Identification of cytochrome P450 3A4 modification site with reactive metabolite using linear ion trap-Fourier transform mass spectrometry

Covalent binding of reactive metabolites to cytochrome P450s (P450s) often causes their mechanism-based inactivation (MBI), resulting in drug-drug interactions or toxicity. The detection and identification of the P450 sites to which reactive metabolites bind would elucidate MBI mechanisms. We describe a proteomic approach using nano-LC/linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to characterize the binding of a reactive metabolite of raloxifene, which is a known P450 3A4 inhibitor, to the P450 3A4 isozyme. LTQ-FT analyses revealed that the metabolic reaction of raloxifene in a reconstituted P450 3A4 system formed a reactive metabolite adduct to P450 3A4 apoprotein, accompanied by a mass shift of 471 Da relative to intact P450 3A4 apoprotein. The reaction mixtures were digested with trypsin, and then the tryptic digests were analyzed by nano-LC-MS/MS. This technique revealed that VWGFYDGQQPVLAITDPDMIK (position 71-91) was a tryptic peptide modified by the reactive metabolite derived from raloxifene. The site of adduction with the reactive metabolite was further postulated to be the nucleophilic OH group of Tyr-75 of P450 3A4. A proteomic approach using LTQ-FT can yield direct information on the P450 3A4 modification site without radiolabeled compounds. In addition, this information can elucidate mechanisms involved in the covalent binding of reactive metabolites and the inactivation of P450 3A4.     

In vitro approaches to investigate mechanism-based inactivation of CYP enzymes

Mechanism-based inactivation (MBI) of human drug-metabolising CYP enzymes is an important consideration in the preclinical ADME evaluation of new drug candidates. In this report, the in vitro approaches used to investigate MBI of CYP enzymes are described, with an emphasis on the characterisation required to assess potential drug-drug interactions. Recent disparities in MBI data between in vitro test systems are also reviewed, highlighting the limitations of Escherichia coli-expressed human recombinant CYP in the prediction of drug-drug interactions that arise via MBI.     

Clinically important drug interactions potentially involving mechanism-based inhibition of cytochrome P450 3A4 and the role of therapeutic drug monitoring

Cytochrome P450 (CYP) 3A4 is the most abundant enzyme of CYPs in the liver and gut that metabolizes approximately 50% currently available drugs. A number of important drugs have been identified as substrates, inducers, and/or inhibitors of CYP3A4. The substrates of CYP3A4 considerably overlap with those of P-glycoprotein. Both CYP3A4 and P-glycoprotein are subject to inhibition and induction by a number of factors. Mechanism-based inhibition of CYP3A4 is characterized by NADPH-, time-, and concentration-dependent enzyme inactivation occurring when some xenobiotics or drugs are converted by CYPs to reactive metabolites. Such an inhibition of CYP3A4 is caused by chemical modification of the heme, the protein, or both as a result of covalent binding of modified heme to the protein. To date, the identified clinically important mechanism-based CYP3A4 inhibitors mainly include macrolide antibiotics (eg, clarithromycin and erythromycin), anti-HIV agents (eg, ritonavir and delavirdine), antidepressants (eg, fluoxetine and fluvoxamine), calcium channel blockers (eg, verapamil and diltiazem), steroids and their modulators (eg, gestodene and mifepristone), and several herbal and dietary components. The inactivation of CYP3A4 by drugs often causes unfavorable and long-lasting drug-drug interactions and probably fatal toxicity, depending on many factors associated with the enzyme, drugs, and the patients. Clinicians are encouraged to have a sound knowledge of drug-induced, mechanism-based CYP3A4 inhibition; take proper cautions, and perform close monitoring for possible drug interactions when using drugs that are mechanism-based CYP3A4 inhibitors. To minimize drug-drug interactions involving mechanism-based CYP3A4 inhibition, it is necessary to choose safe drug combination regimens, adjust drug dosages appropriately, and conduct therapeutic drug monitoring for drugs with narrow therapeutic indices.     

Mechanism-based inactivation of human cytochrome P450 1A2 and 3A4 isoenzymes by anti-tumor triazoloacridinone C-1305

1.?5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, is a promising anti-tumor therapeutic agent with high activity against several experimental tumors.

2.?It was determined to be a potent and selective inhibitor of liver microsomal and human recombinant cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1305 might modulate the effectiveness of other drugs used in multidrug therapy.

3.?The objective of this study was to investigate the mechanism of the observed C-1305-mediated inactivation of CYP1A2 and CYP3A4.

4.?Our findings indicated that C-1305 produced a time- and concentration-dependent decrease in 7-ethoxycoumarin O-deethylation (CYP1A2, K_I?=?10.8?±?2.14?μM) and testosterone 6β-hydroxylation (CYP3A4, K_I = 9.1?±?2.82?μM). The inactivation required the presence of NADPH, was unaffected by a nucleophilic trapping agent (glutathione) and a reactive oxygen species scavenger (catalase), attenuated by a CYP-specific substrate (7-ethoxycoumarin or testosterone), and was not reversed by potassium ferricyanide. The estimated partition ratios of 1086 and 197 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively.

5.?In conclusion, C-1305 inhibited human recombinant CYP1A2 and CYP3A4 isoenzymes by mechanism-based inactivation. The obtained knowledge about specific interactions between C-1305 and/or its metabolites, and CYP isoforms would be useful for predicting the possible drug–drug interactions in potent multidrug therapy.     

Inhibition of human CYP2B6 by N,N',N''-triethylenethiophosphoramide is irreversible and mechanism-based

The chemotherapeutic agent N,N',N'-triethylenethiophosphoramide (thioTEPA) is frequently used in high-dose chemotherapy regimens including cyclophosphamide. Previous studies demonstrated partial inhibition by thioTEPA of the cytochrome P4502B6 (CYP2B6)-catalyzed 4-hydroxylation of cyclophosphamide, which is required for its bioactivation. The aim of our study was to investigate the detailed mechanism of CYP2B6 inhibition by thioTEPA. Using human liver microsomes and recombinant P450 enzymes we confirmed potent inhibition of CYP2B6 enzyme activity determined with bupropion as substrate. ThioTEPA was found to inhibit CYP2B6 activity in a time- and concentration-dependent manner. The loss of CYP2B6 activity was NADPH-dependent and could not be restored by extensive dialysis. The maximal rates of inactivation (K(inact)) were 0.16 min(-1) in human liver microsomes and 0.17 min(-1) in membrane preparations expressing recombinant CYP2B6. The half-maximal inactivator concentrations (K(I)) were 3.8 microM in human liver microsomes and 2.2 microM in recombinant CYP2B6. Inhibition was attenuated by the presence of alternative active site ligands but not by nucleophilic trapping agents or reactive oxygen scavengers, further supporting mechanism-based action. Inactivated CYP2B6 did not lose its ability to form a CO-reduced complex suggesting a modification of the apoprotein, which is common for sulfur-containing compounds. Pharmacokinetic consequences of irreversible inactivation are more complicated than those of reversible inactivation, because the drug's own metabolism can be affected and drug interactions will not only depend on dose but also on duration and frequency of application. These findings contribute to better understanding of drug interactions with thioTEPA.     

Risk assessment for drug-drug interaction caused by metabolism-based inhibition of CYP3A using automated in vitro assay systems and its application in the early drug discovery process.

The CYP3A family is a major drug metabolism enzyme in humans. Metabolism-based inhibition of CYP3A might cause clinically significant drug-drug interactions (DDIs). To assess the risk of DDIs caused by metabolism-based inhibition (MBI) of CYP3A, we established an automated single time- and concentration-dependent inhibition assay. To create a diagram to assess DDI risk of compounds in the early discovery stage, we classified 171 marketed drugs by the possibility of the occurrence of in vivo DDI caused by MBI from the relationship between the inactivation activity determined in the MBI screening, the therapeutic blood or plasma concentration, and the in vivo DDI information. This analysis revealed that the DDI risk depends on both the MBI potential and the blood concentration of a compound, and provided the criteria of the DDI risk. In the assay, three compounds (midazolam, nifedipine, and testosterone) were compared as CYP3A probe substrates. The results show that the evaluation for MBI does not depend on the probe substrates used in the assay. In addition, we established an automated assay to distinguish quasi-irreversible and irreversible binding to CYP3A in which the quasi-irreversible inhibitors such as diltiazem, verapamil, and nicardipine were dissociated from CYP3A by the addition of potassium ferricyanide, whereas the irreversible inhibitors such as clozapine, delavirdine, and mibefradil were not. It provides useful information related to chemical structures likely to cause MBI. By using these MBI assays supported by an extensive database of marketed compounds, a systematic MBI evaluation paradigm was established and has been incorporated into our drug discovery process.     

Enzyme kinetics for clinically relevant CYP inhibition

In vitro cytochrome P450 (CYP)-associated metabolic studies have been considered cost-effective for predicting the potential clinical drug-drug interactions (DDIs), one of the major attritions in drug development. The breakthroughs during the past decade in understanding the biochemistry of CYP-mediated biotransformation and molecular biology of CYP gene regulation in humans have provided the scientific bases for such endeavors in early drug development. In this review, the enzyme kinetics of CYP inhibitions is described, with the primary focus on the ones proven with clinical relevance, namely the competitive inhibition and mechanism-based inactivation (MBI). Competitive CYP inhibition, the most often detected reversible inhibition, is well understood and has been studied extensively both in vitro and in clinical setting. Recently, MBI has received increasing attention. It has been recognized that MBI could occur more often than anticipated, due in part to the redox cycling-allied enzymatic action of CYPs. As commonly as an irreversible inhibition, MBI would inactivate the target proteins, and thus would be generally considered of high potential for causing clinical DDI. Moreover, the reversible inhibitions other than the competitive, namely noncompetitive, uncompetitive and mixed, were also documented for the important drug-metabolizing CYP members, particularly CYP1A2 and CYP2C9. Finally, the unusual kinetic interactions, which did not follow the Michaelis-Menten (M-M) kinetics, were detected in vitro for the majority of drug-metabolizing CYP members, and manifested for CYP3A4. However, the clinical relevance of the interactions involving the unusual CYP kinetics has not yet been fully understood. Nonetheless, the reversibility and inhibitory potency should be considered as the major determinants of the clinical relevance, particularly in combination with the therapeutic exposure levels. With rapid expansion of knowledge and technology, the evaluation of the clinically relevant CYP-associated DDIs in vitro is not only desirable but also achievable.     

Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs

Consistent with its highest abundance in humans, cytochrome P450 (CYP) 3A is responsible for the metabolism of about 60% of currently known drugs. However, this unusual low substrate specificity also makes CYP3A4 susceptible to reversible or irreversible inhibition by a variety of drugs. Mechanism-based inhibition of CYP3A4 is characterised by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-, time- and concentration-dependent enzyme inactivation, occurring when some drugs are converted by CYP isoenzymes to reactive metabolites capable of irreversibly binding covalently to CYP3A4. Approaches using in vitro, in silico and in vivo models can be used to study CYP3A4 inactivation by drugs. Human liver microsomes are always used to estimate inactivation kinetic parameters including the concentration required for half-maximal inactivation (K(I)) and the maximal rate of inactivation at saturation (k(inact)).Clinically important mechanism-based CYP3A4 inhibitors include antibacterials (e.g. clarithromycin, erythromycin and isoniazid), anticancer agents (e.g. tamoxifen and irinotecan), anti-HIV agents (e.g. ritonavir and delavirdine), antihypertensives (e.g. dihydralazine, verapamil and diltiazem), sex steroids and their receptor modulators (e.g. gestodene and raloxifene), and several herbal constituents (e.g. bergamottin and glabridin). Drugs inactivating CYP3A4 often possess several common moieties such as a tertiary amine function, furan ring, and acetylene function. It appears that the chemical properties of a drug critical to CYP3A4 inactivation include formation of reactive metabolites by CYP isoenzymes, preponderance of CYP inducers and P-glycoprotein (P-gp) substrate, and occurrence of clinically significant pharmacokinetic interactions with coadministered drugs.Compared with reversible inhibition of CYP3A4, mechanism-based inhibition of CYP3A4 more frequently cause pharmacokinetic-pharmacodynamic drug-drug interactions, as the inactivated CYP3A4 has to be replaced by newly synthesised CYP3A4 protein. The resultant drug interactions may lead to adverse drug effects, including some fatal events. For example, when aforementioned CYP3A4 inhibitors are coadministered with terfenadine, cisapride or astemizole (all CYP3A4 substrates), torsades de pointes (a life-threatening ventricular arrhythmia associated with QT prolongation) may occur.However, predicting drug-drug interactions involving CYP3A4 inactivation is difficult, since the clinical outcomes depend on a number of factors that are associated with drugs and patients. The apparent pharmacokinetic effect of a mechanism-based inhibitor of CYP3A4 would be a function of its K(I), k(inact) and partition ratio and the zero-order synthesis rate of new or replacement enzyme. The inactivators for CYP3A4 can be inducers and P-gp substrates/inhibitors, confounding in vitro-in vivo extrapolation. The clinical significance of CYP3A inhibition for drug safety and efficacy warrants closer understanding of the mechanisms for each inhibitor. Furthermore, such inactivation may be exploited for therapeutic gain in certain circumstances.     

Inactivation kinetics and residual activity of CYP3A4 after treatment with erythromycin

This study aimed to characterize the inactivation kinetics of cytochrome P450 3A4 (CYP3A4) by erythromycin, which involves mechanism‐based inhibition (MBI), in detail. In addition to an MBI assay based on the conventional method in which erythromycin and recombinant CYP3A4 were pre‐incubated for 15 min, the study also evaluated the long‐term MBI kinetics of this reaction by pre‐incubation for 120 min. Mechanism‐based inhibition profiles were obtained using three typical substrates, testosterone, midazolam and nifedipine. In the long‐term assay, erythromycin evoked a time‐dependent biphasic reduction in enzyme activity, but some residual activity (α) was detected in the terminal phase. The inactivation rate constant obtained in the presence of 30 μm erythromycin using nifedipine as a substrate was 1.44‐fold higher than that acquired using testosterone, while there was no difference among the α values obtained with the three substrates. In the short‐term assay, time‐dependent monophasic inactivation was observed. To extrapolate these data to in vivo , the extent of the increase in the area under the curve (AUC ratio) induced by erythromycin was estimated from the results of the conventional short‐term experiment and the long‐term experiment examining residual activity. The AUC ratio estimated from the long‐term kinetics (2.92) was closer to the clinically reported values (3.3–4.42). In conclusion, the relatively long‐term evaluation of the kinetics of CYP3A4 inactivation revealed that the enzyme was not fully inactivated by erythromycin. To improve the estimation of the extent of the drug–drug interactions induced by MBI from in vitro data, longer‐term investigations of the target enzyme's inactivation profile might be necessary.     

Inhibition of human liver microsomal CYP by nateglinide

Objectives Nateglinide is metabolized by CYP2C9 and CYP3A4, therefore drug–drug interactions through cytochrome P450 (CYP) inhibition may occur. In this study, we examined the inhibitory effects of nateglinide and its major metabolite N‐[trans‐4‐(1‐hydroxy‐1‐methylethyl)‐cyclohexanecarbonyl]‐d ‐phenylalanine (M1) on various CYP isoforms in human liver microsomes. Methods We used typical substrates (7‐ethoxyresorufin for CYP1A1/2, tolbutamide for CYP2C9, S‐mephenytoin for CYP2C19, bufuralol for CYP2D6, chlorzoxazone for CYP2E1 and midazolam for CYP3A4) in the evaluation of the inhibitory effects, and examined the possibility of mechanism‐based inhibition (MBI) by evaluating the influence of pre‐incubation in the inhibition. Key findings The results showed that nateglinide inhibited CYP2C9 and CYP2C19 with an IC50_app (apparent value of the 50% inhibitory concentration) of 125 μmol/l and 946 μmol/l, respectively, while M1 did not inhibit any of the CYP isoforms. The inhibition constant (K_i) value of the inhibitory effect of nateglinide on CYP2C9 and the 1 + I_in,max,u/K_i value were estimated (where I_in,max,u= the maximum unbound concentration of nateglinide). The 1 + I_in,max,u/K_i value was 1.02 (close to 1), suggesting a low risk of drug–drug interactions. The influence of pre‐incubation on the inhibition by nateglinide of CYP3A4, CYP2C9 and CYP2C19 was examined. The results revealed that the inhibition of CYP by nateglinide was not influenced by pre‐incubation, and that the possibility of MBI is very low. Conclusions The possibility of drug–drug interactions involving nateglinide that might be attributable to CYP inhibition is low.     

Drug-drug interactions via mechanism-based cytochrome P450 inactivation: points to consider for risk assessment from in vitro data and clinical pharmacologic evaluation

This commentary discusses the approaches to, and key considerations in the in vitro-in vivo extrapolation of drug-drug interactions (DDI) resulting from mechanism-based inactivation (MBI) of cytochrome P450 (CYP) enzymes and clinical pharmacologic implications. In vitro kinetic assessment and prediction of DDI produced via reversible inhibition and MBI rely on operationally and conceptually distinct approaches. DDI risk assessment for inactivators requires estimation of maximal inactivation rate (k(inact)) and inactivator potency (KI) in vitro, that need to be considered in context of the biological turnover rate of the enzyme (kdeg) and clinical exposures of the inactivator (I), respectively, to predict interaction magnitude. Risk assessment cannot be performed by a simple comparison of inactivator potency against in vivo exposure since inactivation is both concentration and time-dependent. MBI contour plots tracking combinations of I:KI and k(inact):k(deg) resulting in identical fold-reductions in intrinsic clearance are proposed as a useful framework for DDI risk assessment. Additionally, substrate-specific factors like fraction of the total clearance of the object drug via the enzyme being inactivated (f(m(CYP) )) and the bioavailability fraction across the intestine for CYP3A substrates (F(G)) are important determinants of interaction magnitude. Sensitivity analysis of predicted DDI magnitude to uncertainty in input parameters is recommended to inform confidence in predictions. The time course of reversal of DDI resulting from CYP inactivation is determined by the half-life of the enzyme which is an important consideration in the design and interpretation of clinical DDI studies with inactivators.     

Differential selectivity in carbamazepine-induced inactivation of cytochrome P450 enzymes in rat and human liver

Oxidative metabolism of carbamazepine results in covalent binding of its reactive metabolite to liver microsomal proteins, which has been proposed as an important event in pathogenesis of the hypersensitivity reactions to this drug. Although the proposed reactive metabolites are produced by cytochrome P450 enzymes (P450 or CYP), the impact of the formation of unstable metabolites on the enzyme itself has not been elucidated. The present study examines the alteration of P450 enzyme activities during the metabolism of carbamazepine. Liver microsomes from rats and humans were preincubated with carbamazepine in the presence of NADPH, and subsequently assayed for monooxygenase activities representing several P450s. No evidence was obtained for inactivation of CYP2C11, CYP3A, CYP1A1/2 or CYP2B1/2 in rat liver microsomes during the carbamazepine metabolism, whereas the CYP2D enzyme was inactivated in a manner related to the preincubation time. Interestingly, under the same protocol human liver microsomes did not exhibit inactivation of CYP2D6, as well as there being no CYP2C8, CYP2C9 or CYP3A4 inactivation, whereas CYP1A2 was inactivated. Reduced glutathione could not protect against the observed inactivation of the P450s. These results suggest that CYP2D enzyme(s) in rats and CYP1A2 in humans biotransform carbamazepine into reactive metabolites, resulting in inactivation of the enzyme themselves, and raise the possibility that the P450 isoforms participate in toxicity induced by the drug in both animal species.     

Identification of the residue in human CYP3A4 that is covalently modified by bergamottin and the reactive intermediate that contributes to the grapefruit juice effect

Previous studies have demonstrated that bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of CYP3A4 and contributes, in part, to the grapefruit juice-drug interaction. Although the covalent binding of [(14)C]BG to the CYP3A4 apoprotein has been demonstrated by SDS-polyacrylamide gel electrophoresis, the identity of the modified amino acid residue and the reactive intermediate species of BG responsible for the inactivation have not been reported. In the present study, we show that inactivation of CYP3A4 by BG results in formation of a modified apoprotein-3A4 and a GSH conjugate, both exhibiting mass increases of 388 Da, which corresponds to the mass of 6',7'-dihydroxybergamottin (DHBG), a metabolite of BG, plus one oxygen atom. To identify the adducted residue, BG-inactivated 3A4 was digested with trypsin, and the digests were then analyzed by liquid chromatography-tandem mass spectrometry (MS/MS). A mass shift of 388 Da was used for the SEQUEST database search, which revealed a mass increase of 388 Da for the peptide with the sequence (272)LQLMIDSQNSK(282), and MS/MS analysis of the adducted peptide demonstrated that Gln273 is the residue modified. Mutagenesis studies showed that the Gln273 to Val mutant was resistant to inactivation by BG and DHBG and did not generate two of the major metabolites of BG formed by 3A4 wild type. In conclusion, we have determined that the reactive intermediate, oxygenated DHBG, covalently binds to Gln273 and thereby contributes to the mechanism-based inactivation of CYP3A4 by BG.     

Acyclic nucleoside phosphonates: a study on cytochrome P450 gene expression

Abstract

1.?Nucleotide analogues comprise an important class of drugs used in treatment of viral infections but also cancer. These drugs affect the structural integrity of DNA and activate different pathways and processes in the cell and may directly or indirectly influence the drug metabolizing system. Adefovir dipivoxil (AD) and tenofovir disoproxil (TD) are nucleotide analogues approved for the treatment of chronic hepatitis B and/or HIV/AIDS infection. 2.?To evaluate the risk of their drug–drug interactions on the level of drug metabolism, an effect of both compounds on cytochromes P450 expression was studied using cDNA microarrays, real-time RT-PCR and immunoblotting. Mice were given intraperitoneally 25?mg/kg of AD or TD, respectively. As a positive control, a combination of prototypic cytochromes P450 (CYP) inducers, phenobarbital and β-naphthoflavone was chosen. 3.?The data obtained showed a significant CYP induction in the positive control group, but no clinically significant induction of CYP genes by AD or TD was observed. Our results support the evidence of safety of AD and TD with respect to drug–drug interactions based on enzyme induction. These findings are important as a plethora of new antivirals of different types are being tested and introduced to clinical practice, mostly to be used in combinations.     

Time-dependent CYP inhibition

Time-dependent inhibition (TDI) of CYP refers to a change in potency during an in vitro incubation or dosing period in vivo. Potential mechanisms include the formation of inhibitory metabolites and mechanism-based inhibition (MBI). In vitro experiments are configured to assess TDI and MBI is inferred, at least initially. MBI is more profound after multiple-dosing and the recovery period is independent of continued drug exposure. Advances in in vitro-in vivo extrapolations for competitive inhibition and the potential relationship between MBI and reactive metabolite-mediated toxicity, have redirected emphasis to CYP TDI. In contrast, with reversible inhibition, strategies for projecting the risks from TDI are less developed and the traditional I/K(i) model often yields a dramatic underprediction. This review explores the contribution of TDI to drug-drug interactions and idiosyncratic drug toxicity.     

Therapeutic drugs that behave as mechanism-based inhibitors of cytochrome P450 3A4

Cytochrome P450 (CYP) 3A4 is not only the most abundant isoform in human liver but also metabolizes approximately 60% of the therapeutic drugs. This feature renders CYP3A4 highly susceptible to both reversible and irreversible (mechanism-based) inhibition. The latter is characterized by NADPH-, time- and concentration-dependent enzyme inactivation, occurring when some drugs are converted by CYPs to reactive metabolites. Mechanism-based inactivation of CYP3A4 by drugs can be due to the chemical modification of the heme, the protein, or both as a result of covalent binding of modified heme to the protein. The clinical pharmacokinetic effect of a CYP3A4 inactivator is a function of its KI, kinact and partition ratio and the synthesis rate of new or replacement enzyme. Predicting drug-drug interactions involving CYP3A4 inactivation is possible when proper pharmacokinetic principles are followed. However, the prediction may become difficult, since the clinical outcomes due to CYP3A4 inactivation depend on many factors associated with the enzyme, drugs and the patients. A number of clinically important drugs have been identified to be mechanism-based CYP3A4 inhibitors. These include antibiotics (e.g. erythromycin and isoniazid), anticancer drugs (e.g. tamoxifen), antidepressants (e.g. fluoxetine and midazolam), anti-HIV agents (e.g. ritonavir and delavirdine), antihypertensives (e.g. dihydralazine and verapamil), steroids and their receptor modulators (e.g. gestodene and raloxifene), and some herbal constituents (e.g. bergamottin and glabridin). Compared to reversible inhibition, mechanism-based inhibitors of CYP3A4 more frequently cause unfavorable drug-drug interactions, as the inactivated CYP3A4 has to be replaced by newly synthesized CYP3A4 protein. Most CYP3A4 inactivators are also PgP substrates/inhibitors, confounding the in vitro-in vivo extrapolation. Clinicians should have good knowledge on these CYP3A4 inactivators and avoid their combination use.     

Structure-based Drug Metabolism Predictions for Drug Design

Significant progress has been made in structure-based drug design by pharmaceutical companies at different stages of drug discovery such as identifying new hits, enhancing molecule binding affinity in hit-to-lead, and reducing toxicities in lead optimization. Drug metabolism is a major consideration for modifying drug clearance and also a primary source for drug metabolite-induced toxicity. With major cytochrome P450 structures identified and characterized recently, structure-based drug metabolism prediction becomes increasingly attractive. In silico methods based on molecular and quantum mechanics such as docking, molecular dynamics and ab initio chemical reactivity calculations bring us closer to understand drug metabolism and predict drug–drug interactions. In this study, we review important progress in drug metabolism and common in silico techniques adopted to predict drug regioselectivity, stereoselectivity, reactive metabolites, induction, inhibition and mechanism-based inactivation, as well as their implementation in hit-to-lead drug discovery.     

Cytochrome P450 Inactivation by Pharmaceuticals and Phytochemicals: Therapeutic Relevance

One of the major clinical concerns is possible drug interactions that can be the result of abrogation of the P450 pathway(s) of metabolism causing toxicity due to elevated exposures of other drugs metabolized by these pathways. When the P450 substrate is catalytically activated to a reactive intermediate, this transient molecule may react with available nucleophilic residues from the enzyme – thereby resulting in the inactivation of the P450. The effects of CYP inactivation on the pharmacokinetics of co-administered drugs or on the inactivator itself depend on complex factors involving the molecular entities, the kinetics of inactivation (K_I, k_inact), the partition ratio, the zero-order synthesis rate of new enzyme, multiple pathways of metabolism (competing pathways), the dose or exposure, and specific patient characteristics. This review summarizes the catalytic efficiencies of many inactivator drugs along with any consequent clinical relevance. The chemical agents described have been ranked for the kinetic efficiency of inactivation and contrasted with the known clinically relevant drug interactions. This will allow judicious consideration of the many factors that influence the importance of CYP inactivation and their relative contribution to systemic clearance of co-administered drugs. This study allows an improved characterization and dissection of potential physiological interactions with various drugs and nutrients. Knowing more about selective inactivation of cytochrome P450 by common xenobiotics, drugs and phytochemicals allows better understanding of expected interactions with chemotherapeutics and other xenobiotics.     

Analysis of inhibition kinetics of three beverage ingredients,bergamottin, dihydroxybergamottin and resveratrol,on CYP2C9 activity

Some grapefruit juice (GFJ) ingredients and resveratrol, a fruit-derived phytoalexin, are known to inhibit cytochrome P450 (CYP) 2C9. However, their inhibition modes and detailed inhibition kinetics remain undetermined. This study aimed to investigate the inhibitory effects of two GFJ ingredients, bergamottin (BG) and dihydroxybergamottin (DHB), and resveratrol on CYP2C9 activity in vitro. DHB inhibited CYP2C9 activity, as assessed by warfarin 7-hydroxylation, in a preincubation time-dependent manner (i.e., mechanism-based inhibition; MBI), in the same manner as CYP2C19 and CYP3A4. The maximal inactivation rate (k_inact,max) was 0.0638 min⁻¹ and 0.12- and 0.26-fold of that for CYP2C19 and CYP3A4, respectively. BG showed both MBI and time-independent competitive inhibition. Resveratrol showed non-competitive inhibition with an inhibition constant (K_i) of 3.64 μM. Unlike the inhibition of CYP2C19 and CYP3A4, resveratrol did not induce MBI. These findings are important for estimating the risk of drug interactions between CYP2C9 substrates and some beverages. (146 words)     

Covalent binding of 14C- and 35S-labeled thiocarbamides in rat hepatic microsomes.

The covalent binding of a series of 14C- or 35S-labeled benzimidazole-2-thione (MBI) derivatives to rat liver microsomal proteins was studied to determine the mechanisms of hepatic monooxygenase oxidation of model anti-hyperthyroid compounds. All thiocarbamides tested (including methimazole) produced an NADPH-dependent loss of cytochrome P450 (P450) chromophore which could be prevented by the addition of glutathione (GSH). The covalent binding of MBI to liver microsomal proteins from dexamethasone (DEX)-pretreated rats was enhanced 10-fold with NADPH, unaffected by P450 inactivation with 1-aminobenzotriazole (ABT) and attenuated by GSH addition. Heat treatment of microsomes to inactivate the flavin-containing monooxygenase (FMO) decreased the observed binding. Equivalent amounts of [35S]- and [14C]MBI were covalently bound to hepatic microsomal proteins, suggesting retention of both the carbon and sulfur portions of the molecule in the MBI/protein adduct. Thiophilic reagents effected release of covalently bound [14C]- and [35S]MBI in equal amounts suggesting the presence of disulfide bonds between an MBI-derived sulfenic acid and microsomal protein thiols. Coincubation with bovine serum albumin (BSA) resulted in NADPH-dependent binding of [14C]-MBI to BSA sulfhydryls which was blocked by prior treatment of BSA with iodoacetamide. 1-Methyl-benzimidazole-2-thione (MMBI) also covalently bound to microsomal proteins and BSA but at levels lower than with MBI. P450, however, appeared to be more important than FMO in the metabolism of MMBI based on the effects of microsome heat pretreatment or ABT addition. In addition, ca. 1.5-fold more 35S- than 14C-label became bound. The covalent binding of [35S]1,3-dimethyl-benzimidazole-2-thione (DMMBI) to microsomal proteins was ca. six times greater than that of [14C]DMMBI. ABT, catalase and superoxide dismutase had a minimal effect on [35S]DMMBI binding, while FMO inactivation decreased binding by ca. 30%. These findings suggest that both monooxygenases contribute significantly to the hepatic metabolism of thiocarbamides. However, FMO activates thiocarbamides primarily to sulfenic acids, whereas P450 appears to produce both sulfenic acid and other reactive sulfur-derived metabolites. Thiol groups of P450 and other proteins are the molecular targets for these reactive species formed during the hepatic metabolism of anti-hyperthyroid drugs.