CYP2C8) inhibitor for clinical investigation by the U.S. Food and Drug Administration and the European Medicines Agency. However, gemfibrozil drug-drug interactions (DDIs) are complex; its major circulating metabolite, gemfibrozil 1-O-ß-glucuronide (Gem-Glu), exhibits time-dependent inhibition of CYP2C8, and both parent and metabolite also behave as moderate inhibitors of organic anion transporting polypeptide 1B1 (OATP1B1) in vitro. Additionally, parent and metabolite also inhibit renal transport mediated by OAT3. Here, in vitro inhibition data for gemfibrozil and Gem-Glu were used to assess their impact on the pharmacokinetics of several victim drugs (including rosiglitazone, pioglitazone, cerivastatin, and repaglinide) by employing both static mechanistic and dynamic physiologically based pharmacokinetic (PBPK) models. Of the 48 cases evaluated using the static models, about 75% and 98% of the DDIs were predicted within 1.5- and 2-fold of the observed values, respectively, when incorporating the interaction potential of both gemfibrozil and its 1-O-ß-glucuronide. Moreover, the PBPK model was able to recover the plasma profiles of rosiglitazone, pioglitazone, cerivastatin, and repaglinide under control and gemfibrozil treatment conditions. Analyses suggest that Gem-Glu is the major contributor to the DDIs, and its exposure needed to bring about complete inactivation of CYP2C8 is only a fraction of that achieved in the clinic after a therapeutic gemfibrozil dose. Overall, the complex interactions of gemfibrozil can be quantitatively rationalized, and the learnings from this analysis can be applied in support of future predictions of gemfibrozil DDIs.
Assuntos
Proteínas de Transporte/metabolismo , Enzimas/metabolismo , Genfibrozila/farmacologia , Genfibrozila/farmacocinética , Hipolipemiantes/farmacologia , Hipolipemiantes/farmacocinética , Algoritmos , Área Sob a Curva , Simulação por Computador , Citocromo P-450 CYP2C8/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacologia , Interações Medicamentosas , Glucuronídeos/metabolismo , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , Modelos Biológicos , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismoRESUMO
Gemfibrozil (GEM), which decreases serum triglycerides and low density lipoprotein, perpetrates drug-drug interactions (DDIs) with several drugs. These DDIs are primarily attributed to the inhibition of drug transporters and metabolic enzymes, particularly cytochrome P450 (CYP) 2C8 by the major circulating metabolite gemfibrozil 1-O-ß-glucuronide (GG). Here, we characterized the transporter-mediated hepatic disposition of GEM and GG using sandwich-cultured human hepatocytes (SCHH) and transporter-transfect systems. Significant active uptake was noted in SCHH for the metabolite. GG, but not GEM, showed substrate affinity to organic anion transporting polypeptide (OATP) 1B1, 1B3, and 2B1. In SCHH, glucuronidation was characterized affinity constants (Km) of 7.9 and 61.4 µM, and biliary excretion of GG was observed. Furthermore, GG showed active basolateral efflux from preloaded SCHH and ATP-dependent uptake into membrane vesicles overexpressing multidrug resistance-associated protein (MRP) 2, MRP3, and MRP4. A mathematical model was developed to estimate hepatic uptake and efflux kinetics of GEM and GG based on SCHH studies. Collectively, the hepatic transporters play a key role in the disposition and thus determine the local concentrations of GEM and more so for GG, which is the predominant inhibitory species against CYP2C8 and OATP1B1.
Assuntos
Genfibrozila/metabolismo , Glucuronídeos/metabolismo , Hepatócitos/metabolismo , Hipolipemiantes/metabolismo , Fígado/metabolismo , Transporte Biológico , Cromatografia Líquida , Células HEK293 , Humanos , Modelos Teóricos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Espectrometria de Massas em TandemRESUMO
Quantitative prediction of complex drug-drug interactions (DDIs) involving hepatic transporters and cytochromes P450 (P450s) is challenging. We evaluated the extent of DDIs of nine victim drugs-which are substrates to organic anion-transporting polypeptide 1B1 and undergo P450 metabolism or biliary elimination-caused by five perpetrator drugs, using in vitro data and the proposed extended net-effect model. Hepatobiliary transport and metabolic clearance estimates were obtained from in vitro studies. Of the total of 62 clinical interaction combinations assessed using the net-effect model, 58 (94%) could be predicted within a 2-fold error, with few false-negative predictions. Model predictive performance improved significantly when in vitro active uptake clearance was corrected to recover in vivo clearance. The basic R-value model yielded only 63% predictions within 2-fold error. This study demonstrates that the interactions involving transporter-enzyme interplay need to be mechanistically assessed for quantitative rationalization and prospective prediction.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Hepatócitos/metabolismo , Modelos Biológicos , Transportadores de Ânions Orgânicos/metabolismo , Anti-Infecciosos/farmacocinética , Anti-Infecciosos/farmacologia , Anti-Hipertensivos/farmacocinética , Anti-Hipertensivos/farmacologia , Células Cultivadas , Feminino , Hepatócitos/efeitos dos fármacos , Humanos , Hipolipemiantes/farmacocinética , Hipolipemiantes/farmacologia , MasculinoRESUMO
A previously developed physiologically based pharmacokinetic model for hepatic transporter substrates was extended to an organic anion transporting polypeptide substrate, telmisartan. Predictions used in vitro data from sandwich culture human hepatocyte and human liver microsome assays. We have developed a novel method to calibrate partition coefficients (Kps) between nonliver tissues and plasma on the basis of published human positron emission tomography (PET) data to decrease the uncertainty in tissue distribution introduced by in silico-predicted Kps. With in vitro data-predicted hepatic clearances, published empirical scaling factors, and PET-calibrated Kps, the model could accurately recapitulate telmisartan pharmacokinetic (PK) behavior before 2.5 hours. Reasonable predictions also depend on having a model structure that can adequately describe the drug disposition pathways. We showed that the elimination phase (2.5-12 hours) of telmisartan PK could be more accurately recapitulated when enterohepatic recirculation of parent compound derived from intestinal deconjugation of glucuronide metabolite was incorporated into the model. This study demonstrated the usefulness of the previously proposed physiologically based modeling approach for purely predictive intravenous PK simulation and identified additional biologic processes that can be important in prediction.
Assuntos
Benzimidazóis/farmacocinética , Benzoatos/farmacocinética , Hepatócitos/metabolismo , Microssomos Hepáticos/metabolismo , Simulação por Computador , Humanos , Modelos Biológicos , Transportadores de Ânions Orgânicos/metabolismo , TelmisartanRESUMO
BACKGROUND AND OBJECTIVE: Physiologically based pharmacokinetic (PBPK) models are valuable for translating in vitro absorption, distribution, metabolism, and excretion (ADME) data to predict clinical pharmacokinetics, and can enable discovery and early clinical stages of pharmaceutical research. However, in predicting pharmacokinetics of organic anion transporting polypeptide (OATP) 1B substrates based on in vitro transport and metabolism data, PBPK models typically require additional empirical in vitro-to-in vivo scaling factors (ESFs) in order to accurately recapitulate observed clinical profiles. As model simulation is very sensitive to ESFs, a critical evaluation of ESF estimation is prudent. Previously studies have applied classic 'two-stage' and 'naïve pooled data' approaches in identifying a set of compound independent ESFs. However, the 'two-stage' approach has the parameter identification issue in separately fitting data for individual compounds, while the 'naïve pooled data' approach ignores interstudy variability, leading to potentially biased ESF estimates. METHODS: In this study, we have applied a nonlinear mixed-effect approach in estimating ESF of the PBPK model and incorporated additional data from 86 runs of in vitro uptake assay and 49 clinical studies of 12 training compounds in model development to further enhance the translation of in vitro data to predict the pharmacokinetics of OATP1B substrate drugs. To test predication accuracy of the model, a 'leave-one-out' analysis has been performed. RESULTS: The established model can reasonably describe the clinical observations, with both mean values and interstudy variabilities quantified for ESF and volume of distribution parameters. The mean estimates are largely consistent with values in the previous reports. The interstudy variabilities of these parameters are estimated to be at least 50% (as coefficient of variation). Most compounds can be reasonably predicted in the 'leave-one-out' analysis. CONCLUSION: This study improves the confidence in predicting the pharmacokinetics of OATP1B substrates in individual studies of small sample sizes, and quantifies the variability associated with the prediction.
Assuntos
Transportador 1 de Ânion Orgânico Específico do Fígado , Modelos Biológicos , Dinâmica não Linear , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Simulação por ComputadorRESUMO
Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.
Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , Inibidores de Proteases , SARS-CoV-2 , Humanos , Animais , Camundongos , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , Antivirais/farmacocinética , Antivirais/uso terapêutico , Antivirais/química , Administração Oral , Inibidores de Proteases/farmacologia , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/uso terapêutico , Inibidores de Proteases/química , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Ratos , COVID-19/virologiaRESUMO
PURPOSE: Quantitative prediction of complex drug-drug interactions (DDIs) is challenging. Repaglinide is mainly metabolized by cytochrome-P-450 (CYP)2C8 and CYP3A4, and is also a substrate of organic anion transporting polypeptide (OATP)1B1. The purpose is to develop a physiologically based pharmacokinetic (PBPK) model to predict the pharmacokinetics and DDIs of repaglinide. METHODS: In vitro hepatic transport of repaglinide, gemfibrozil and gemfibrozil 1-O-ß-glucuronide was characterized using sandwich-culture human hepatocytes. A PBPK model, implemented in Simcyp (Sheffield, UK), was developed utilizing in vitro transport and metabolic clearance data. RESULTS: In vitro studies suggested significant active hepatic uptake of repaglinide. Mechanistic model adequately described repaglinide pharmacokinetics, and successfully predicted DDIs with several OATP1B1 and CYP3A4 inhibitors (<10% error). Furthermore, repaglinide-gemfibrozil interaction at therapeutic dose was closely predicted using in vitro fraction metabolism for CYP2C8 (0.71), when primarily considering reversible inhibition of OATP1B1 and mechanism-based inactivation of CYP2C8 by gemfibrozil and gemfibrozil 1-O-ß-glucuronide. CONCLUSIONS: This study demonstrated that hepatic uptake is rate-determining in the systemic clearance of repaglinide. The model quantitatively predicted several repaglinide DDIs, including the complex interactions with gemfibrozil. Both OATP1B1 and CYP2C8 inhibition contribute significantly to repaglinide-gemfibrozil interaction, and need to be considered for quantitative rationalization of DDIs with either drug.