RESUMEN
The propensity for aldehyde oxidase (AO) substrates to be implicated in drug-drug interactions (DDIs) is not well understood due to the dearth of potent inhibitors that elicit in vivo inhibition of AO. Although there is only one reported instance of DDI that has been ascribed to the inhibition of AO to date, the supporting evidence for this clinical interaction is rather tenuous, and its veracity has been called into question. Our group recently reported that the epidermal growth factor receptor inhibitor erlotinib engendered potent time-dependent inhibition of AO with inactivation kinetic constants in the same order of magnitude as its free circulating plasma concentrations. At the same time, it was previously reported that the concomitant administration of erlotinib with the investigational drug OSI-930 culminated in a an approximately twofold increase in its systemic exposure. Although the basis underpinning this interaction remains unclear, the structure of OSI-930 contains a quinoline motif that is amenable to oxidation at the electrophilic carbon adjacent to the nitrogen atom by molybdenum-containing hydroxylases like AO. In this study, we conducted metabolite identification that revealed that OSI-930 undergoes AO metabolism to a mono-oxygenated 2-oxo metabolite and assessed its formation kinetics in human liver cytosol. Additionally, reaction phenotyping in human hepatocytes revealed that AO contributes nearly 50% to the overall metabolism of OSI-930. Finally, modeling the interaction between erlotinib and OSI-930 using a mechanistic static model projected an â¼1.85-fold increase in the systemic exposure of OSI-930, which accurately recapitulated clinical observations. SIGNIFICANCE STATEMENT: This study delineates an aldehyde oxidase (AO) metabolic pathway in the investigational drug OSI-930 for the first time and confirmed that it represented a major route of metabolism through reaction phenotyping in human hepatocytes. Our study provided compelling mechanistic and modeling evidence for the first instance of an AO-mediated clinical drug-drug interaction stemming from the in vivo inhibition of the AO-mediated quinoline 2-oxidation pathway in OSI-930 by erlotinib.
Asunto(s)
Aldehído Oxidasa , Interacciones Farmacológicas , Clorhidrato de Erlotinib , Humanos , Aldehído Oxidasa/metabolismo , Aldehído Oxidasa/antagonistas & inhibidores , Clorhidrato de Erlotinib/farmacología , Clorhidrato de Erlotinib/farmacocinética , Interacciones Farmacológicas/fisiología , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Aldehyde oxidase (AO) is a molybdenum cofactor-containing cytosolic enzyme that has gained prominence due to its involvement in the developmental failure of several drug candidates in first-in-human trials. Unlike cytochrome P450s (P450) and glucuronosyltransferase, AO substrates have been plagued by poor in vitro to in vivo extrapolation, leading to low systemic exposures and underprediction of human dose. However, apart from measuring a drug's AO clearance rates, it is also important to determine the relative contribution to metabolism by this enzyme (fm,AO). Although hydralazine is the most well-studied time-dependent inhibitor (TDI) of AO and is frequently employed for AO reaction phenotyping in human hepatocytes to derive fm,AO, multiple studies have expressed concerns pertaining to its utility in providing accurate estimates of fm,AO values due to its propensity to significantly inhibit P450s at the concentrations typically used for reaction phenotyping. In this study, we characterized icotinib, a cyclized analog of erlotinib, as a potent TDI of AO-inactivating human liver cytosolic zoniporide 2-oxidation equipotently with erlotinib with a maximal inactivate rate/inactivator concentration at half maximal inactivation rate (K I) ratio of 463 and 501 minute-1mM-1 , respectively. Moreover, icotinib also exhibits selectivity against P450 and elicits significantly weaker inhibition against human liver microsomal UGT1A1/3 as compared with erlotinib. Finally, we evaluated icotinib as an inhibitor of AO for reaction phenotyping in cryopreserved human hepatocytes and demonstrated that it can yield more accurate prediction of fm,AO compared with hydralazine and induce sustained suppression of AO activity at higher cell densities, which will be important for reaction phenotyping endeavors of low clearance drugs SIGNIFICANCE STATEMENT: In this study, we characterized icotinib as a potent time-dependent inhibitor of AO with ample selectivity margins against the P450s and UGT1A1/3 and demonstrated its utility for reaction phenotyping in human hepatocytes to obtain accurate estimates of fm,AO for victim DDI risk predictions. We envisage the adoption of icotinib in place of hydralazine in AO reaction phenotyping.
Asunto(s)
Aldehído Oxidasa , Hepatocitos , Fenotipo , Quinazolinas , Humanos , Aldehído Oxidasa/metabolismo , Aldehído Oxidasa/antagonistas & inhibidores , Éteres Corona , Inhibidores Enzimáticos/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Quinazolinas/farmacologíaRESUMEN
The biochemical basis for substrate dependences in apparent inhibition constant values (Ki) remains unknown. Our study aims to elucidate plausible structural determinants underpinning these observations. In vitro steady-state inhibition assays conducted using human recombinant CYP3A4 enzyme and testosterone substrate revealed that fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and pemigatinib noncompetitively inhibited CYP3A4 with apparent Ki values of 10.2 ± 1.1 and 3.3 ± 0.9 µM, respectively. However, when rivaroxaban was adopted as the probe substrate, there were 2.0- and 3.2-fold decreases in its apparent Ki values. To glean mechanistic insights into this phenomenon, erdafitinib and pemigatinib were docked to allosteric sites in CYP3A4. Subsequently, molecular dynamics (MD) simulations of apo- and holo-CYP3A4 were conducted to investigate the structural changes induced. Comparative structural analyses of representative MD frames extracted by hierarchical clustering revealed that the allosteric inhibition of CYP3A4 by erdafitinib and pemigatinib did not substantially modulate its active site characteristics. In contrast, we discovered that allosteric binding of the FGFR inhibitors reduces the structural flexibility of the F-F' loop region, an important gating mechanism to regulate access of the substrate to the catalytic heme. We surmised that the increased rigidity of the F-F' loop engenders a more constrained entrance to the CYP3A4 active site, which in turn impedes access to the larger rivaroxaban molecule to a greater extent than testosterone and culminates in more potent inhibition of its CYP3A4-mediated metabolism. Our findings suggest a potential mechanism to rationalize probe substrate dependencies in Ki arising from the allosteric noncompetitive inhibition of CYP3A4.
Asunto(s)
Citocromo P-450 CYP3A , Rivaroxabán , Humanos , Citocromo P-450 CYP3A/metabolismo , Sitio Alostérico , Simulación de Dinámica Molecular , Testosterona/metabolismoRESUMEN
Infigratinib (INF) is a fibroblast growth factor receptor inhibitor that was recently United States Food and Drug Administration-approved for the treatment of advanced or metastatic cholangiocarcinoma. We previously established that INF inhibited and inactivated cytochrome P450 3A4 (CYP3A4). Here, in a follow up to our previous study, we identified for the first time that INF also elicited potent competitive inhibition and mechanism-based inactivation of CYP2J2 with kinetic parameters K i, K I, k inact, and a partition ratio of 1.94 µM, 0.10 µM, 0.026 minute-1, and â¼3, respectively, when rivaroxaban was harnessed as the probe substrate. Inactivation was revealed to exhibit cofactor-dependency and was attenuated by an alternative substrate (astemizole) and direct inhibitor (nilotinib) of CYP2J2. Additionally, the nature of inactivation was unlikely to be pseudo-irreversible and instead arose from covalent modification due to the lack of substantial enzyme activity recovery after dialysis and chemical oxidation, as well as the lack of a resolvable Soret band in spectral scans. Glutathione trapping confirmed that the identity of the putative reactive intermediate implicated in the covalent inactivation of both CYP2J2 and CYP3A4 was identical and likely attributable to an electrophilic p-benzoquinonediimine intermediate of INF. Finally, mechanistic static modeling revealed that by integrating the previously arcane inhibition and inactivation kinetic parameters of CYP2J2-mediated rivaroxaban hydroxylation by INF illuminated in this work, together with those previously documented for CYP3A4, a 49% increase in the systemic exposure of rivaroxaban was projected. Our modeling results predicted a potential risk of metabolic drug-drug interactions between the clinically relevant combination of rivaroxaban and INF in the setting of cancer. SIGNIFICANCE STATEMENT: This study reported that INF elicits potent reversible inhibition and mechanism-based inactivation of CYP2J2. Furthermore, static modelling predicted that its coadministration with the direct oral anticoagulant rivaroxaban may potentially culminate in a metabolic drug-drug interaction (DDI) leading to an increased risk of major bleeding. As rivaroxaban is steadily gaining prominence as the anticoagulant of choice in the treatment of cancer-associated venous thromboembolism, the DDI projections reported here are clinically relevant and warrant further investigation via physiologically based pharmacokinetic modelling and simulation.
Asunto(s)
Citocromo P-450 CYP3A , Rivaroxabán , Anticoagulantes , Citocromo P-450 CYP2J2 , Citocromo P-450 CYP3A/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Compuestos de Fenilurea , Pirimidinas , Rivaroxabán/farmacocinéticaRESUMEN
Futibatinib (FUT) is a potent inhibitor of fibroblast growth factor receptor (FGFR) 1-4 that is currently under clinical investigation for intrahepatic cholangiocarcinoma. Unlike its predecessors, FUT possesses an acrylamide warhead, which enables it to bind covalently to a free cysteine residue in the FGFR kinase domain. However, it remains uninterrogated if this electrophilic α, ß -unsaturated carbonyl scaffold could also directly or indirectly engender off-target covalent binding to nucleophilic centers on other cellular proteins. Here, we discovered that FUT inactivated both CYP3A isoforms with inactivator concentration at half-maximum inactivation rate constant, maximum inactivation rate constant, and partition ratios of 12.5 and 51.4 µ M, 0.25 and 0.06 minutes-1, and â¼52 and â¼58 for CYP3A4 and CYP3A5, respectively. Along with its time-, concentration-, and cofactor-dependent inhibitory profiles, FUT also exhibited several cardinal features that were consistent with mechanism-based inactivation. Moreover, the nature of inactivation was unlikely to be pseudo-irreversible and instead arose from the covalent modification of the cytochrome P450 apoprotein and/or its heme moiety due to the lack of substantial enzyme activity recovery following dialysis and chemical oxidation, as well as the absence of the diagnostic Soret peak in spectral analyses. Finally, utilizing glutathione (GSH) trapping and high-resolution mass spectrometry, we illuminated that while the acrylamide moiety in FUT could nonenzymatically conjugate to GSH via Michael addition, it was not implicated in the covalent inactivation of CYP3A. Rather, we surmised that it likely stemmed from the metabolic activation of its acrylamide covalent warhead to a highly electrophilic epoxide intermediate that could covalently modify CYP3A and culminate in its catalytic inactivation. SIGNIFICANCE STATEMENT: In this study, we reported for the first time the inactivation of CYP3A by futibatinib (FUT). Furthermore, using FUT as an exemplary targeted covalent inhibitor, our study revealed the propensity for its acrylamide Michael acceptor moiety to be metabolically activated to a highly electrophilic epoxide. Due to the growing resurgence of covalent inhibitors and the well-established toxicological ramifications associated with epoxides, we advocate that closer scrutiny be adopted when profiling the reactive metabolites of compounds possessing an α, ß -unsaturated carbonyl scaffold.
Asunto(s)
Citocromo P-450 CYP3A , Compuestos Epoxi , Acrilamida , Activación Metabólica , Citocromo P-450 CYP3A/metabolismo , Pirazoles , Pirimidinas , Pirroles , Diálisis RenalRESUMEN
We recently established the mechanism-based inactivation (MBI) of cytochrome P450 3A (CYP3A) by the fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and infigratinib. Serendipitously, our preliminary data have also revealed that pemigatinib (PEM), another clinically approved FGFR1-3 inhibitor, similarly elicited time-dependent inhibition of CYP3A. This was rather unexpected, as it was previously purported that PEM did not pose any metabolism-dependent liabilities due to the absence of glutathione-related conjugates in metabolic profiling experiments conducted in human liver microsomes. Here, we confirmed that PEM inhibited both CYP3A isoforms in a time-, concentration-, and cofactor-dependent manner consistent with MBI, with inactivator concentration at half-maximum rate constant, maximum inactivation rate constant, and partition ratio of 8.69 and 11.95 µM, 0.108 and 0.042 min-1, and approximately 44 and approximately 47 for CYP3A4 and CYP3A5, respectively. Although the rate of inactivation was diminished by coincubation with an alternative substrate or direct inhibitor of CYP3A, the inclusion of nucleophilic trapping agents afforded no such protection. Furthermore, the lack of catalytic activity recovery following dialysis and oxidation with potassium ferricyanide coupled with the absence of a spectrally resolvable peak in the Soret region collectively implied that the underlying mechanism of inactivation was not elicited via the formation of pseudo-irreversible metabolite-intermediate complexes. Finally, utilizing cyanide trapping and high-resolution mass spectrometry, we illuminated the direct and sequential oxidative bioactivation of PEM and its major O-desmethylated metabolite at its distal morpholine moiety to reactive iminium ion hard electrophilic species that could covalently inactivate CYP3A via MBI. SIGNIFICANCE STATEMENT: This study reports for the first time the covalent MBI of CYP3A by PEM and deciphered its bioactivation pathway involving the metabolic activation of PEM and its major O-desmethylated metabolite to reactive iminium ion intermediates. Following which, a unique covalent docking methodology was harnessed to unravel the structural and molecular determinants underpinning its inactivation. Findings from this study lay the foundation for future investigation of clinically relevant drug-drug interactions between PEM and concomitant substrates of CYP3A.
Asunto(s)
Inhibidores del Citocromo P-450 CYP3A , Citocromo P-450 CYP3A , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/metabolismo , Humanos , Microsomas Hepáticos/metabolismo , Morfolinas , Pirimidinas , Pirroles , Diálisis RenalRESUMEN
Febuxostat is a second-line xanthine oxidase inhibitor that undergoes extensive hepatic metabolism to yield its major acyl-ß-D-glucuronide metabolite (febuxostat AG). It was recently reported that febuxostat inhibited organic anion transporter 3 (OAT3)-mediated uptake of enalaprilat. Here, we investigated the inhibition of febuxostat and febuxostat AG on OAT3 in transfected human embryonic kidney 293 cells. Our transporter inhibition assays confirmed the potent noncompetitive and competitive inhibition of OAT3-mediated estrone-3-sulfate transport by febuxostat and febuxostat AG with corresponding apparent Ki values of 0.55 and 6.11 µM respectively. After accounting for probe substrate-dependency and protein binding effects, mechanistic static modelling with the direct factor Xa anticoagulant rivaroxaban estimated a 1.47-fold increase in its systemic exposure when co-administered with febuxostat based on OAT3 interaction which in turn exacerbates the bleeding risk from baseline for patients with atrial fibrillation by 1.51-fold. Taken together, our results suggested that the concomitant usage of febuxostat with rivaroxaban may potentially culminate in a clinically-significant drug-drug interaction and result in an increased risk of bleeding as a result of its OAT3 inhibition.
Asunto(s)
Glucurónidos , Rivaroxabán , Interacciones Farmacológicas , Febuxostat/farmacología , Células HEK293 , Humanos , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Rivaroxabán/farmacologíaRESUMEN
Benzbromarone (BBR), a potent uricosuric agent for the management of gout, is known to cause fatal fulminant hepatitis. Although the mechanism of BBR-induced idiosyncratic hepatotoxicity remains unelucidated, cytochrome P450 enzyme-mediated bioactivation of BBR to electrophilic reactive metabolites is commonly regarded as a key molecular initiating event. However, apart from causing aberrant toxicities, reactive metabolites may result in mechanism-based inactivation (MBI) of cytochrome P450. Here, we investigated and confirmed that BBR inactivated CYP3A4 in a time-, concentration-, and NADPH-dependent manner with K I, k inact, and partition ratio of 11.61 µM, 0.10 minutes-1, and 110, respectively. Coincubation with ketoconazole, a competitive inhibitor of CYP3A4, attenuated the MBI of CYP3A4 by BBR, whereas the presence of glutathione and catalase did not confer such protection. The lack of substantial recovery of enzyme activity postdialysis and after oxidation with potassium ferricyanide, combined with the absence of a Soret peak in spectral difference scans, implied that MBI of CYP3A4 by BBR did not occur through the formation of quasi-irreversible metabolite-intermediate complexes. Analysis of the reduced CO-difference spectrum revealed an â¼44% reduction in ferrous-CO binding and hinted that inactivation is mediated via irreversible covalent adduction to both the prosthetic heme moiety and the apoprotein. Finally, our in silico covalent docking analysis further suggested the modulation of substrate binding to CYP3A4 via the covalent adduction of epoxide-derived reactive intermediates of BBR to two key cysteine residues (Cys239 and Cys58) vicinal to the entrance of the orthosteric binding site. SIGNIFICANCE STATEMENT: Although the bioactivation of benzbromarone (BBR) to reactive metabolites has been well characterized, its potential to cause mechanism-based inactivation (MBI) of cytochrome P450 has not been fully investigated. This study reports the MBI of CYP3A4 by BBR via irreversible covalent adduction and develops a unique covalent docking methodology to predict the structural molecular determinants underpinning the inactivation for the first time. These findings lay the groundwork for future investigation of clinically relevant drug-drug interactions implicating BBR and mechanisms of BBR-induced idiosyncratic hepatotoxicity.
Asunto(s)
Benzbromarona/farmacología , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Simulación del Acoplamiento Molecular/métodos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Uricosúricos/farmacologíaRESUMEN
Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and ß of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares â¼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.
Asunto(s)
Benzbromarona/química , Inhibidores del Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/química , Benzbromarona/metabolismo , Benzbromarona/farmacología , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/farmacología , Humanos , Hidroxilación/efectos de los fármacos , Hidroxilación/fisiología , Concentración 50 Inhibidora , Midazolam/metabolismo , Midazolam/farmacología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Rivaroxabán/metabolismo , Rivaroxabán/farmacología , Testosterona/metabolismo , Testosterona/farmacologíaRESUMEN
Infigratinib (INF) is a promising selective inhibitor of fibroblast growth factor receptors 1-3 that has recently been accorded both orphan drug designation and priority review status by the US Food and Drug Administration for the treatment of advanced cholangiocarcinoma. Its propensity to undergo bioactivation to electrophilic species was recently expounded upon. However, other than causing aberrant idiosyncratic toxicities, these reactive intermediates may elicit mechanism-based inactivation of cytochrome P450 enzymes. In this study, we investigated the interactions between INF and the most abundant hepatic CYP3A. Our findings revealed that, apart from being a potent noncompetitive reversible inhibitor of CYP3A4, INF inactivated CYP3A4 in a time-, concentration- and NADPH-dependent manner with inactivator concentration at half-maximum inactivation rate constant, maximum inactivation rate constant, and partition ratio of 4.17 µM, 0.068 minute-1, and 41, respectively, when rivaroxaban was employed as the probe substrate. Coincubation with testosterone (alternative CYP3A substrate) or ketoconazole (direct CYP3A inhibitor) attenuated the rate of inactivation, whereas the inclusion of glutathione and catalase did not confer such protection. The lack of enzyme activity recovery after dialysis for 4 hours and oxidation with potassium ferricyanide, coupled with the absence of the characteristic Soret peak signature collectively substantiated that inactivation of CYP3A4 by INF was not mediated by the formation of quasi-irreversible metabolite-intermediate complexes but rather through irreversible covalent adduction to the prosthetic heme and/or apoprotein. Finally, glutathione trapping and high-resolution mass spectrometry experimental results unraveled two plausible bioactivation mechanisms of INF arising from the generation of a p-benzoquinonediimine and epoxide reactive intermediate. SIGNIFICANCE STATEMENT: The potential of INF to cause MBI of CYP3A4 was unknown. This study reports the reversible noncompetitive inhibition and irreversible covalent MBI of CYP3A4 by INF and proposes two potential bioactivation pathways implicating p-benzoquinonediimine and epoxide reactive intermediates, following which a unique covalent docking methodology was harnessed to elucidate the structural and molecular determinants underscoring its inactivation. Findings from this study lay the groundwork for future investigation of clinically relevant drug-drug interactions between INF and concomitant substrates of CYP3A4.
Asunto(s)
Inhibidores del Citocromo P-450 CYP3A/farmacocinética , NADP/metabolismo , Compuestos de Fenilurea/farmacocinética , Pirimidinas/farmacocinética , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Antineoplásicos/farmacocinética , Colangiocarcinoma/tratamiento farmacológico , Interacciones Farmacológicas , Humanos , Inactivación Metabólica , Tasa de Depuración Metabólica , Redes y Vías MetabólicasRESUMEN
Erdafitinib (ERD) is a first-in-class pan inhibitor of fibroblast growth factor receptor 1-4 that has garnered global regulatory approval for the treatment of advanced or metastatic urothelial carcinoma. Although it has been previously reported that ERD elicits time-dependent inhibition (TDI) of cytochrome P450 (P450) 3A4 (CYP3A4), the exact biochemical nature underpinning this observation remains obfuscated. Moreover, it is also uninterrogated if CYP3A5-its highly homologous counterpart-could be susceptible to such interactions. Mechanism-based inactivation (MBI) of P450 is a unique subset of TDI that hinges on prior bioactivation of the drug to a reactive intermediate and possesses profound clinical and toxicological implications due to its irreversible nature. Here, we investigated and confirmed that ERD inactivated both CYP3A isoforms in a time-, concentration-, and NADPH-dependent manner with KI, kinact, and partition ratio of 4.01 and 10.04 µM, 0.120 and 0.045 min-1, and 32 and 55 for both CYP3A4 and CYP3A5, respectively, when rivaroxaban was employed as the probe substrate. Co-incubation with an alternative substrate or direct inhibitor of CYP3A attenuated the rate of inactivation, whereas the addition of glutathione or catalase did not induce such protection. The lack of enzyme activity recovery following dialysis for 4 h and oxidation with potassium ferricyanide combined with the lack of a Soret peak in spectral scans collectively substantiated that ERD is an irreversible covalent MBI of CYP3A. Finally, glutathione trapping and high-resolution mass spectrometry experiments illuminated a plausible bioactivation mechanism of ERD by CYP3A arising from metabolic epoxidation of its quinoxaline ring.
Asunto(s)
Inhibidores del Citocromo P-450 CYP3A/farmacología , Pirazoles/farmacología , Quinoxalinas/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/química , Humanos , NADP/metabolismo , Pirazoles/química , Quinoxalinas/químicaAsunto(s)
Acetamidas , Antineoplásicos , Vacuna BCG , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Quinolinas , Neoplasias de la Vejiga Urinaria , Acetamidas/farmacología , Acetamidas/uso terapéutico , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Vacuna BCG/farmacología , Vacuna BCG/uso terapéutico , Quinurenina/sangre , Ratones , Neoplasias Experimentales/química , Neoplasias Experimentales/tratamiento farmacológico , Quinolinas/farmacología , Quinolinas/uso terapéutico , Vejiga Urinaria/química , Vejiga Urinaria/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/química , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
Hepatocytes are one of the most physiologically relevant in vitro liver systems for human translation of clearance and drug-drug interactions (DDI). However, the cell membranes of hepatocytes can limit the entry of certain compounds into the cells for metabolism and DDI. Passive permeability through hepatocytes can be different in vitro and in vivo, which complicates the human translation. Permeabilized hepatocytes offer a useful tool to probe mechanistic understanding of permeability-limited metabolism and DDI. Incubation with saponin of 0.01% at 0.5 million cells/mL and 0.05% at 5 million cells/mL for 5 min at 37°C completely permeabilized the plasma membrane of hepatocytes, while leaving the membranes of subcellular organelles intact. Permeabilized hepatocytes maintained similar enzymatic activity as intact unpermeabilized hepatocytes and can be stored at -80°C for at least 7 months. This approach reduces costs by preserving leftover hepatocytes. The relatively low levels of saponin in permeabilized hepatocytes had no significant impact on the enzymatic activity. As the cytosolic contents leak out from permeabilized hepatocytes, cofactors need to be added to enable metabolic reactions. Cytosolic enzymes will no longer be present if the media are removed after cells are permeabilized. Hence permeabilized hepatocytes with and without media removal may potentially enable reaction phenotyping of cytosolic enzymes. Although permeabilized hepatocytes work similarly as human liver microsomes and S9 fractions experimentally requiring addition of cofactors, they behave more like hepatocytes maintaining enzymatic activities for over 4 h. Permeabilized hepatocytes are a great addition to the drug metabolism toolbox to provide mechanistic insights.
Asunto(s)
Hígado , Saponinas , Humanos , Hígado/metabolismo , Hepatocitos/metabolismo , Descubrimiento de Drogas , Microsomas Hepáticos , Saponinas/farmacología , Saponinas/metabolismoRESUMEN
CYP3A is one of the most important classes of enzymes and is involved in the metabolism of over 70% drugs. While several selective CYP3A4 inhibitors have been identified, the search for a selective CYP3A5 inhibitor has turned out to be rather challenging. Recently, several selective CYP3A5 inhibitors have been identified through high-throughput screening of ~ 11,000 compounds and hit expansion using human recombinant enzymes. We set forth to characterize the three most selective CYP3A5 inhibitors in a more physiologically relevant system of human liver microsomes to understand if these inhibitors can be used for reaction phenotyping studies in drug discovery settings. Gomisin A and T-5 were used as selective substrate reactions for CYP3A4 and CYP3A5 to determine IC50 values of the two enzymes. The results showed that clobetasol propionate and loteprednol etabonate were potent and selective CYP3A5 reversible inhibitors with selectivity of 24-fold against CYP3A4 and 39-fold or more against the other major CYPs. The selectivity of difluprednate in HLM is much weaker than that in the recombinant enzymes due to hydrolysis of the acetate group in HLM. Based on the selectivity data, loteprednol etabonate can be utilized as an orthogonal approach, when experimental fraction metabolized of CYP3A5 is greater than 0.5, to understand CYP3A5 contribution to drug metabolism and its clinical significance. Future endeavors to identify even more selective CYP3A5 inhibitors are warranted to enable accurate determination of CYP3A5 contribution to metabolism versus CYP3A4.
Asunto(s)
Inhibidores del Citocromo P-450 CYP3A , Citocromo P-450 CYP3A , Humanos , Inhibidores del Citocromo P-450 CYP3A/farmacología , Citocromo P-450 CYP3A/metabolismo , Etabonato de Loteprednol , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/metabolismoRESUMEN
Selective chemical inhibitors are critical for reaction phenotyping to identify drug-metabolizing enzymes that are involved in the elimination of drug candidates. Although relatively selective inhibitors are available for the major cytochrome P450 enzymes (CYP), they are quite limited for the less common CYPs and non-CYPs. To address this gap, we developed a multiplexed high throughput screening (HTS) assay using 20 substrate reactions of multiple enzymes to simultaneously monitor the inhibition of enzymes in a 384-well format. Four 384-well assay plates can be run at the same time to maximize throughput. This is the first multiplexed HTS assay for drug-metabolizing enzymes reported. The HTS assay is technologically enabled with state-of-the-art robotic systems and highly sensitive modern LC-MS/MS instrumentation. Virtual screening is utilized to identify inhibitors for HTS based on known inhibitors and enzyme structures. Screening of ~4600 compounds generated many hits for many drug-metabolizing enzymes including the two time-dependent and selective aldehyde oxidase inhibitors, erlotinib and dibenzothiophene. The hit rate is much higher than that for the traditional HTS for biological targets due to the promiscuous nature of the drug-metabolizing enzymes and the biased compound selection process. Future efforts will focus on using this method to identify selective inhibitors for enzymes that do not currently have quality hits and thoroughly characterizing the newly identified selective inhibitors from our screen. We encourage colleagues from other organizations to explore their proprietary libraries using a similar approach to identify better inhibitors that can be used across the industry.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masas en Tándem , Humanos , Cromatografía Liquida , Sistema Enzimático del Citocromo P-450 , Hepatocitos , Inhibidores Enzimáticos/farmacologíaRESUMEN
Accurate diagnosis of the most common histological subtypes of small B-cell lymphomas is challenging due to overlapping morphological features and limitations of ancillary testing, which involves a large number of immunostains and molecular investigations. In addition, a common diagnostic challenge is to distinguish reactive lymphoid hyperplasia that do not require additional stains from such lymphomas that need ancillary investigations. We investigated if tissue-specific microRNA (miRNA) expression may provide potential biomarkers to improve the pathology diagnostic workflow. This study seeks to distinguish reactive lymphoid proliferation (RL) from small B-cell lymphomas, and to further distinguish the four main subtypes of small B-cell lymphomas. Two datasets were included: a discovery cohort (n = 100) to screen for differentially expressed miRNAs and a validation cohort (n = 282) to develop classification models. The models were evaluated for accuracy in subtype prediction. MiRNA gene set enrichment was also performed to identify differentially regulated pathways. 306 miRNAs were detected and quantified, resulting in 90-miRNA classification models from which smaller panels of miRNAs biomarkers with good accuracy were derived. Bioinformatic analysis revealed the upregulation of known and other potentially relevant signaling pathways in such lymphomas. In conclusion, this study suggests that miRNA expression profiling may serve as a promising tool to aid the diagnosis of common lymphoid lesions.
RESUMEN
Futibatinib (FUT) is a potent irreversible inhibitor of fibroblast growth factor receptor 1-4 currently under clinical investigation for the treatment of cholangiocarcinoma. However, there remains a paucity of information pertaining to its hepatic metabolism. In this study, our overarching aims were to systematically develop and validate a novel ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analytical method to quantify FUT for the subsequent application to the metabolic stability assay. Chromatographic separation was achieved on a C18 column and a gradient elution system comprising 0.1% formic acid in water (A) and acetonitrile (B). Positive electrospray ionization in conjunction with multiple reaction monitoring (MRM) mode was harnessed for the selective and sensitive quantification of FUT (m/z 419.2 â 296.0) and erdafitinib (m/z 447.0 â 362.0; internal standard). The retention time was 1.49 min for FUT and 1.29 min for erdafitinib. The calibration curve was linear from 0.003 to 3 µM (r2 > 0.99) and the lower limit of quantification was 0.003 µM. The intra-day and inter-day precision (% RSD) and accuracy (% bias) were all < 11.4% and < 11.3% respectively. Quality control samples were determined to be stable under several conditions routinely employed in sample preparation and UPLC-MS/MS analyses. Moreover, the liver microsomal matrix did not adversely affect the quantification of FUT. Following which, the in vitro microsomal intrinsic clearance (CLint) of FUT was calculated from our metabolic stability assay to be 29.3 µL/min/mg, thereby suggesting that it was a medium clearance drug. Finally, extrapolating the CLint with human scaling factors yielded an estimated in vitro hepatic intrinsic clearance value of 2075 mL/min. Our study reports the first UPLC-MS/MS method and offers a specific, sensitive and rapid means of determining FUT human liver microsomal stability.
Asunto(s)
Microsomas Hepáticos , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida , Humanos , Hígado , Inhibidores de Proteínas Quinasas , Pirazoles , Pirimidinas , Pirroles , Receptores de Factores de Crecimiento de Fibroblastos , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
Metabolic activation of drugs by cytochrome P450 enzymes (P450) to chemically reactive electrophiles is commonly regarded as a key molecular-initiating event underpinning idiosyncratic drug-induced liver injury. However, apart from precipitating toxicities, these labile intermediates can be sequestered within the P450 active site and engender a unique form of irreversible inhibition known as mechanism-based inactivation (MBI) which bears profound clinical implications (i.e., drug-drug interactions, autoinhibition of hepatic elimination, time-dependent and/or nonlinear pharmacokinetics). Consequently, there has been considerable attempts to develop medicinal chemistry strategies to attenuate or abolish metabolic activation and its deleterious downstream effects (i.e., MBI). In this review, we will first summarize the fundamental aspects and consequences of P450 metabolic activation with a focus on MBI. Following which, we will share our recent discoveries on the arcane metabolic activation pathways of an emerging class of tyrosine kinase inhibitors known as the fibroblast growth factor receptor (FGFR) inhibitors which in turn unravelled mechanistic insights into the biochemical basis and pharmacokinetic implications of its MBI. Finally, we will discuss, using relevant examples from the literature as well as from our laboratory, limitations of existing chemical approaches to minimize metabolic activation and highlight a promising new paradigm which involves the rational deuteration of a drug molecule at its known bioactivation 'hot-spot' to shunt metabolism away from these aberrant pathways and reduce reactive metabolite formation.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Inhibidores de Proteínas Quinasas , Activación Metabólica , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Receptores de Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
Atypical kinetics are observed in metabolic reactions catalyzed by cytochrome P450 enzymes (P450). Yet, this phenomenon is regarded as experimental artifacts in some instances despite increasing evidence challenging the assumptions of typical Michaelis-Menten kinetics. As P450 play a major role in the metabolism of a wide range of substrates including drugs and endogenous compounds, it becomes critical to consider the impact of atypical kinetics on the accuracy of estimated kinetic and inhibitory parameters which could affect extrapolation of pharmacological and toxicological implications. The first half of this book chapter will focus on atypical non-Michaelis-Menten kinetics (e.g. substrate inhibition, biphasic and sigmoidal kinetics) as well as proposed underlying mechanisms supported by recent insights in mechanistic enzymology. In particular, substrate inhibition kinetics in P450 as well as concurrent drug inhibition of P450 in the presence of substrate inhibition will be further discussed. Moreover, mounting evidence has revealed that despite the high degree of sequence homology between CYP3A isoforms (i.e. CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different susceptibilities and potencies of mechanism-based inactivation (MBI) with a common drug inhibitor. These experimental observations pertaining to the presence of these atypical isoform- and probe substrate-specific complexities in CYP3A isoforms by several clinically-relevant drugs will therefore be expounded and elaborated upon in the second half of this book chapter.
Asunto(s)
Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450 , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , CinéticaRESUMEN
Deuteration is a chemical modification strategy that has recently gained traction in drug development. The replacement of one or more hydrogen atom(s) in a drug molecule with its heavier stable isotope deuterium can enhance its metabolic stability and pharmacokinetic properties. However, it remains uninterrogated if rational deuteration at bioactivation "hot-spots" could attenuate its associated toxicological consequences. Here, our preliminary screening with benzofuran antiarrhythmic agents first revealed that dronedarone and its major metabolite N-desbutyldronedarone elicited a greater loss of viability and cytotoxicity in human hepatoma G2 (HepG2) cells as compared with amiodarone and its corresponding metabolite N-desethylamiodarone. A comparison of dronedarone and its in-house synthesized deuterated analogue (termed poyendarone) demonstrated that deuteration could attenuate its in vitro toxicity in HepG2 cells by modulating the extent of mitochondrial dysfunction, reducing the dissipation of mitochondrial membrane potential, and evoking a distinct apoptotic kinetic signature. Furthermore, although pretreatment with the CYP3A inducer rifampicin or the substitution of glucose with galactose in the growth media significantly augmented the loss of cell viability elicited by dronedarone and poyendarone, a lower loss of cell viability was consistently observed in poyendarone across all concentrations. Taken together, our preliminary investigations suggested that the rational deuteration of dronedarone at its benzofuran ring reduces aberrant cytochrome P450 3A4/5-mediated bioactivation, which attenuated its mitochondrial toxicity in human hepatic HepG2 cells.