Metabolic stability and determination of cytochrome P450 isoenzymes' contribution to the metabolism of medetomidine in dog liver microsomes

Medetomidine is a potent and selective α2‐adrenergic agonist. The activation of α2‐adrenergic receptor mediates a variety of effects including sedation, analgesia, relief of anxiety, vasoconstriction and bradycardia. However, our main interest is the sedative effects of medetomidine when used as a premedicant prior surgery in companion animals, especially in dogs. Recently, data suggested that following intravenous infusion at six dosing regiments non‐linear pharmacokinetics was observed. Major causes of non‐linear pharmacokinetics are the elimination of the drug not following a simple first‐order kinetics and/or the elimination half‐life changing due to saturation of an enzyme system. The goal of this study was to establish the metabolic stability and determine the metabolic pathway of medetomidine in dog liver microsomes. Consequently, Michaelis–Menten parameters (V_max, K_m), T_1/2 and CL_i were determined. The incubations were performed in a microcentrifuge tube and containing various concentrations of medetomidine (10–5000 nm ), 1 mg/mL of microsomal proteins suspended in 0.1 m phosphate buffer, pH 7.4. Microsomal suspensions were preincubated with NADPH (1 mm ) for 5 min at 37°C prior to fortification with medetomidine. Samples were taken at various time points for kinetic information and the initial velocity (v_i) was determined after 10 min incubation. The reaction was stopped by the addition of an internal standard solution (100 ng/mL of dextrometorphan in acetone). Medetomidine concentrations were determined using a selective and sensitive HPLC‐ESI/MS/MS method. Using non‐linear regression, we determined a K_m value of 577 nm , indicating relatively low threshold enzyme saturation consistent with previous in vivo observation. The metabolic stability was determined at a concentration of 100 nm (?K_m) and the observed T_1/2 was 90 min with a CL_i of 0.008 mL/min indicating moderately low clearance in dog liver microsomes, also consistent with previous in vivo data. Moreover, results suggest that principally medetomidine is metabolized by the CYP3A with a small contribution from CYP2D and CYP2E. The participation of CYP3A is an important discovery since medetomidine is used as a premedicant in combination with fentanyl, ketamine and/or midazolam. These findings combined with a low K_m value may indicate that medetomidine can competitively inhibit the metabolism of these drugs and consequently significantly impair metabolic clearance. Copyright © 2009 John Wiley & Sons, Ltd.     

Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP‐HPLC method to serve as activity marker

In this study, a simple and reliable reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was established and validated to analyze S‐mephenytoin 4‐hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co‐expressing CYP2C19 and NADPH‐CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP‐HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP‐HPLC assay showed good linearity (r² = 1.00) with 4‐hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10^?2 μm . Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (K_m, V_max and K_i) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co‐expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP‐HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabolism of methylstenbolone studied with human liver microsomes and the uPA+/+‐SCID chimeric mouse model

Anti‐doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono‐hydroxylated derivatives (U1–U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di‐hydroxylated metabolites (U11–U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC‐MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method. Copyright © 2014 John Wiley & Sons, Ltd.     

Corydaline inhibits multiple cytochrome P450 and UDP-glucuronosyltransferase enzyme activities in human liver microsomes

Corydaline is a bioactive alkaloid with various antiacetylcholinesterase, antiallergic, and antinociceptive activities found in the medicinal herb Corydalis Tubers. The inhibitory potential of corydaline on the activities of seven major human cytochrome P450 and four UDP-glucuronosyltransferase enzymes in human liver microsomes was investigated using LC-tandem MS. Corydaline was found to inhibit CYP2C19-catalyzed S-mephenytoin-4'-hydroxylatoin and CYP2C9-catalyzed diclofenac 4-hydroxylation, with K(i) values of 1.7 and 7.0 mM, respectively. Corydaline also demonstrated moderate inhibition of UGT1A1-mediated 17b-estradiol 3-glucuronidation and UGT1A9-mediated propofol glucuronidation with K(i) values of 57.6 and 37.3 mM, respectively. In the presence of corydaline, CYP3A-mediated midazolam hydroxylation showed a decrease with increasing preincubation time in a dose-dependent manner with K(i) values of 30.0 mM. These in vitro results suggest that corydaline should be evaluated for potential pharmacokinetic drug interactions in vivo due to potent inhibition of CYP2C19 and CYP2C9.     

Computational Prediction of Inhibitors and Inducers of the Major Isoforms of Cytochrome P450

Human cytochrome P450 enzymes (CYPs) are heme-containing monooxygenases. This superfamily of drug-metabolizing enzymes is responsible for the metabolism of most drugs and other xenobiotics. The inhibition of CYPs may lead to drug–drug interactions and impair the biotransformation of drugs. CYP inducers may decrease the bioavailability and increase the clearance of drugs. Based on the freely available databases ChEMBL and PubChem, we have collected over 70,000 records containing the structures of inhibitors and inducers together with the IC50 values for the inhibitors of the five major human CYPs: 1A2, 3A4, 2D6, 2C9, and 2C19. Based on the collected data, we developed (Q)SAR models for predicting inhibitors and inducers of these CYPs using GUSAR and PASS software. The developed (Q)SAR models could be applied for assessment of the interaction of novel drug-like substances with the major human CYPs. The created (Q)SAR models demonstrated reasonable accuracy of prediction. They have been implemented in the web application P450-Analyzer that is freely available via the Internet.     

In vitro metabolism of corydaline in human liver microsomes and hepatocytes using liquid chromatography-ion trap mass spectrometry

Corydaline is a pharmacologically active isoquinoline alkaloid isolated from Corydalis tubers. It exhibits the antiacetylcholinesterase, antiallergic, antinociceptive, and gastric emptying activities. The purposes of this study were to establish in vitro metabolic pathways of corydaline in human liver microsomes and hepatocytes by identification of their metabolites using liquid chromatography-ion trap mass spectrometry. Human liver microsomal incubation of corydaline in the presence of an NADPH-generating system resulted in the formation of nine metabolites, namely, four O-desmethylcorydaline [M1 (yuanhunine), M2 (9-O-desmethylcorydaline), M3 (isocorybulbine), and M4 (corybulbine)], three di-O-desmethylcorydaline [M5 (9,10-di-O-desmethylcorydaline), M6 (2,10-di-O-desmethylcorydaline), and M7 (3,10-di-O-desmethylcorydaline)], M8 (hydroxyyuanhunine), and M9 (hydroxycorydaline). Incubation of corydaline in human hepatocytes produced four metabolites including M1, M5, M6, and M9. O-Demethylation and hydroxylation were the major metabolic pathways for the metabolism of corydaline in human liver microsomes and hepatocytes.     

细胞色素P450酶电化学生物传感器的构建及其药物代谢应用

药物代谢过程是药物在体内产生药效和毒性的主要过程,发展廉价、方便、快速、高通量的体外药物代谢研究方法对新药的开发和设计、给药的方法和剂量、临床药物的检测等都有重要的指导意义. 细胞色素P450酶（CYP450酶）在药物的I相反应中起到关键作用,以电极代替辅酶NADPH提供CYP450酶催化反应过程中需要的两个电子,构建CYP450酶电化学生物传感器可实现药物的初步筛选. 大量研究表明,CYP450酶在电极表面合适的固定方法与电极材料可有效提高传感器的检测性能. 本文主要综述近年来CYP450酶电化学生物传感器的构建及其在药物代谢研究方面的应用,并展望其研发前景.     

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b₅ (cyt‐b₅), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.     

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.     

Direct inhibition of Re Du Ning Injection and its active compounds on human liver cytochrome P450 enzymes by a cocktail method

The aim of this study was to investigate the direct inhibitory effects of Re Du Ning Injection (RDN) and its active compounds on the major cytochrome P450 enzyme (CYP) isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomes by ‘a cocktail method’. The activity of each CYP isform was represented as the formation rate of the specific metabolite from relevant substrate. Then a sensitive and specific ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and validated to simultaneously analyze the seven metabolites. RDN (0.035–2.26 mg/mL) showed a strong inhibitiory effect on CYP2C8, followed by CYP2C9, CYP2B6, CYP2C19, CYP1A2 and CYP3A4. The IC₅₀ value for each enzyme was 0.19, 0.66, 0.72, 1.27, 1.66 and 2.13 mg/mL, respectively. RDN competitively inhibited the activities of CYP1A2 (K _i = 1.22 mg/mL), CYP2B6 (K _i = 0.65 mg/mL) and CYP3A4 (K _i = 0.88 mg/mL); it also exhibited mixed inhibition of CYP2C8, CYP2C9 and CYP2C19 with a K _i value of 0.26, 0.64 and 0.82 mg/mL, respectively. However, the activity of CYP2D6 was not significantly inhibited even by 2.26 mg/mL RDN. Moreover, the data of nine active compounds on the CYPs showed that cryptochlorogenin acid, sochlorogenic acid B and sochlorogenic acid C were the major contributors to the inhibitory effect of RDN on CYP2C8, while the inhibitory effect of RDN on CYP2C9 might be caused by sochlorogenic acid A and sochlorogenic acid C. Moreover, neochlorogenic acid might be the major contributor to the inhibitory effect on CYP2B6. All of the findings suggested that drug–drug interactions may occur and great caution should be taken when RDN is combined with drugs metabolized by these CYPs.     

Structural identification of SAR-943 metabolites generated by human liver microsomes in vitro using mass spectrometry in combination with analysis of fragmentation patterns

SAR-943 (32-deoxo rapamycin) is a proliferation signal inhibitor via interaction with the mammalian target of rapamycin (mTOR). Most importantly, SAR-943 has improved chemical stability compared to rapamycin (sirolimus) and is currently under investigation as a drug coated on coronary stents. It was the goal of this study to identify the SAR-943 metabolites generated after incubation with human liver microsomes using high-resolution mass spectrometry (MS) and MS/iontrap (MS(n)) and comparison of fragmentation patterns of the metabolites with those of SAR-943 and other known rapamycin derivatives. Our study showed that SAR-943 is mainly hydroxylated and/or demethylated by human liver microsomes. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl and 27-O-desmethyl SAR-943; hydroxylated metabolites: hydroxy piperidine SAR-943, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 46-hydroxy and 49-hydroxy SAR-943; didemethylated metabolites: 16,39-O-didesmethyl and 27,39-O-didesmethyl SAR-943; demethylated-hydroxylated metabolites: 39-O-desmethyl, 23- or 24-hydroxy and 39-O-desmethyl, hydroxy piperidine SAR-943 and dihydroxylated metabolites: 12-,23- or 24-dihydroxy SAR-943. In addition, several other demethylated-hydroxylated and dihydroxylated metabolites were detected. However, their exact structures could not be identified.     

A sensitive and high‐throughput LC‐MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates

A sensitive and high‐throughput LC‐MS/MS method was established and validated for the simultaneous quantification of seven probe substrate‐derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy‐bupropion (CYP2B6), n‐desethyl‐amodiaquine (CYP2C8), 4′‐hydroxy‐diclofenac (CYP2C9), 4′‐hydroxy‐mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1′‐hydroxy‐midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well‐known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC₅₀ values of these inhibitors determined on this cocktail assay were highly correlated (R² > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89–113.35%) and between‐day (RSD <13.95%) and within‐day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze–thaw cycles. This seven‐CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds’ potential inhibition of the seven major CYPs in drug development settings. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of stable and reactive metabolites of piperine formed on incubation with human liver microsomes

Black pepper, though commonly employed as a spice, has many medicinal properties. It consists of volatile oils, alkaloids, pungent resins, etc., of which piperine is a major constituent. Though safe at low doses, piperine causes alteration in the activity of drug metabolising enzymes and transporters at high dose and is known to precipitate liver toxicity. It has a potential to form reactive metabolite(s) (RM) owing to the presence of structural alerts, such as methylenedioxyphenyl (MDP), α, β‐unsaturated carbonyl group (Michael acceptor), and piperidine. The present study was designed to detect and characterize stable and RM(s) of piperine formed on in vitro incubation with human liver microsomes. The investigation of RMs was done with the aid of trapping agents, viz, glutathione (GSH) and N‐acetylcysteine (NAC). The samples were analysed by ultra‐high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC‐HRMS) using Thermo Scientific Q Exactive Plus Orbitrap. Full scan MS followed by data‐dependent MS² (Full MS‐ddMS²) mode was used to establish mass spectrometric fragmentation pathways of protonated piperine and its metabolites. In total, four stable metabolites and their isomers (M1a‐c, M2a‐b, M3a‐c, and M4a‐b) were detected. Their formation involved removal of carbon (3, M1a‐c), hydroxylation (2, M2a‐b), hydroxylation with hydrogenation (3, M3a‐c), and dehydrogenation (2, M4a‐b). Out of these metabolites, M1, M2, and M3 are reported earlier in the literature, but their isomers and two M4 variants are novel. In addition, six novel conjugates of RMs, including three GSH conjugates of m/z 579 and three NAC conjugates of m/z 435, were also observed.     

Microcalorimetric studies on catalase reaction and inhibition of catalase by cyanide ion

As Chance et al. [1, 2 and 3] proposed, the decomposition of hydrogen peroxide catalyzed by catalase is an overall first-order reaction. In this paper, we have studied this enzyme-catalyzed reaction with a thermokinetic method. The rate constant and the molar reaction enthalpy of this reaction have been measured. At 310.15 K and pH=8.2, k_cat=1.75×10⁶ l mol⁻¹ s⁻¹, Δ_rH_m=88.99 kJ mol⁻¹. Furthermore, we have studied the competitive inhibition of catalase by cyanide ion and reported some correlated parameters.     

Cytochrome P450 2C9 polymorphism: Effect of amino acid substitutions on protein flexibility in the presence of tamoxifen

Tamoxifen is a prodrug and cytochrome P450 2C9 (CYP2C9) has a significant role in the formation of a therapeutically more potent metabolite (4-hydroxytamoxifen) than tamoxifen. Since CYP2C9 exhibits genetic polymorphism, it may contribute to different phenotypic drug response. Moreover, it may be misleading if the possibility of heterogeneous clinical observations of pharmacogenetic investigations is ignored. Above all, clinical investigation of all the polymorphic variants is beyond the scope of a pharmacogenetic study. Therefore, in order to understand the genotype-phenotype association, it is aimed to study the interatomic interactions of amino acid substitutions in CYP2C9 variants in the presence of tamoxifen. Computational structural biology approach was adopted to study the effect of amino acid substitutions of polymorphic variants of CYP2C9 R144C (*2), I359 L (*3), D360E (*5), R150H (*8), R335W (*11) and L90 P (*13) on the flexibility of the enzyme in the presence of tamoxifen. The mutations were selected based on previously determined associations on genotype and clinical outcome of drugs.Against the above plane, docking of tamoxifen was performed with the crystal structure representing the wild-type form of the enzyme. The docked conformation of tamoxifen was favourable for 4-hydroxylation with the site of metabolism within 5 Å of oxyferrylheme consistent with the drug metabolism pathway of tamoxifen. Further, the effect of amino acid substitutions CYP2C9 variants on the protein flexibility in the presence of tamoxifen in 4-hydroxy orientation was evaluated by molecular dynamics (MD) simulations.Distinct protein flexibility modulations between variants were observed in F/G segment constituting the substrate access/egress channels, helix B' involved with substrate specificity and helix I associated with the holding of substrates. Root Mean Square Fluctuation analysis of the trajectories of variants exhibited fluctuations in F/G segment, B’ and I helix. Dominant motions in the structure were identified by performing Principal Component Analysis on trajectories and the porcupine plot depicted displaced F/G segment in variants.Thus, the interatomic interaction study of CYP2C9 variants in the presence of tamoxifen predicts the plausible effect of the investigated variants on the therapeutic outcome of tamoxifen. It is presumed that the observations of the study would be meaningful to understand tamoxifen pharmacogenetics.     

An NH2-terminal truncated cytochrome P450 CYP3A4 showing catalytic activity is present in the cytoplasm of human liver cells

Cytochrome P450 3A4 (CYP3A4), is the dominant human liver hemoprotein enzyme localized in the endoplasmic reticulum (ER), and is responsible for the metabolism of more than 50% of clinically relevant drugs. While we were studying CYP3A4 expression and activity in human liver, we found that anti-CYP3A4 antibody cross-reacted with a lower band in liver cytoplasmic fraction. We assessed the activities of CYP3A4 and its truncated form in the microsomal and cytoplasmic fraction, respectively. In the cytoplasmic fraction, truncated CYP3A4 showed catalytic activity when reconstituted with NADPH-cytochrome P-450 reductase and cytochrome b5. In order to determine which site was deleted in the truncated form in vitro, we transfected cells with N-terminal tagged or C-terminal tagged human CYP3A4 cDNA. The truncated CYP3A4 is the N-terminal deleted form and was present in the soluble cytoplasmic fraction. Our result shows, for the first time, that N-terminal truncated, catalytically active CYP3A4 is present principally in the cytoplasm of human liver cells.     

Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes

In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- and intraday validations were satisfactory. Since the implementation of the LC/MS/MS assays, our sample throughput has increased by over 40-fold.     

In vitro metabolites characterization of ponatinib in human liver microsomes using ultra-high performance liquid chromatography combined with Q-Exactive-Orbitrap tandem mass spectrometry

Ponatinib is an oral drug for the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia, which has been reported to increase the risk of hepatotoxicity. The aim of this study was to characterize the metabolites of ponatinib in human liver microsomes as well as its reactive metabolites. Ponatinib was incubated with human liver microsomes in the presence of NADPH and trapping agents (glutathione or potassium cyanide). The metabolites were characterized by liquid chromatography in combination with Q-Exactive-Orbitrap-MS. Under the current conditions, six metabolites were detected and structurally identified on the basis of their accurate masses, fragmentation patterns, and retention times. M3 (N-demethylation) was unambiguously identified by matching its retention time and fragment ions with those of its reference standard. N-demethylation and oxygenation were proved to be the predominant metabolic pathways of ponatinib. In addition, two reactive metabolites (cyano adducts) were detected in human liver microsomes in the presence of potassium cyanide and NADPH, suggesting that ponatinib underwent CYP450-mediated metabolic activation, which could be one of the causative mechanisms for its hepatotoxicity. The current study provides new information regarding the metabolic profiles of ponatinib and would be helpful in understanding the effectiveness and toxicity of ponatinib, especially the mechanism of hepatotoxicity.     

In vitro and in vivo metabolism studies of dimethazine

The use of anabolic steroids is prohibited in sports. Effective control is done by monitoring their metabolites in urine samples collected from athletes. Ethical objections however restrict the use of designer steroids in human administration studies. To overcome these problems alternative in vitro and in vivo models were developed to identify metabolites and to assure a fast response by anti‐doping laboratories to evolutions on the steroid market. In this study human liver microsomes and an uPA^+/+‐SCID chimeric mouse model were used to elucidate the metabolism of a steroid product called ‘Xtreme DMZ’. This product contains the designer steroid dimethazine (DMZ), which consists of two methasterone molecules linked by an azine group. In the performed stability study, degradation from dimethazine to methasterone was observed. By a combination of LC‐High Resolution Mass Spectrometry (HRMS) and GC‐MS(/MS) analysis methasterone and six other dimethazine metabolites (M1–M6), which are all methasterone metabolites, could be detected besides the parent compound in both models. The phase II metabolism of dimethazine was also investigated in the mouse urine samples. Only metabolites M1 and M2 were exclusively detected in the glucuro‐conjugated fraction; all other compounds were also found in the free fraction. For effective control of DMZ misuse in doping control samples, screening for methasterone and methasterone metabolites should be sufficient. Copyright © 2016 John Wiley & Sons, Ltd.