In vitro and in vivo human metabolism of the synthetic cannabinoid AB‐CHMINACA

N‐[(1S)‐1‐(aminocarbonyl)‐2‐methylpropyl]‐1‐(cyclohexylmethyl)‐1H‐indazole‐3‐carboxamide (AB‐CHMINACA) is a recently introduced synthetic cannabinoid. At present, no information is available about in vitro or in vivo human metabolism of AB‐CHMINACA. Therefore, biomonitoring studies to screen AB‐CHMINACA consumption lack any information about the potential biomarkers (e.g. metabolites) to target. To bridge this gap, we investigated the in vitro metabolism of AB‐CHMINACA using human liver microsomes (HLMs). Formation of AB‐CHMINACA metabolites was monitored using liquid chromatography coupled to time‐of‐flight mass spectrometry. Twenty‐six metabolites of AB‐CHMINACA were detected including seven mono‐hydroxylated and six di‐hydroxylated metabolites and a metabolite resulting from N‐dealkylation of AB‐CHMINACA, all produced by cytochrome P450 (CYP) enzymes. Two carboxylated metabolites, likely produced by amidase enzymes, and five glucuronidated metabolites were also formed. Five mono‐hydroxylated and one carboxylated metabolite were likely the major metabolites detected. The involvement of individual CYPs in the formation of AB‐CHMINACA metabolites was tested using a panel of seven human recombinant CYPs (rCYPs). All the hydroxylated AB‐CHMINACA metabolites produced by HLMs were also produced by the rCYPs tested, among which rCYP3A4 was the most active enzyme. Most of the in vitro metabolites of AB‐CHMINACA were also present in urine obtained from an AB‐CHMINACA user, therefore showing the reliability of the results obtained using the in vitro metabolism experiments conducted to predict AB‐CHMINACA in vivo metabolism. The AB‐CHMINACA metabolites to target in biomonitoring studies using urine samples are now reliably identified and can be used for routine analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

塞来昔布对阿戈美拉汀抑制作用的体内外研究

目的：研究塞来昔布对阿戈美拉汀的体内外抑制作用及机制。方法：鼠肝微粒体体外实验得到塞来昔布对阿戈美拉汀的抑制机制,并在大鼠体内得到验证。因此,可通过人肝脏微粒体和重组CYP2C9.1蛋白体外实验预测塞来昔布在人体中对阿戈美拉汀的抑制作用和可能机制。结果：塞来昔布表现出明显抑制作用,该作用在CYP2C9.1中呈现浓度依赖性,而在鼠肝微粒体中表现为非竞争性抑制。结论：塞来昔布联合阿戈美拉汀进行抑郁治疗时可能会对后者产生非竞争抑制,这一作用能允许降低阿戈美拉汀的使用剂量,从而降低不良反应的发生,提高治疗的安全性。     

治疗黑色素瘤的新型二芳基衍生物04022a在不同种属肝微粒体中的稳定性及代谢产物分析

目的 采用SD大鼠、人、比格犬的肝微粒体酶,考察新型二芳基衍生物04022a在3个种属肝微粒体中的代谢稳定性,比较代谢的种属差异,并对产生的代谢产物进行筛选与结构表征。方法 将04022a溶解在由还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)启动的肝微粒体孵育体系中,孵育0、15、30、60、90、120 min后,加入等体积的冰甲醇终止反应。采用高效液相色谱法测定04022a的质量浓度,计算各个时间点的剩余药物百分比、体外半衰期(t_1/2)和固有清除率(CL_int)。采用超高效液相色谱串联飞行时间质谱法对04022a在肝微粒体中的代谢产物进行检测与分析。结果 孵育120 min时,04022a在大鼠、人、比格犬肝微粒体中的剩余药物百分比分别为88.30%、82.15%、98.63%。t_1/2分别为770、364、3 465 min, CL_int分别为0.001 8、0.003 8、0.000 4 mL·(min·mg)^-1。初步判断04022a在3个种属共有5个体外代谢产物。结...     

No effect of CYP450 and P-glycoprotein on hydroxyurea in vitro metabolism

Our objectives were (1) to study the HU metabolism via human cytochromes and (2) to test if HU is a substrate of P-gp. HU metabolism was investigated by determining the appearance of urea and HU decreasing upon incubation with human liver microsomes. Quantification was determined using HPLC coupled with UV-detection at 449 nm. Our method was linear between 5 and 1000 μ m , precise (coefficients of variation ranging from 1.7 to 9.9%), accurate (97.7–103.9%). The limit of quantification was 7 μ m . The ATPase activity of human P-gp membranes was determined by measuring inorganic phosphate liberation. HU and urea measurements in microsomes were not different between 0 and 60 min whatever HU concentration used from 30 to 300 μ m . The presence of NADPH in the medium has no effect on HU and urea measurements. In the absence of verapamil, the ATPase activity was unaffected by HU at concentrations of 10, 30, 100 and 300 μ m . HU is unlikely to cause clinically relevant drug interactions with the substrates of these enzymes/transporters. However, it will be necessary to validate these in vitro data in patients with sickle cell anemia to evaluate the impact of genetic polymorphisms of these enzymes in a black population.     

In vitro studies on flubromazolam metabolism and detection of its metabolites in authentic forensic samples

Flubromazolam is a triazole benzodiazepine with high potency and long‐lasting central nervous system depressant effects; however, limited data about its pharmacokinetics are available. Here, we report in vitro studies of the human flubromazolam metabolism analyzed by liquid chromatography high‐resolution mass spectrometry (LC‐HRMS). In vitro investigations were carried out in pooled human liver microsomes (pHLM) and recombinant cytochrome P450 (CYP)‐enzymes. To confirm those metabolites detected in vitro , authentic samples obtained from two forensic cases were also analyzed by LC‐HRMS. Additionally, determination of the unbound fraction of flubromazolam in pHLM and in plasma was performed by equilibrium dialysis with subsequent prediction of its hepatic clearance (CL_H ) using well‐stirred and parallel‐tube models. Additional findings obtained by routine screening methods of these forensic cases are also reported. Studies using incubations with nicotinamide adenine dinucleotide phosphate‐fortified pHLM with or without uridine 5'‐diphosphoglucuronic acid and incubations with CYP‐enzymes identified the main metabolic pathway of flubromazolam as hydroxylation on the α‐ and/or 4‐position mediated by CYP3A4 and CYP3A5, with subsequent glucuronidation of the hydroxylated metabolites as well as of the parent drug. Further, α‐hydroxy‐flubromazolam and its corresponding glucuronide were detected in vivo together with the N ‐glucuronide of flubromazolam. The predicted CL _H of flubromazolam using the well‐stirred and parallel‐tube models were 0.42 and 0.43 mL/min/kg, respectively. Based on the data presented here, flubromazolam is primarily metabolized by CYP3A4/5 with a high protein‐binding and a predicted low clearance. Analysis of authentic samples suggested that analytical targets for flubromazolam should be the compound itself and α‐hydroxy‐flubromazolam. Copyright © 2016 John Wiley & Sons, Ltd.     

TM208在大鼠肝微粒体中代谢产物与细胞色素P450亚型代谢酶鉴别研究

在大鼠肝微粒体的体外代谢中研究作用于TM208的细胞色素P450亚型代谢酶。以不含细胞色素P450化学抑制剂的样品为对照，研究不同细胞色素P450亚型选择性化学抑制剂对TM208代谢转化率的影响。CYP2D和CYP2B的选择性抑制剂对TM208的代谢表现出浓度依赖性较强抑制作用，CYP1A的选择性抑制剂对TM208的代谢表现出一定抑制作用。CYP3A的选择性抑制剂对TM208的代谢没有表现出明显的抑制作用。TM208在人鼠肝微粒体体外代谢中主要通过CYP2D和CYP2B两种细胞色素P450亚型代谢酶参与代谢。     

Reduced (+/-)-3,4-methylenedioxymethamphetamine ("Ecstasy") metabolism with cytochrome P450 2D6 inhibitors and pharmacogenetic variants in vitro

"Ecstasy" [(+/-)-3,4-methylenedioxymethamphetamine or MDMA] is a CNS stimulant, whose use is increasing despite evidence of long-term neurotoxicity. In vitro, the majority of MDMA is demethylenated to (+/-)-3,4-dihydroxymethamphetamine (DHMA) by the polymorphic cytochrome P450 2D6 (CYP2D6). We investigated the demethylenation of MDMA and dextromethorphan (DEX), as a comparison drug, in reconstituted microsomes expressing the variant CYP2D6 alleles (*)2, (*)10, and (*)17, all of which have been linked to decreased enzyme activity. With MDMA, intrinsic clearances (V(max)/K(m)) in CYP2D6.2, CYP2D6.17, and CYP2D6.10 were reduced 15-, 13-, and 135-fold, respectively, compared with wild-type CYP2D6.1. With DEX, intrinsic clearances were reduced by 37-, 51-, and 164-fold, respectively. It was evident that CYP2D6.17 displayed substrate-specific changes in drug affinity (K(m)). Compounds potentially used with MDMA [fluoxetine, paroxetine, (-)-cocaine] demonstrated significant inhibition of MDMA metabolism in both human liver and CYP2D6.1-expressing microsomes. These data demonstrate that individuals possessing the CYP2D6(*)2, (*)17, and, particularly, (*)10 alleles may show significantly reduced MDMA metabolism. These individuals, and those taking CYP2D6 inhibitors, may demonstrate altered acute and/or long-term MDMA-related toxicity.     

Nonspecific binding of drugs to human liver microsomes

AIMS: To characterize the nonspecific binding to human liver microsomes of drugs with varying physicochemical characteristics, and to develop a model for the effect of nonspecific binding on the in vitro kinetics of drug metabolism enzymes. METHODS: The extent of nonspecific binding to human liver microsomes of the acidic drugs caffeine, naproxen, tolbutamide and phenytoin, and of the basic drugs amiodarone, amitriptyline and nortriptyline was investigated. These drugs were chosen for study on the basis of their lipophilicity, charge, and extent of ionization at pH 7.4. The fraction of drug unbound in the microsomal mixture, fu(mic), was determined by equilibrium dialysis against 0.1 M phosphate buffer, pH 7.4. The data were fitted to a standard saturable binding model defined by the binding affinity KD, and the maximum binding capacity Bmax. The derived binding parameters, KD and Bmax, were used to simulate the effects of saturable nonspecific binding on in vitro enzyme kinetics. RESULTS: The acidic drugs caffeine, tolbutamide and naproxen did not bind appreciably to the microsomal membrane. Phenytoin, a lipophilic weak acid which is mainly unionized at pH 7. 4, was bound to a small extent (fu(mic) = 0.88) and the binding did not depend on drug concentration over the range used. The three weak bases amiodarone, amitriptyline and nortriptyline all bound extensively to the microsomal membrane. The binding was saturable for nortriptyline and amitriptyline. Bmax and KD values for nortriptyline at 1 mg ml-1 microsomal protein were 382 +/- 54 microM and 147 +/- 44 microM, respectively, and for amitriptyline were 375 +/- 23 microM and 178 +/- 33 microM, respectively. Bmax, but not KD, varied approximately proportionately with the microsome concentration. When KD is much less than the Km for a reaction, the apparent Km based on total drug can be corrected by multiplying by fu(mic). When the substrate concentration used in a kinetic study is similar to or greater than the KD (Km >/= KD), simulations predict complex effects on the reaction kinetics. When expressed in terms of total drug concentrations, sigmoidal reaction velocity vs substrate concentration plots and curved Eadie Hofstee plots are predicted. CONCLUSIONS: Nonspecific drug binding in microsomal incubation mixtures can be qualitatively predicted from the physicochemical characteristics of the drug substrate. The binding of lipophilic weak bases is saturable and can be described by a standard binding model. If the substrate concentrations used for in vitro kinetic studies are in the saturable binding range, complex effects are predicted on the reaction kinetics when expressed in terms of total (added) drug concentration. Sigmoidal reaction curves result which are similar to the Hill plots seen with cooperative substrate binding.     

Stereoselective glucuronidation of formoterol by human liver microsomes

AIMS: Formoterol is a beta2-adrenoceptor agonist marketed as a racemic mixture of the active (R; R)- and inactive (S; S)-enantiomers (rac-formoterol). The drug produces prolonged bronchodilation by inhalation but there is significant interpatient variability in duration of effect. Previous work has shown that in humans formoterol is metabolized by conjugation with glucuronic acid but little is known about the stereoselectivity of this reaction. The aim of the present study was to investigate the glucuronidation of formoterol enantiomers in vitro by human liver microsomes. METHODS: The kinetics of formation of formoterol glucuronides during incubation of racemate and of single formoterol enantiomers with human liver microsomes (n=9) was characterized by chiral h.p.l.c. assay. RESULTS: The kinetics of glucuronidation of the two formoterol enantiomers obeyed the Michaelis-Menten equation. Glucuronidation of formoterol was stereoselective and occurred more than two times faster for (S; S)-formoterol than for (R; R)-formoterol. In incubations with single formoterol enantiomers, the median (n=9) Km values for (R; R)-glucuronide and (S; S)-glucuronide were 827.6 and 840.4 microm, respectively, and the median V max values were 2625 and 4304 pmol min-1 mg-1, respectively. Corresponding values determined in incubations with rac-formoterol were 357.2 and 312.1 microm and 1435 and 2086 pmol min-1 mg-1 for (R; R)- and (S; S)-glucuronide, respectively. Interindividual variation was large with the ratio of V max/Km (S; S/R; R) ranging from 0.57 to 6.90 for incubations with rac-formoterol. CONCLUSIONS: Our study demonstrates that glucuronidation of formoterol by human liver microsomes is stereoselective and subject to high interindividual variability. These findings suggest that clearance of formoterol in humans is subject to variable stereoselectivity which could explain the variation in duration of bronchodilation produced by inhaled formoterol in patients with asthma.     

The effects of 2,2 diethylallylacetamide on hepatic cytochromes in rats and in vitro

The application of 0.15 and 0.3% of diethylallylacetamide (DA) in the drinking water to rats during 10 days increased the relative liver weight, the yield of hepatic microsomes, and cytochromes P-450 and b₅.Single s.c. doses of 400 mg DA per kg reduced cytochrome P-450 concentrations in the hepatic endoplasmic reticulum of rats. This effect was considerably more pronounced in rats pretreated with phenobarbital. The activity of 5-aminolaevulinic acid synthesis in liver mitochondria, and total liver porphyrins increased.In incubations of metabolizing hepatic microsomes from rabbits pretreated with phenobarbital 75% of 0.1 mM DA was degraded after 1 h. The aerobic incubation of 0.1 mM DA with rabbit hepatic microsomes in the presence of NADPH produced 50% destruction of cytochrome P-450 within 1 h. Addition of EDTA revealed that a part of this destruction cannot be explained by accelerated lipid peroxidation.This work was supported by the Deutsche Forschungsgemeinschaft.