Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes

Pinostrobin (PI, 5‐hydroxy‐7‐methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450‐selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro . High‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug‐metabolizing CYP450 isozymes in vitro . In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 μm ). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.     

Insights on cytochrome p450 enzymes and inhibitors obtained through QSAR studies

The cytochrome P450 (CYP) superfamily of heme enzymes play an important role in the metabolism of a large number of endogenous and exogenous compounds, including most of the drugs currently on the market. Inhibitors of CYP enzymes have important roles in the treatment of several disease conditions such as numerous cancers and fungal infections in addition to their critical role in drug-drug interactions. Structure activity relationships (SAR), and three-dimensional quantitative structure activity relationships (3D-QSAR) represent important tools in understanding the interactions of the inhibitors with the active sites of the CYP enzymes. A comprehensive account of the QSAR studies on the major human CYPs 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and a few other CYPs are detailed in this review which will provide us with an insight into the individual/common characteristics of the active sites of these enzymes and the enzyme-inhibitor interactions.     

Comparison of metabolic soft spot predictions of CYP3A4, CYP2C9 and CYP2D6 substrates using MetaSite and StarDrop

Metabolite identification study plays an important role in determining the sites of metabolic liability of new chemical entities (NCEs) in drug discovery for lead optimization. Here we compare the two predictive software, MetaSite and StarDrop, available for this purpose. They work very differently but are used to predict the site of oxidation by major human cytochrome P450 (CYP) isoforms. Neither software can predict non-CYP catalyzed metabolism nor the rates of metabolism. For the purpose of comparing the two software packages, we tested known probe substrate for these enzymes, which included 12 substrates of CYP3A4 and 18 substrates of CYP2C9 and CYP2D6 were analyzed by each software and the results were compared. It is possible that these known substrates were part of the training set but we are not aware of it. To assess the performance of each software we assigned a point system for each correct prediction. The total points assigned for each CYP isoform experimentally were compared as a percentage of the total points assigned theoretically for the first choice prediction for all substrates for each isoform. Our results show that MetaSite and StarDrop are similar in predicting the correct site of metabolism by CYP3A4 (78% vs 83%, respectively). StarDrop appears to do slightly better in predicting the correct site of metabolism by CYP2C9 and CYP2D6 metabolism (89% and 93%, respectively) compared to MetaSite (63% and 70%, respectively). The sites of metabolism (SOM) from 34 in-house NCEs incubated in human liver microsomes or human hepatocytes were also evaluated using two prediction software packages and the results showed comparable SOM predictions. What makes this comparison challenging is that the contribution of each isoform to the intrinsic clearance (Clint) is not known. Overall the software were comparable except for MetaSite performing better for CYP2D6 and that MetaSite has a liver model that is absent in StarDrop that predicted with 82% accuracy.     

Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated β-cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis–Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.     

Coexpression of CPR from Various Origins Enhances Biotransformation Activity of Human CYPs in S. pombe

Cytochrome P450 enzymes (CYPs or P450s) are the most important enzymes involved in the phase I metabolism of drugs (and other xenobiotics) in humans, and the corresponding drug metabolites are needed as reference substances for their structural confirmation and for pharmacological or toxicological characterization. We have previously shown that biotechnological synthesis of such metabolites is feasible by whole-cell biotransformation with human CYPs recombinantly expressed in the fission yeast Schizosaccharomyces pombe. It was the aim of this study to compare the activity of seven human microsomal CYPs (CYP2C9, CYP2D6, CYP3A4, CYP3A5, CYP3A7, CYP17, and CYP21) upon coexpression with NADPH-cytochrome P450 oxidoreductases (CPRs) from various origins, namely, human CPR (hCPR) and its homologues from fission yeast (ccr1) and the bishop’s weed Ammi majus (AmCPR), respectively. For this purpose, 28 recombinant strains were needed, with five of them having been constructed previously and 23 strains being newly constructed. Bioconversion experiments showed that coexpression of a CPR does not only influence the reaction rate but, in some cases, also exerts an influence on the metabolite pattern. For CYP3A enzymes, coexpression of hCPR yielded the best results, while for another two, hCPR was equally helpful as ccr1 (both CYP17 and CYP21) or AmCPR (CYP17 only), respectively. Interestingly, CYP2D6 displayed its highest activity when coexpressed with ccr1 and CYP2C9 with AmCPR. These results corroborate the view of CPR as a well-suited bio-brick in synthetic biology for the construction of artificial enzyme complexes.     

Analysis of frankincense from various Boswellia species with inhibitory activity on human drug metabolising cytochrome P450 enzymes using liquid chromatography mass spectrometry after automated on-line extraction

In our search for herbal remedies with inhibitory activity on cytochrome P450 (CYP) enzymes, we identified extracts of the gum-resin of Boswellia carteri, Boswellia frereana, Boswellia sacra and Boswellia serrata as equally potent, non-selective inhibitors of the major drug metabolising CYP enzymes 1A2/2C8/2C9/2C19/2D6 and 3A4. LC/LC/ESI-MS fingerprint analyses of the boswellic acids 11-keto-beta-boswellic acid, alpha-boswellic acid, beta-boswellic acid and their 3-O-acylated derivatives were used for the authentication of the commercially obtained frankincense samples. Although the boswellic acids could be identified as moderate to potent inhibitors of the applied CYP enzymes, they are not the major CYP inhibitory principle of frankincense.     

Capillary electrophoretic investigation of the enantioselective metabolism of propafenone by human cytochrome P-450 SUPERSOMES: Evidence for atypical kinetics by CYP2D6 and CYP3A4

An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). Using in vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N-dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.     

Constituents of Zingiber aromaticum and their CYP3A4 and CYP2D6 inhibitory activity

A new sesquiterpene 2,9-humuladien-6-ol-8-one (1) was isolated from methanol extract of Zingiber aromaticum, along with 15 known compounds. The structures of the isolated compounds were elucidated on the basis of spectroscopic analyses. The isolated compounds were tested for their inhibitory activity on the metabolism mediated by cytochrome P450 3A4 (CYP3A4) and CYP2D6.     

High-throughput screening of inhibitory potential of nine cytochrome P450 enzymes in vitro using liquid chromatography/tandem mass spectrometry

The early detection of potential drug-drug interactions is an important issue of drug discovery that has led to the development of high-throughput screening (HTS) methods for potential drug interactions. We developed a HTS method for potential interactions of inhibitory drugs for nine human P450 enzymes using cocktail incubation and tandem mass spectrometry in vitro. This new method involves incubation of two cocktail doses and single cassette analysis. The two cocktail doses in vitro were developed to minimize solvent effects and mutual drug interactions among substrates: cocktail A was composed of phenacetin for CYP1A2, coumarin for CYP2A6, paclitaxel for CYP2C8, S-mephenytoin for CYP2C19, dextromethorphan for CYP2D6, and midazolam for CYP3A4; and cocktail B was composed of three substrates including bupropion for CYP2B6, tolbutamide for CYP2C9, and chlorzoxazone for CYP2E1. In the incubation study of these cocktails, the reaction mixtures were pooled and simultaneously analyzed using liquid chromatography/tandem mass spectrometry employing a fast gradient. The method was validated by comparing the inhibition data obtained from the incubation of each individual probe substrate alone with data from the new method. The IC50 value of each inhibitor in the cocktail agreed well with that of the individual probe drug as well as with values previously reported in the literature. As a HTS method for potential interactions of the inhibition of these nine P450 enzymes, this new method will be useful in the drug discovery process and for the mechanistic understanding of drug interactions.     

Predictive models for cytochrome p450 isozymes based on quantitative high throughput screening data

The human cytochrome P450 (CYP450) isozymes are the most important enzymes in the body to metabolize many endogenous and exogenous substances including environmental toxins and therapeutic drugs. Any unnecessary interactions between a small molecule and CYP450 isozymes may raise a potential to disarm the integrity of the protection. Accurately predicting the potential interactions between a small molecule and CYP450 isozymes is highly desirable for assessing the metabolic stability and toxicity of the molecule. The National Institutes of Health Chemical Genomics Center (NCGC) has screened a collection of over 17,000 compounds against the five major isozymes of CYP450 (1A2, 2C9, 2C19, 2D6, and 3A4) in a quantitative high throughput screening (qHTS) format. In this study, we developed support vector classification (SVC) models for these five isozymes using a set of customized generic atom types. The CYP450 data sets were randomly split into equal-sized training and test sets. The optimized SVC models exhibited high predictive power against the test sets for all five CYP450 isozymes with accuracies of 0.93, 0.89, 0.89, 0.85, and 0.87 for 1A2, 2C9, 2C19, 2D6, and 3A4, respectively, as measured by the area under the receiver operating characteristic (ROC) curves. The important atom types and features extracted from the five models are consistent with the structural preferences for different CYP450 substrates reported in the literature. We also identified novel features with significant discerning power to separate CYP450 actives from inactives. These models can be useful in prioritizing compounds in a drug discovery pipeline or recognizing the toxic potential of environmental chemicals.     

Prediction of cytochrome P450 3A4, 2D6, and 2C9 inhibitors and substrates by using support vector machines

Statistical learning methods have been used in developing filters for predicting inhibitors of two P450 isoenzymes, CYP3A4 and CYP2D6. This work explores the use of different statistical learning methods for predicting inhibitors of these enzymes and an additional P450 enzyme, CYP2C9, and the substrates of the three P450 isoenzymes. Two consensus support vector machine (CSVM) methods, "positive majority" (PM-CSVM) and "positive probability" (PP-CSVM), were used in this work. These methods were first tested for the prediction of inhibitors of CYP3A4 and CYP2D6 by using a significantly higher number of inhibitors and noninhibitors than that used in earlier studies. They were then applied to the prediction of inhibitors of CYP2C9 and substrates of the three enzymes. Both methods predict inhibitors of CYP3A4 and CYP2D6 at a similar level of accuracy as those of earlier studies. For classification of inhibitors of CYP2C9, the best CSVM method gives an accuracy of 88.9% for inhibitors and 96.3% for noninhibitors. The accuracies for classification of substrates and nonsubstrates of CYP3A4, CYP2D6, and CYP2C9 are 98.2 and 90.9%, 96.6 and 94.4%, and 85.7 and 98.8%, respectively. Both CSVM methods are potentially useful as filters for predicting inhibitors and substrates of P450 isoenzymes. These methods generally give better accuracies than single SVM classification systems, and the performance of the PP-CSVM method is slightly better than that of the PM-CSVM method.     

Preparation, characterization, and substrate metabolism of gold-immobilized cytochrome P450 2C9

The cytochrome P450 enzymes represent an important class of heme-containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological-based chips for standardizing cytochrome P450 metabolism or for high-throughput screening of pharmaceutical agents. Previous studies have generally immobilized P450 enzymes in a matrix or on a surface. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely, superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed.     

Structural determinants of steroids for cytochrome P450 3A4-mediated metabolism

Cytochrome P450 3A4 (CYP3A4), a major member of cytochrome P450 isoenzymes, metabolizes the majority of steroids in 6β-position. For the purpose of determining requisite structural features of a series of structurally related steroids for CYP3A4-mediated metabolism, three-dimensional pharmacophore modeling as well as electrotopological state map were conducted for 15 steroids. Though prior studies speculated that the chemical reactivity of the allylic 6β-position might have a greater influence on CYP3A4 selective 6-hydroxylation than steric constraints in the enzyme, our results reveal that for CYP3A4 steroidal substrates, it is not the chemical reactivity of atoms at 6β-site, but the pharmacophoric features, i.e. the two hydrophobic rings together with two H-bond donors, that act as the key factors responsible for determining the CYP3A4 selective 6-hydroxylation of steroids.     

Molecular docking,PKPD, and assessment of toxicity of few chalcone analogues as EGFR inhibitor in search of anticancer agents

In the present work, molecular docking of the chalcone analogues with receptor EGFR carried out using erlotinib as reference drug is reported. About 15 chalcone analogues were analyzed CHL(1–15). Molecules CHL2, CHL3, CHL9, CHL11, and CHL15 found strong affinity for receptor EGFR exhibiting binding energies ??7.7 kcal/mol, ??7.5 kcal/mol, ??7.6 kcal/mol, ??7.9 kcal/mol, and ??8.1 kcal/mol, respectively, when erlotinib a reference drug exhibits binding energy ??7.6 kcal/mol. Toxicity for molecules was assessed against the cytochromes P450 (CYP) and P-gp using Swiss ADMET. Molecule CHL9 could be a suitable lead compound inhibitor to CYP1A2 followed by CHL2 inhibitor of CYP1A2 and CYP2C9 and CHL15 with a most stable binding affinity of ??8.1 kcal/mol, inhibiting CYP1A2, CYP2C19, and CYP2D6. CHL3 has a binding affinity of ??7.5 kcal/mol, inhibiting all the 05 CYP enzymes (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4). CHL11 has a binding affinity of ??7.9 kcal/mol, inhibiting CYP1A2, CYP2C19, and CYP2C9. Considering inhibition of CYP family enzymes by molecules, further here we have perform the enrichment analysis to these CYP family enzymes and reported the metabolic pathways which were probably affected by inhibition of these enzymes using EnrichR online enrichment analysis server. The current predictions over these 15 chalcone derivatives will be needed to further investigate in vivo and in vitro conditions to identify the optimum therapeutic efficacy and least toxicity.

     

Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies

An automated in-capillary assay requiring very small quantities of reagents was developed for performing in vitro cytochrome P450 (CYP450) drug metabolism studies. The approach is based on the following: (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sandwich mode, within a capillary; (ii) mixing the reagents by diffusion across the interfaces between the injected solutions; (iii) collection of the capillary content at the end of the in-capillary assay; and (iv) off-line analysis of the incubation mixture by ultrahigh pressure liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). After optimizing the injection sequence of the reagents, the in-capillary approach was applied to the quantitative determination of the kinetics of drug metabolism reactions catalyzed by three CYP450 isozymes involved in human drug metabolism: CYP1A2, CYP2D6, and CYP3A4. It was demonstrated that this in-capillary method was able to provide similar kinetic parameters for CYP450 activity (e.g., Michaelis constants and turnover values) as the classical in vitro method, with a drastic reduction of reagent consumption.     

The Use of Immobilized Cytochrome P4502C9 in PMMA-Based Plug Flow Bioreactors for the Production of Drug Metabolites

Cytochrome P450 enzymes play a key role in the metabolism of pharmaceutical agents. To determine metabolite toxicity, it is necessary to obtain P450 metabolites from various pharmaceutical agents. Here, we describe a bioreactor that is made by immobilizing cytochrome P450 2C9 (CYP2C9) to a poly(methyl methacrylate) surface and, as an alternative to traditional chemical synthesis, can be used to biosynthesize P450 metabolites in a plug flow bioreactor. As part of the development of the CYP2C9 bioreactor, we have studied two different methods of attachment: (1) coupling via the N-terminus using N-hydroxysulfosuccinimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and (2) using the Ni(II) chelator 1-acetato-4-benzyl-triazacyclononane to coordinate the enzyme to the surface using a C-terminal histidine tag. Additionally, the propensity for metabolite production of the CYP2C9 proof-of-concept bioreactors as a function of enzyme attachment conditions (e.g., time and enzyme concentration) was examined. Our results show that the immobilization of CYP2C9 enzymes to a PMMA surface represents a viable and alternative approach to the preparation of CYP2C9 metabolites for toxicity testing. Furthermore, the basic approach can be adapted to any cytochrome P450 enzyme and in a high-throughput, automated process.     

UHPLC‐Q‐TOF‐MS/MS‐based screening and characterization of metabolites of cnidilin in human liver microsomes

Cnidilin is an active natural furocoumarin ingredient originating from well‐known traditional Chinese medicine Radix Angelicae Dahuricae . In the present study, an efficient approach was developed for the screening and identification of cnidilin metabolites using ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry. In this approach, an on‐line data acquisition method multiple mass defect filter combined with dynamic background subtraction was developed to trace all probable metabolites. Based on this analytical strategy, a total of 24 metabolites of cnidilin were detected in human liver microsomal incubation samples and the metabolic pathways were proposed. The results indicated that oxidation was the main biotransformation route for cnidilin in human liver microsomes. In addition, the specific cytochrome P450 (CYP) enzymes involved in the metabolism of cnidilin were identified using chemical inhibition and CYP recombinant enzymes. The results showed that CYP1A2 and CYP3A4 might be the major enzymes involved in the metabolism of cnidilin in human liver microsomes. The relationship between cnidilin and the CYP450 enzymes could provide us a theoretical basis of the pharmacological mechanism.