Metabolite Intermediate Complex Research Articles

Direct and Sequential Bioactivation of Pemigatinib to Reactive Iminium Ion Intermediates Culminates in Mechanism-Based Inactivation of Cytochrome P450 3A.

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.

Infigratinib Is a Reversible Inhibitor and Mechanism-Based Inactivator of Cytochrome P450 3A4.

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.

Mechanism-Based Inactivation of Cytochrome P450 3A4 by Benzbromarone.

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 ∼44% 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.

Numerical analysis of time-dependent inhibition kinetics: comparison between rat liver microsomes and rat hepatocyte data for mechanistic model fitting

Time-dependent inhibition (TDI) may confound drug interaction predictions. Recently, models were generated for an array of TDI kinetic schemes using numerical analysis of microsomal assays. Additionally, a distinct terminal inactivation step was identified for certain mechanism based inhibitors (MBI) following reversible metabolite intermediate complex (MIC) formation. Longer hepatocyte incubations potentially allow analysis of slow TDI and terminal inactivation. In the experiments presented here, we compared the quality of TDI parameterization by numerical analysis between hepatocyte and microsomal data. Rat liver microsomes (RLM), suspended rat hepatocytes (SRH) and sandwich-cultured rat hepatocytes (SCRH) were incubated with the prototypical CYP3A MBI troleandomycin and the substrate midazolam. Data from RLM provided a better model fit as compared to SRH. Increased CYP3A expression after dexamethasone (DEX) induction improved the fit for RLM and SRH. A novel sequential kinetic scheme, defining inhibitor metabolite production prior to MIC formation, improved the fit compared to direct MIC formation. Furthermore, terminal inactivation rate constants were parameterized for RLM and SRH samples with DEX-induced CYP3A. The low expression of CYP3A and experimental error in SCRH resulted in poor data for model fitting. Overall, RLM generated data better suited for elucidation of TDI mechanisms by numerical analysis.

Metabolism of Sesamin and Drug-Sesamin Interaction

Sesamin, derived from sesame seeds, is known to have various biological effects. Since some of these effects appear to be derived from its metabolites, the elucidation of sesamin metabolism is essential to understanding the molecular mechanism of its effects. In addition, it is important to clarify drug-sesamin interactions in order to address safety concerns, as some food factors are known to affect drug metabolism. Our previous studies revealed that sesamin was sequentially metabolized by cytochrome P450 (CYP) and UDP-glucuronosyltransferase or sulfotransferase. Whereas sesamin metabolism is mainly mediated by CYP2C9 in human liver, sesamin causes a mechanism-based inhibition (MBI) of CYP2C9. However, we found that the metabolite-intermediate complex between CYP2C9 and sesamin was unstable, and the effects of sesamin appeared to be minimal. To confirm this assumption, in vivo studies using rats were conducted. After the administration of sesamin to rats for 3 d, diclofenac (an NSAID) was administered to measure the time course of plasma concentration of diclofenac. No significant differences were observed in the diclofenac Cmax, Tmax, and AUC0-24 h between the group that was administered sesamin and the group that was not. Based on these results, it could be concluded that no significant interaction occurs in people who take sesamin supplements at a standard dose.

Biotransformation of Isoniazid by Cytochromes P450: Analyzing the Molecular Mechanism using Density Functional Theory.

Hydrazide group (-C(O)-NH-NH2) is considered as a structural alert in the drug discovery process because the biotransformation chemistry of this group leads to the generation of toxic radical intermediates. The most important antitubercular drug isoniazid (INH) carries the hydrazide group. The toxicity of INH has been attributed to the protein adduct formation involving isonicotinoyl radical. However, the structures of reactive metabolites (RMs) and metabolite intermediate complexes (MICs), as well as the reaction mechanism for the formation and fate of RMs/MICs, have not been established. This report provides a detailed account of the biotransformation of INH by cytochromes using quantum chemical (QC) methods. Two cycles of cytochrome catalysis are involved in the formation of the most important RM, isonicotinoyl radical. The first cycle requires ∼11 kcal/mol barrier on the oxidation pathway involving the formation of the RM isonicotinoyldiazene. The second cycle involves a barrier of ∼7 kcal/mol for the activation of the diazene intermediate leading to isonicotinic acid via three reaction steps: (i) N-H bond activation, (ii) loss of N2 molecule, and (iii) rebound of isonicotinoyl radical. The RMs on the pathway (diazene, isonicotinoyl radical, N-hydroxy diazene) can react with the porphyrin ring/the amino acids of the cytochrome leading to many MICs (at least nine varieties), which can cause mechanism based inhibition and drug-drug interactions. This QC, molecular docking, and QM/MM study explored all the above reaction pathways and established the 3D structures of the RMs and MICs.

Difference in Mechanism-Based Inhibition of Cytochrome P450 3A4 and 3A5 by a Series of Fluoroquinolone Antibacterial Agents.

A series of fluoroquinolone antibacterial compounds were found to be irreversible (compounds 1-5) and quasi-irreversible (compounds 6-9) inhibitors of CYP3A4. The purpose of this study was to evaluate their mechanism-based inhibition (MBI) potency against CYP3A5. Compounds 1-5 were also irreversible inhibitors of CYP3A5, whereas compounds 6-9 showed neither irreversible nor quasi-irreversible inhibition of CYP3A5. Compounds 6 and 8 did not form a metabolite-intermediate complex with the heme of CYP3A5 during incubation. The structural analysis of the metabolites after incubation of compounds 1 and 6 with CYP3A5 revealed that their metabolites were identical to those produced by CYP3A4, including the precursors of which are speculated to account for the MBI of CYP3A4. The homology modeling of CYP3A5 suggests that four residues around the nitroso intermediate of compound 6 in the substrate-binding pocket of CYP3A4 correspond with the bulkier residues in CYP3A5-especially Phe210 in CYP3A5-which might contribute to the steric hindrance with the nitroso intermediate of compound 6. The substrate-binding pocket structure of CYP3A5 might prevent the nitroso intermediate from coordinate binding with the heme, thereby preventing quasi-irreversible inhibition. Our study may provide new insights into the observable differences between the inhibition of CYP3A4 and CYP3A5.

Analysis of Mechanism-Based Inhibition of CYP 3A4 by a Series of Fluoroquinolone Antibacterial Agents.

A series of fluoroquinolone compounds (compounds 1-9), which contain a common quinolone scaffold, inactivated the metabolic activity of CYP3A. The purpose of this study was to identify mechanism-based inhibition (MBI) among these fluoroquinolone compounds by metabolite profiling to elucidate the association of the substructure and MBI potential. Reversibility of MBI after incubation with potassium ferricyanide differed among the test compounds. Representative quasi-irreversible inhibitors form a metabolite-intermediate (MI) complex with the heme of CYP3A4 according to absorption analysis. Metabolite profiling identified the cyclopropane ring-opened metabolites from representative irreversible inhibitors, suggesting irreversible binding of the carbon-centered radical species with CYP3A4. On the other hand, the oxime form of representative quasi-irreversible inhibitors was identified, suggesting generation of a nitroso intermediate that could form the MI complex. Metabolites of compound 10 with a methyl group at the carbon atom at the root of the amine moiety of compound 8 include the oxime form, but compound 10 did not show quasi-irreversible inhibition. The docking study with CYP3A4 suggested that a methyl moiety introduced at the carbon atom at the root of the primary amine disrupts formation of the MI complex between the heme and the nitroso intermediate because of steric hindrance. This study identified substructures of fluoroquinolone compounds associated with the MBI mechanism; introduction of substituted groups inducing steric hindrance with the heme of P450 can prevent formation of an MI complex. Our series of experiments may be broadly applicable to prevention of MBI at the drug discovery stage.

Inactivation of Human Cytochrome P450 3A4 and 3A5 by Dronedarone and N-Desbutyl Dronedarone.

Dronedarone is an antiarrhythmic agent approved in 2009 for the treatment of atrial fibrillation. An in-house preliminary study demonstrated that dronedarone inhibits cytochrome P450 (CYP) 3A4 and 3A5 in a time-dependent manner. This study aimed to investigate the inactivation of CYP450 by dronedarone. We demonstrated for the first time that both dronedarone and its main metabolite N-desbutyl dronedarone (NDBD) inactivate CYP3A4 and CYP3A5 in a time-, concentration-, and NADPH-dependent manner. For the inactivation of CYP3A4, the inactivator concentration at the half-maximum rate of inactivation and inactivation rate constant at an infinite inactivator concentration are 0.87 µM and 0.039 minute(-1), respectively, for dronedarone, and 6.24 µM and 0.099 minute(-1), respectively, for NDBD. For CYP3A5 inactivation, the inactivator concentration at the half-maximum rate of inactivation and inactivation rate constant at an infinite inactivator concentration are 2.19 µM and 0.0056 minute(-1) for dronedarone and 5.45 µM and 0.056 minute(-1) for NDBD. The partition ratios for the inactivation of CYP3A4 and CYP3A5 by dronedarone are 51.1 and 32.2, and the partition ratios for the inactivation of CYP3A4 and CYP3A5 by NDBD are 35.3 and 36.6. Testosterone protected both CYP3A4 and CYP3A5 from inactivation by dronedarone and NDBD. Although the presence of Soret peak confirmed the formation of a quasi-irreversible metabolite-intermediate complex between dronedarone/NDBD and CYP3A4/CYP3A5, partial recovery of enzyme activity by potassium ferricyanide illuminated an alternative irreversible mechanism-based inactivation (MBI). MBI of CYP3A4 and CYP3A5 was further supported by the discovery of glutathione adducts derived from the quinone oxime intermediates of dronedarone and NDBD. In conclusion, dronedarone and NDBD inactivate CYP3A4 and CYP3A5 via unique dual mechanisms of MBI and formation of the metabolite-intermediate complex. Our novel findings contribute new knowledge for future investigation of the underlying mechanisms associated with dronedarone-induced hepatotoxicity and clinical drug-drug interactions.

Isoniazid mediates the CYP2B6*6 genotype-dependent interaction between efavirenz and antituberculosis drug therapy through mechanism-based inactivation of CYP2A6.

Efavirenz is commonly used to treat patients coinfected with human immunodeficiency virus and tuberculosis. Previous clinical studies have observed paradoxically elevated efavirenz plasma concentrations in patients with the CYP2B6*6/*6 genotype (but not the CYP2B6*1/*1 genotype) during coadministration with the commonly used four-drug antituberculosis therapy. This study sought to elucidate the mechanism underlying this genotype-dependent drug-drug interaction. In vitro studies were conducted to determine whether one or more of the antituberculosis drugs (rifampin, isoniazid, pyrazinamide, or ethambutol) potently inhibit efavirenz 8-hydroxylation by CYP2B6 or efavirenz 7-hydroxylation by CYP2A6, the main mechanisms of efavirenz clearance. Time- and concentration-dependent kinetics of inhibition by the antituberculosis drugs were determined using genotyped human liver microsomes (HLMs) and recombinant CYP2A6, CYP2B6.1, and CYP2B6.6 enzymes. Although none of the antituberculosis drugs evaluated at up to 10 times clinical plasma concentrations were found to inhibit efavirenz 8-hydroxylation by HLMs, both rifampin (apparent inhibition constant [Ki] = 368 μM) and pyrazinamide (Ki = 637 μM) showed relatively weak inhibition of efavirenz 7-hydroxylation. Importantly, isoniazid demonstrated potent time-dependent inhibition of efavirenz 7-hydroxylation in both HLMs (inhibitor concentration required for half-maximal inactivation [KI] = 30 μM; maximal rate constant of inactivation [kinact] = 0.023 min(-1)) and recombinant CYP2A6 (KI = 15 μM; kinact = 0.024 min(-1)) and also formed a metabolite intermediate complex consistent with mechanism-based inhibition. Selective inhibition of the CYP2B6.6 allozyme could not be demonstrated for any of the antituberculosis drugs using either recombinant enzymes or CYP2B6*6 genotype HLMs. In conclusion, the results of this study identify isoniazid as the most likely perpetrator of this clinically important drug-drug interaction through mechanism-based inactivation of CYP2A6.

Characterization of Inhibition Kinetics of (S)-Warfarin Hydroxylation by Noscapine: Implications in Warfarin Therapy

Noscapine is an antitussive and potential anticancer drug. Clinically significant interactions between warfarin and noscapine have been previously reported. In this study, to provide a basis for warfarin dosage adjustment, the inhibition kinetics of noscapine against warfarin metabolism was characterized. Our enzyme kinetics data obtained from human liver microsomes and recombinant CYP2C9 proteins indicate that noscapine is a competitive inhibitor of the (S)-warfarin 7-hydroxylation reaction by CYP2C9. Interestingly, noscapine also inhibited (S)-warfarin metabolism in a NADPH- and time-dependent manner, and removal of unbound noscapine and its metabolites by ultrafiltration did not reverse inhibition of (S)-warfarin metabolism by noscapine, suggesting mechanism-based inhibition of CYP2C9 by noscapine. Spectral scanning of the reaction between CYP2C9 and noscapine revealed the formation of an absorption spectrum at 458 nm, indicating the formation of a metabolite-intermediate complex. Surprisingly, noscapine is a 2- to 3-fold more efficient inactivator of CYP2C9.2 and CYP2C9.3 variants than it is of the wild type, by unknown mechanisms. Based on the inhibitory kinetic data, (S)-warfarin exposure is predicted to increase up to 7-fold (depending on CYP2C9 genotypes) upon noscapine coadministration, mainly due to mechanism-based inactivation of CYP2C9 by noscapine. Together, these results indicate that mechanism-based inhibition of CYP2C9 by noscapine may dramatically alter pharmacokinetics of warfarin and provide a basis for warfarin dosage adjustment when noscapine is coadministered.

Investigation of drug–drug interaction via mechanism‐based inhibition of cytochrome P450 3A by macrolides in dexamethasone‐treated female rats

The in vitro and in vivo inhibition of cytochrome P450 (CYP) 3A with mechanism-based inhibition (MBI) by macrolides was investigated using dexamethasone-treated female rats (DEX-female rats). In the in vitro CYP inhibition studies using erythromycin (ERM) and clarithromycin (CAM), similar inhibition responses were observed between human and DEX-female rat liver microsomes, however, there were fewer effects in intact male rats. The ex vivo study showed that midazolam (MDZ) metabolism in liver microsomes of DEX-female rats was reduced by ERM administration and the inhibitory effect was increased with increasing ERM doses, indicating that metabolite intermediate complex formation caused irreversible inhibition of CYP3A activity in DEX-female rats as well as in humans. In the in vivo studies, ERM and CAM significantly increased the area under the plasma concentration-time curve of MDZ and decreased the total clearance in DEX-female rats. It was concluded that the DDIs via MBI of CYP3A following macrolide administration in humans could be reproduced in female rats, suggesting that DEX-female rats can serve as an in vivo model for assessing this DDI in humans.

CYP Inhibition-Mediated Drug-Drug Interactions

Adverse drug reaction is a frequent cause of drug withdrawals from the market. The drug-drug interaction (DDI) potential of new drug candidates is an increasing safety concern of pharmaceutical companies. DDIs frequently occur between coadministered drugs on a pharmacokinetic ground and sometimes create life-threatening conditions. The unexpected increase of the exposure of a drug is most frequently evoked by the inhibition of metabolic enzymes. While the inhibition of CYPs by new drug candidates is unwanted, one has to recognize that several currently marketed successful drugs with relatively clean record of drug-drug interactions are time-dependent inhibitors of drug metabolic enzymes. Therefore, the correct and high throughput prediction of drug-drug interaction propensity of new chemical entities (NCEs) at an affordable cost is a major interest of pharmaceutical research. False negatives and positives, also the misinterpretation of otherwise flawlessly generated data are equally costly or hazardous. While screening methods for the detection of CYP inhibition are available now, the quantitative estimation of DDI potential and its PK effect leaves an ample room for improvement. This is reflected in the great research activity focused on the establishment of correct in vitro-in vivo correlations. The impressing number of recent publications and methods recommended in this field provide an increasingly solid ground for the selection of lead compounds and development candidates. Keywords: Drug-drug interaction, CYP450, IVIVE, CYPs, of new chemical entities (NCEs), isozymes, cisapride, mibefradil, terfenadine, nefazodone, toxicology, lucocorticoides, biotransformation, Metabolism-based inhibitor (MBI), xenobiotic compounds, CYP monooxygenases, Fluoxetine, paroxetine, fraction metabolized (fm,CYP), conjugases, allosteric feature, Competitive inhibition, metabolite-intermediate complex (MIC), macrolide antibiotics, erythromycin, clarithromycin, troleandomycin, carbamazepine, cyclosporine, midazolam, ritonavir, microsomal monooxygenases, idiosyncratic reactions, Cholestasis, proinflammatory cytokines, antagonize nuclear receptors, ketoconazole, neuropathic pain, poor metabolizer (PM), extensive metabolizer (EM), ultrarapid metabolizer (UM), Ethynyl groups, ethynylestradiol, acetyl salicylic acid, hepatic inlet concentration, metabolite, pregnane X receptor, ultrarapid metabolizer

Mechanism-Based Inactivation of Cytochrome P450 3A4 by Lapatinib

Fatalities stemming from hepatotoxicity associated with the clinical use of lapatinib (Tykerb), an oral dual tyrosine kinase inhibitor (ErbB-1 and ErbB-2) used in the treatment of metastatic breast cancer, have been reported. We investigated the inhibition of CYP3A4 by lapatinib as a possible cause of its idiosyncratic toxicity. Inhibition of CYP3A4 was time-, concentration-, and NADPH-dependent, with k(inact) = 0.0202 min(-1) and K(i) = 1.709 μM. The partition ratio was approximately 50.9. Addition of GSH did not affect the rate of inactivation. Testosterone protected CYP3A4 from inactivation by lapatinib. The characteristic Soret peak associated with a metabolite-intermediate complex was not observed for lapatinib during spectral difference scanning. However, reduced carbon monoxide (CO)-difference spectroscopy did reveal a 43% loss of the spectrally detectable CYP3A4-CO complex in the presence of lapatinib. Incubation of either lapatinib or its dealkylated metabolite with human liver microsomes in the presence of GSH resulted in the formation of a reactive metabolite (RM)-GSH adduct derived from the O-dealkylated metabolite of lapatinib. In addition, coincubation of lapatinib with ketoconazole inhibited the formation of the RM-GSH adduct. In conclusion, we demonstrated for the first time that lapatinib is a mechanism-based inactivator of CYP3A4, most likely via the formation and further oxidation of its O-dealkylated metabolite to a quinoneimine that covalently modifies the CYP3A4 apoprotein and/or heme moiety.

Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: A case study with melanocortin-4 receptor agonists

1-[(2R)-2-({[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl}amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a λmax at approximately 450 nm. The species responsible for this absorbance required the presence of β-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug–drug interaction projections based on the kinact and KI parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug–drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution α to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.

Time‐dependent inhibition of human drug metabolizing cytochromes P450 by tricyclic antidepressants

To investigate time-dependent inhibition (TDI) of human drug metabolizing CYP enzymes by tricyclic antidepressants (TCAs). CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A/CYP3A4 activities were investigated following co- and preincubation with TCAs using human liver microsomes (HLM) and human recombinant CYP proteins (expressed in Escherichia coli) as the enzyme sources. A two-step incubation method was employed to examine the in vitro mechanism-based inactivation (MBI) criteria. Potential metabolite-intermediate complex (MIC) formation was studied by spectral analysis. TCAs generally exhibited significant TDI of recombinant CYP1A2, CYP2C19 and CYP2D6 (>10% positive inhibition differences between co- and preincubation conditions). TDI of recombinant CYP2C9 was minor (<10%), and was minor or absent in experiments utilizing recombinant CYP3A4 or HLM as the enzyme sources. Where observed, TDI of recombinant CYP occurred via alkylamine MIC formation, but evidence to support similar behaviour in HLM was limited. Indeed, only secondary amine TCAs reduced the apparent P450 content of HLM (3-6%) consistent with complexation. As a representative TCA, nortriptyline fulfilled the in vitro MBI criteria using recombinant CYP2C19 and CYP3A4 (K(I) and k(inact) values of 4 microm and 0.19 min(-1), and 70 microm and 0.06 min(-1)), but not with the human liver microsomal enzymes. TCAs appear to have minimal potential for MBI of human liver microsomal CYP enzymes involved in drug metabolism. HLM and recombinant CYP (expressed in E. coli) are not equivalent enzyme sources for evaluating the TDI associated with some drugs.

An evaluation of potential mechanism-based inactivation of human drug metabolizing cytochromes P450 by monoamine oxidase inhibitors, including isoniazid.

To characterize potential mechanism-based inactivation (MBI) of major human drug-metabolizing cytochromes P450 (CYP) by monoamine oxidase (MAO) inhibitors, including the antitubercular drug isoniazid. Human liver microsomal CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities were investigated following co- and preincubation with MAO inhibitors. Inactivation kinetic constants (KI and kinact) were determined where a significant preincubation effect was observed. Spectral studies were conducted to elucidate the mechanisms of inactivation. Hydrazine MAO inhibitors generally exhibited greater inhibition of CYP following preincubation, whereas this was less frequent for the propargylamines, and tranylcypromine and moclobemide. Phenelzine and isoniazid inactivated all CYP but were most potent toward CYP3A and CYP2C19. Respective inactivation kinetic constants (KI and kinact) for isoniazid were 48.6 microm and 0.042 min-1 and 79.3 microm and 0.039 min-1. Clorgyline was a selective inactivator of CYP1A2 (6.8 microm and 0.15 min-1). Inactivation of CYP was irreversible, consistent with metabolite-intermediate complexation for isoniazid and clorgyline, and haeme destruction for phenelzine. With the exception of phenelzine-mediated CYP3A inactivation, glutathione and superoxide dismutase failed to protect CYP from inactivation by isoniazid and phenelzine. Glutathione partially slowed (17%) the inactivation of CYP1A2 by clorgyline. Alternate substrates or inhibitors generally protected against CYP inactivation. These data are consistent with mechanism-based inactivation of human drug-metabolizing CYP enzymes and suggest that impaired metabolic clearance may contribute to clinical drug-drug interactions with some MAO inhibitors.

Metabolite-cytochrome P450 complex formation by methylenedioxyphenyl lignans of Piper cubeba: Mechanism-based inhibition

Five methylenedioxyphenyl lignans, (–)-clusin ( 1), (–)-dihydroclusin ( 2), (–)-yatein ( 3), (–)-hinokinin ( 4), and (–)-dihydrocubebin ( 5), were isolated from Piper cubeba as potent and selective inhibitors against cytochrome P450 3A4 (CYP3A4). In this study, we investigated the mechanism of inhibition of CYP3A4 by these lignans and the possibility of their mechanism-based inhibition. Using [ N-methyl- 14C]erythromycin as a substrate, all lignans appear to be showed mixed-type of inhibition with apparent K i of 1.96–4.07 μM. Furthermore, all lignans ( 1– 5) inhibited CYP3A4 in a time-, concentration-, and NADPH-dependent manners and thus appear to be the mechanism-based inhibitors of CYP3A4. The apparent inactivation parameter, K I for these compounds were in the range of 0.054–0.373 μM, whereas the k inact values were 0.225–0.320 min −1. Among them, (–)-clusin ( 1) and (–)-dihydroclusin ( 2) were found to be the most potent CYP3A4 inactivator with apparent K I and k inact values of 0.082, 0.054 μM and 0.253, 0.310 min −1, respectively. Spectral scanning of microsomes with these lignans yielded an absorbance at 455 nm, suggesting that all of them appear to inactivate the cytochrome P450 via the formation of a metabolite intermediate complex. This pattern is consistent with the metabolism of the methylenedioxyphenyl compounds. These results indicate that (–)-clusin ( 1), (–)-dihydroclusin ( 2), (–)-yatein ( 3), (–)-hinokinin ( 4), and (–)-dihydrocubebin ( 5) are effective mechanism-based inhibitors of CYP3A4.

Identification and characterization of potent CYP3A4 inhibitors in Schisandra fruit extract.

Schisandra fruit, a Schisandraceae family herb, is used as a component in Kampo medicines (developed from Chinese medicines, but established in Japan). It can act as a sedative and antitussive, improve hepatic function, and give a general tonic effect. An extract of Schisandra fruit has been shown with a potent inhibitory effect on human liver microsomal erythromycin N-demethylation activity mediated by cytochrome P450 3A4 (CYP3A4). The present study was conducted to identify Schisandra fruit components having inhibitory effects on CYP3A4 by surveying the effect on human liver microsomal erythromycin N-demethylation activity. Known components of Schisandra fruit, gomisins B, C, G, and N and gamma-shizandrin, showed inhibitory effects on N-demethylation activity. Among these components, gomisin C displayed the most potent and competitive inhibitory effect, with a Ki value of 0.049 microM. Furthermore, the inhibitory effect of gomisin C was stronger than that of ketoconazole (Ki = 0.070 microM), a known potent CYP3A4 inhibitor. Gomisin C, however, inhibited CYP1A2-, CYP2C9-, CYP2C19-, and CYP2D6-dependent activities only to a limited extent (IC50 values >10 microM). Moreover, gomisin C inactivated human liver microsomal erythromycin N-demethylation activity in a time- and concentration-dependent manner. The inactivation kinetic parameters k(inact) and K(I) were 0.092 min(-1) and 0.399 microM, respectively. The human liver microsomal erythromycin N-demethylation activity inactivated by gomisin C did not recover on dialysis of the microsomes. Spectral scanning of CYP3A4 with gomisin C yielded an absorbance at 455 nm, suggesting that gomisin C inactivated the cytochrome P450 via the formation of a metabolite intermediate complex. This pattern is consistent with the metabolism of the methylenedioxy substituent in gomisin C. These results indicate that gomisin C is a mechanism-based inhibitor that not only competitively inhibits but irreversibly inactivates CYP3A4.

Mechanism-based inactivation of human cytochrome P4502C8 by drugs in vitro.

Studies were conducted to evaluate the potential mechanism-based inactivation of recombinant and human liver microsomal CYP2C8 by clinically used drugs. Several tricyclic antidepressants, calcium channel blockers, monoamine oxidase inhibitors, and various other known CYP3A4 inhibitors exhibited greater inhibition of CYP2C8 (paclitaxel 6alpha-hydroxylation) following preincubation, consistent with mechanism-based inactivation. Inactivation of recombinant CYP2C8 by phenelzine, amiodarone, verapamil, nortriptyline, fluoxetine, and isoniazid was of the pseudo-first order type and was characterized by respective inactivation kinetic constants (KI and kinact) of 1.2 microM and 0.243 min(-1), 1.5 microM and 0.079 min(-1), 17.5 microM and 0.065 min(-1), 49.9 microM and 0.036 min(-1), 294 microM and 0.083 min(-1), and 374 microM and 0.042 min(-1). Spectral scanning of recombinant CYP2C8 demonstrated the formation of metabolite-intermediate complexes with verapamil, nortriptyline, fluoxetine, and isoniazid, but not amiodarone. In contrast, inactivation by phenelzine resulted from heme destruction by free radicals. Studies with human liver microsomes (HLMs) revealed that nortriptyline, verapamil, and fluoxetine were not mechanism-based inactivators (MBIs) of CYP2C8. Simultaneous inactivation of CYP2C8 and CYP3A4 (paclitaxel 3'-phenyl-hydroxylation) was observed using amiodarone, isoniazid, and phenelzine with the efficiency of inactivation greater for the CYP3A4 pathway. With the exception of phenelzine, glutathione and superoxide dismutase failed to protect CYP2C8 (recombinant and HLMs) or CYP3A4 from inactivation by MBIs. However, the alternate CYP2C8 substrate, torsemide, prevented CYP2C8 inactivation in all cases. These data are consistent with mechanism-based inactivation of CYP2C8 by a range of commonly prescribed drugs, several of which have been implicated in clinically important drug-drug interactions.