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.     

In vitro metabolism of nobiletin,a polymethoxy-flavonoid,by human liver microsomes and cytochrome P450

1. Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs.

2. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4′-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4′-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL.

3. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4′-OH-NBL.

4. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

     

Synthesis of side chain specifically deuterated (−)‐Δ9‐tetrahydrocannabinols

(?)‐Δ⁹‐Tetrahydrocannabinols specifically deuterated at the n‐pentyl side chain were prepared using the corresponding resorcinols as key intermediates. To obtain the deuterated resorcinols we developed conditions under which no deuterium scrambling or loss was observed. The methodology allows for the preparative scale synthesis of deuterated resorcinols and corresponding (?)‐Δ⁹‐tetrahydrocannabinols. Copyright © 2002 John Wiley & Sons, Ltd.     

Effects of anticancer drugs on the metabolism of the anticancer drug 5,6-dimethylxanthenone-4-acetic (DMXAA) by human liver microsomes

AIMS: To investigate the effects of various anticancer drugs on the major metabolic pathways (glucuronidation and 6-methylhydroxylation) of DMXAA in human liver microsomes. METHODS: The effects of various anticancer drugs at 100 and 500 microM on the formation of DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) in human liver microsomes were determined by high performance liquid chromatography (h.p.l.c.). For those anticancer drugs showing significant inhibition of DMXAA metabolism, the inhibition constants (Ki) were determined. The resulting in vitro data were extrapolated to predict in vivo changes in DMXAA pharmacokinetics. RESULTS: Vinblastine, vincristine and amsacrine at 500 microM significantly (P < 0.05) inhibited DMXAA glucuronidation (Ki = 319, 350 and 230 microM, respectively), but not 6-methylhydroxylation in human liver microsomes. Daunorubicin and N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA) at 100 and 500 microM showed significant (P < 0.05) inhibition of DMXAA 6-methylhydroxylation (Ki = 131 and 0.59 microM, respectively), but not glucuronidation. Other drugs such as 5-fluoroucacil, paclitaxel, tirapazamine and methotrexate exhibited little or negligible inhibition of the metabolism of DMXAA. Pre-incubation of microsomes with the anticancer drugs (100 and 500 microM) did not enhance their inhibitory effects on DMXAA metabolism. Prediction of DMXAA-drug interactions in vivo based on these in vitro data indicated that all the anticancer drugs investigated except DACA appear unlikely to alter the pharmacokinetics of DMXAA, whereas DACA may increase the plasma AUC of DMXAA by 6%. CONCLUSIONS: These results indicate that alteration of the pharmacokinetics of DMXAA appears unlikely when used in combination with other common anticancer drugs. However, this does not rule out the possibility of pharmacokinetic interactions with other drugs used concurrently with this combination of anticancer drugs.     

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.     

2,5-Dihydroxy-4-methoxybenzophenone: a novel major in vitro metabolite of benzophenone-3 formed by rat and human liver microsomes

1.?When benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) was incubated with liver microsomes of untreated rats in the presence of NADPH, the 5-hydroxylated metabolite, 2,5-dihydroxy-4-methoxybenzophenone (5-OH-BP-3), was formed as a major novel metabolite of BP-3. The 4-desmethylated metabolite, 2,4-dihydroxybenzophenone (2,4-diOH-BP), previously reported as the major in vivo metabolite of BP-3, was also detected. However, the amount of 5-OH-BP-3 formed in vitro was about the same as that of 2,4-diOH-BP.

2.?The oxidase activity affording 5-OH-BP-3 was inhibited by SKF 525-A and ketoconazole, and partly by quinidine and sulfaphenazole. The oxidase activity affording 2,4-diOH-BP was inhibited by SKF 525-A, ketoconazole and α-naphthoflavone, and partly by sulfaphenazole.

3.?The oxidase activity affording 5-OH-BP-3 was enhanced in liver microsomes of dexamethasone-, phenobarbital- and 3-methylcholanthrene-treated rats. The activity affording 2,4-diOH-BP was enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats.

4.?When examined recombinant rat cytochrome P450 isoforms catalyzing the metabolism of BP-3, 5-hydroxylation was catalyzed by P450 3A2, 1A1, 2B1, 2C6 and 2D1, while 4-desmethylation was catalyzed by P450 2C6 and 1A1.     

In vitro metabolic studies using homogenized horse liver in place of horse liver microsomes

The study of the metabolism of drugs, in particular steroids, by both in vitro and in vivo methods has been carried out in the authors' laboratory for many years. For in vitro metabolic studies, the microsomal fraction isolated from horse liver is often used. However, the process of isolating liver microsomes is cumbersome and tedious. In addition, centrifugation at high speeds (over 100 000 g) may lead to loss of enzymes involved in phase I metabolism, which may account for the difference often observed between in vivo and in vitro results. We have therefore investigated the feasibility of using homogenized horse liver instead of liver microsomes with the aim of saving preparation time and improving the correlation between in vitro and in vivo results. Indeed, the preparation of the homogenized horse liver was very simple, needing only to homogenize the required amount of liver. Even though no further purification steps were performed before the homogenized liver was used, the cleanliness of the extracts obtained, based on gas chromatography‐mass spectrometry (GC‐MS) analysis, was similar to that for liver microsomes. Herein, the results of the in vitro experiments carried out using homogenized horse liver for five anabolic steroids—turinabol, methenolone acetate, androst‐4‐ene‐3,6,17‐trione, testosterone, and epitestosterone—are discussed. In addition to the previously reported in vitro metabolites, some additional known in vivo metabolites in the equine could also be detected. As far as we know, this is the first report of the successful use of homogenized liver in the horse for carrying out in vitro metabolism experiments. Copyright © 2011 John Wiley & Sons, Ltd.     

In vitro characterization of potential CYP‐ and UGT‐derived metabolites of the psychoactive drug 25B‐NBOMe using LC‐high resolution MS

One of the main challenges posed by the emergence of new psychoactive substances is their identification in human biological samples. Trying to detect the parent drug could lead to false‐negative results when the delay between consumption and sampling has been too long. The identification of their metabolites could then improve their detection window in biological matrices. Oxidative metabolism by cytochromes P450 and glucuronidation are two major detoxification pathways in humans. In order to characterize possible CYP‐ and UGT‐dependent metabolites of the 2‐(4‐bromo‐2,5‐dimethoxy‐phenyl)‐N‐[(2‐methoxyphenyl)methyl]ethanamine (25B‐NBOMe), a synthetic psychoactive drug, analyses of human liver microsome (HLM) incubates were performed using an ultra‐high performance liquid chromatography system coupled with a quadrupole‐time of flight mass spectrometry detector (UHPLC‐Q‐TOF/MS). On‐line analyses were performed using a Waters OASIS HLB column (30 x 2.1 mm, 20 µm) for the automatic sample loading and a Waters ACQUITY HSS C18 column (150 x 2 mm, 1.8 µm) for the chromatographic separation. Twenty‐one metabolites, consisting of 12 CYP‐derived and 9 UGT‐derived metabolites, were identified. O‐Desmethyl metabolites were the most abundant compounds after the phase I process, which appears to be in accordance with data from previously published NBOMe‐intoxication case reports. Although other important metabolic transformations, such as sulfation, acetylation, methylation or glutathione conjugation, were not studied and artefactual metabolites might have been produced during the HLM incubation process, the record of all the metabolite MS spectra in our library should enable us to characterize relevant metabolites of 25B‐NBOMe and allow us to detect 25B‐MBOMe users. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro identification of metabolites of verapamil in rat liver microsomes

AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 μmol/L and 5.0 μmol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS^n), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eightmetabolites, including two novel metabolites (M4 and MS), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1 - M4), N-dealkylated derivatives of verapamil (MS-MT), and N, O-didemethyl-verapamil (MS). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.     

In vitro metabolism study of saikosaponin d and its derivatives in rat liver microsomes

1.?Saikosaponins, one of the representative bioactive ingredients in Radix Bupleuri, possess hepatoprotective, anti-inflammatory, antiviral, antitumor, and other pharmacological activities. Up to now, few studies focused on the further metabolism of saikosaponins and their secondary metabolites absorbed into the circulatory system.

2.?To understand the in vivo efficacy of saikosaponin d, the in vitro metabolism of saikosaponin d, and its two derivatives formed in the gastrointestinal tract, prosaikogenin G and saikogenin G was investigated in rat liver microsomes, respectively.

3.?Fifteen metabolites were detected using high-performance liquid chromatography hybrid ion trap and time-of-flight mass spectrometry and triple-quadrupole mass spectrometry, and the predominant metabolic reactions were hydroxylation, carboxylation and combinations of these steps on the aglycone moiety.

4.?The metabolic pathways of saikosaponin d, prosaikogenin G, and saikogenin G were proposed in vitro and the results contribute to the understanding of saikosaponins in vivo metabolism.     

−(−)Gossypol promotes the apoptosis of bladder cancer cells in vitro

Dysregulation of Bcl2 family member proteins has been associated with poor chemotherapeutic response in bladder cancer, suggesting that agents targeting these crucial proteins may provide an interventional strategy to slow or halt bladder cancer progression and metastasis. In this study, we investigated whether the cottonseed polyphenol, -(-)gossypol, a BH3 mimetic, can reduce the expression of pro-survival, or increase the expression of pro-apoptotic, Bcl2 family proteins and thereby effectively sensitize otherwise resistant bladder cancer cells to the standard chemotherapeutic drugs gemcitabine, paclitaxel and carboplatin. These studies show that gossypol induced apoptosis in both chemosensitive UM-UC2 and chemoresistant resistant UM-UC9 bladder cancer cells in vitro in a dose and time dependent manner via a caspase mediated death signaling pathway. Moreover, in combined treatments, gossypol synergized with gemcitabine and carboplatin to induce apoptosis in chemoresistant bladder cancer cells. This effect was associated with the down-regulation the Bcl-xl and Mcl-1 pro-survival Bcl2 family proteins and up-regulation of the Bim and Puma BH3-only Bcl2 family proteins. Overall, these studies show that gossypol sensitizes bladder cancer cells to standard chemotherapeutic drugs and may provide a promising new strategy for bladder cancer treatment.     

Study of the in vitro and in vivo metabolism of the tryptamine 5‐MeO‐MiPT using human liver microsomes and real case samples

The synthetic tryptamine 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (5‐MeO‐MiPT) has recently been abused as a hallucinogenic drug in Germany and Switzerland. This study presents a case of 5‐MeO‐MiPT intoxication and the structural elucidation of metabolites in pooled human liver microsomes (pHLM), blood, and urine. Microsomal incubation experiments were performed using pHLM to detect and identify in vitro metabolites. In August 2016, the police encountered a naked man, agitated and with aggressive behavior on the street. Blood and urine samples were taken at the hospital and his premises were searched. The obtained blood and urine samples were analyzed for in vivo metabolites of 5‐MeO‐MiPT using liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS). The confiscated pills and powder samples were qualitatively analyzed using Fourier transform infrared (FTIR), gas chromatography–mass spectrometry (GC–MS), LC‐HRMS/MS, and nuclear magnetic resonance (NMR). 5‐MeO‐MiPT was identified in 2 of the seized powder samples. General unknown screening detected cocaine, cocaethylene, methylphenidate, ritalinic acid, and 5‐MeO‐MiPT in urine. Seven different in vitro phase I metabolites of 5‐MeO‐MiPT were identified. In the forensic case samples, 4 phase I metabolites could be identified in blood and 7 in urine. The 5 most abundant metabolites were formed by demethylation and hydroxylation of the parent compound. 5‐MeO‐MiPT concentrations in the blood and urine sample were found to be 160 ng/mL and 3380 ng/mL, respectively. Based on the results of this study we recommend metabolites 5‐methoxy‐N‐isopropyltryptamine (5‐MeO‐NiPT), 5‐hydroxy‐N‐methyl‐N‐isopropyltryptamine (5‐OH‐MiPT), 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine‐N‐oxide (5‐MeO‐MiPT‐N‐oxide), and hydroxy‐5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (OH‐5‐MeO‐MiPT) as biomarkers for the development of new methods for the detection of 5‐MeO‐MiPT consumption, as they have been present in both blood and urine samples.     

Assay of caffeine metabolism in vitro by human liver microsomes using radio-high-performance liquid chromatography

The low turnover of caffeine in vitro by human liver microsomes makes the study of the metabolic pathways of this compound difficult. Analytical methods with high sensitivity and specificity are needed for the detection of its metabolic products. A method based on the on-line radiometric determination of [8C-³H]caffeine and its principal metabolite (paraxanthine) in man has been developed using reversed-phase high-performance liquid chromatography. The method has been successfully employed in preliminary studies of the kinetics of this reaction.     

Effects of (+)catechin and (−)epicatechin on heterocyclic amines‐induced oxidative DNA damage

The aim of the present study was to evaluate the protective effect of (+)‐catechin and (?)‐epicatechin against 2‐amino‐3,8‐ dimethylimidazo[4,5‐f]quinoxaline (8‐MeIQx), 2‐amino‐3,4,8‐trimethylimidazo[4,5‐f]‐quinoxaline (4,8‐diMeIQx) and 2‐amino‐1‐methyl‐6‐phenyl‐imidazo[4,5‐b]pyridine (PhIP)‐induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single‐cell gel electrophoresis or comet assay. Increasing concentrations of 8‐MeIQx, 4,8‐diMeIQx and PhIP induced a significant increase in DNA strand breaks and oxidized purines and pyrimidines in a dose‐dependent manner. Among those, PhIP (300 µm ) exerted the highest genotoxicity. (+)‐Catechin exerted protection against oxidized purines induced by 8‐MeIQx, 4,8‐diMeIQx and PhIP. Oxidized pyrimidines and DNA strand breaks induced by PhIP were also prevented by (+)‐catechin. Otherwise, (?)‐epicatechin protected against the oxidized pyrimidines induced by PhIP and the oxidized purines induced by 8‐MeIQx and 4,8‐diMeIQx. One feasible mechanism by which (+)‐catechin and (?)‐epicatechin exert their protective effect towards heterocyclic amines‐induced oxidative DNA damage may be by modulation of phase I and II enzyme activities. The ethoxyresorufin O‐deethylation (CYP1A1) activity was moderately inhibited by (+)‐catechin, while little effect was observed by (?)‐epicatechin. However, (+)‐catechin showed the greatest increase in UDP‐glucuronyltransferase activity. In conclusion, our results clearly indicate that (+)‐catechin was more efficient than (?)‐epicatechin in preventing DNA damage (strand breaks and oxidized purines/pyrimidines) induced by PhIP than that induced by 8‐MeIQx and 4,8‐diMeIQx. Copyright © 2010 John Wiley & Sons, Ltd.     

Diazepam–omeprazole inhibition interaction: an in vitro investigation using human liver microsomes

1 The metabolism of diazepam to its primary metabolites 3-hydroxydiazepam (3HDZ) and nordiazepam (NDZ) was evaluated in human liver microsomes. The 3HDZ pathway was the major route of metabolism representing 90% of total metabolism with a V _max /K _m ratio of 0.50–7.26  μl  min⁻¹  mg ⁻¹ protein.
2 Inhibition of the two metabolic pathways of diazepam by omeprazole was investigated. The NDZ pathway was not affected by omeprazole whilst a K _i of 201±89  μm was obtained for the 3HDZ pathway ( K _m /K _i ratio of 3.0±0.9).
3 Inhibitory effects of omeprazole sulphone on the 3HDZ and NDZ pathways were also investigated. Omeprazole sulphone inhibited both pathways with similar K_is of 121±45 and 188±73  μm respectively ( K _m /K _i ratios of 5.2±2.3 and 3.3±1.5 respectively).
4 These in vitro data provide direct evidence for cytochrome P450 inhibition as the mechanism for the well documented diazepam-omeprazole clinical interaction and indicate that omeprazole sulphone, as well as the parent drug, contribute to the inhibition effect.     

The metabolic fate of two new psychoactive substances − 2‐aminoindane and N‐methyl‐2‐aminoindane − studied in vitro and in vivo to support drug testing

The aim of this study was to characterize the in vitro and in vivo metabolism of 2‐aminoindane (2,3‐dihydro‐1H‐inden‐2‐amine, 2‐AI), and N‐methyl‐2‐aminoindane (N‐methyl‐2,3‐dihydro‐1H‐inden‐2‐amine, NM‐2‐AI) after incubations using pooled human liver microsomes (pHLMs), pooled human liver S9 fraction (pS9), and rat urine after oral administration. After analysis using liquid chromatography coupled to high‐resolution mass spectrometry, pHLM incubations revealed that 2‐AI was left unmetabolized, while NM‐2‐AI formed a hydroxylamine and diastereomers of a metabolite formed after hydroxylation in beta position. Incubations using pS9 led to the formation of an acetyl conjugation in the case of 2‐AI and merely a hydroxylamine for NM‐2‐AI. Investigations on rat urine showed that 2‐AI was hydroxylated also forming diasteromers as described for NM‐2‐AI or acetylated similar to incubations using pS9. All hydroxylated metabolites of NM‐2‐AI except the hydroxylamine were found in rat urine as additional sulfates. Assuming similar patterns in humans, urine screening procedures might be focused on the parent compounds but should also include their metabolites. An activity screening using human recombinant N‐acetyl transferase (NAT) isoforms 1 and 2 revealed that 2‐AI was acetylated exclusively by NAT2, which is polymorphically expressed.     

Studies on the metabolism of the fentanyl‐derived designer drug butyrfentanyl in human in vitro liver preparations and authentic human samples using liquid chromatography‐high resolution mass spectrometry (LC‐HRMS)

Increasing numbers of new psychoactive substances (NPS) among them fentanyl derivatives has been reported by the European monitoring centre for drugs and drug addiction (EMCDDA). Butyrfentanyl is a new fentanyl derivative whose potency ratio was found to be seven compared to morphine and 0.13 compared to fentanyl. Several case reports on butyrfentanyl intoxications have been described. Little is known about its pharmacokinetic properties including its metabolism. However, knowledge of metabolism is essential for analytical detection in clinical and forensic toxicology. Therefore, in vitro and in vivo phase I and phase II metabolites of butyrfentanyl were elucidated combining liquid chromatography with a qTOF high resolution mass spectrometer. Human liver microsomes and recombinant cytochrome P450 enzymes (CYP) were used for in vitro assays. Authentic blood and urine samples from a fatal intoxication case were available for in vivo comparison. Butyrfentanyl was shown to undergo extensive metabolism. Six pathways could be postulated with hydroxylation and N ‐dealkylation being the major ones in vitro . In vivo , hydroxylation of the butanamide side chain followed by subsequent oxidation to the carboxylic acid represented the major metabolic step in the authentic case. Initial screening experiments with the most relevant CYPs indicated that mainly CYP2D6 and 3A4 were involved in the primary metabolic steps. Altered CYP2D6 and CYP3A4 status might cause a different metabolite pattern, making the inclusion of metabolites of different pathways recommendable when applying targeted screening procedures in clinical and forensic toxicology. Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro metabolism of the synthetic cannabinoid 3,5‐AB‐CHMFUPPYCA and its 5,3‐regioisomer and investigation of their thermal stability

Recently, the pyrazole‐containing synthetic cannabinoid N ‐(1‐amino‐3‐methyl‐1‐oxobutan‐2‐yl)‐1‐(cyclohexylmethyl)‐3‐(4‐fluorophenyl)‐1H ‐pyrazole‐5‐carboxamide (3,5‐AB‐CHMFUPPYCA) has been identified as a ‘research chemical’ both in powdered form and as an adulterant present in herbal preparations. Urine is the most common matrix used for abstinence control and the extensive metabolism of synthetic cannabinoids requires implementation of targeted analysis. The present study describes the investigation of the in vitro phase I metabolism of 3,5‐AB‐CHMFUPPYCA and its regioisomer 5,3‐AB‐CHMFUPPYCA using pooled human liver microsomes. Metabolic patterns of both AB‐CHMFUPPYCA isomers were qualitatively similar and dominated by oxidation of the cyclohexylmethyl side chain. Biotransformation to monohydroxylated metabolites of high abundance confirmed that these species might serve as suitable targets for urine analysis. Furthermore, since synthetic cannabinoids are commonly administered by smoking and because some metabolites can also be formed as thermolytic artefacts, the stability of both isomers was assessed under smoking conditions. Under these conditions, pyrolytic cleavage of the amide bond occurred that led to approximately 3 % conversion to heat‐induced degradation products that were also detected during metabolism. These artefactual ‘metabolites’ could potentially bias in vivo metabolic profiles after smoking and might have to be considered for interpretation of metabolite findings during hair analysis. This might be relevant to the analysis of hair samples where detection of metabolites is generally accepted as a strong indication of drug use rather than a potential external contamination. Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro metabolism of an estrogen‐related receptor γ modulator,GSK5182, by human liver microsomes and recombinant cytochrome P450s

GSK5182 (4‐[(Z)‐1‐[4‐(2‐dimethylaminoethyloxy)phenyl]‐hydroxy‐2‐phenylpent‐1‐enyl]phenol) is a specific inverse agonist for estrogen‐related receptor γ, a member of the orphan nuclear receptor family that has important functions in development and homeostasis. This study was performed to elucidate the metabolites of GSK5182 and to characterize the enzymes involved in its metabolism. Incubation of human liver microsomes with GSK5182 in the presence of NADPH resulted in the formation of three metabolites, M1, M2 and M3. M1 and M3 were identified as N‐desmethyl‐GSK5182 and GSK5182 N‐oxide, respectively, on the basis of liquid chromatography‐tandem mass spectrometric (LC‐MS/MS) analysis. M2 was suggested to be hydroxy‐GSK5182 through interpretation of its MS/MS fragmentation pattern. In addition, the specific cytochrome P450 (P450) and flavin‐containing monooxygenase (FMO) isoforms responsible for GSK5182 oxidation to the three metabolites were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 and FMO isoforms. GSK5182 N‐demethylation and hydroxylation is mainly mediated by CYP3A4, whereas FMO1 and FMO3 contribute to the formation of GSK5182 N‐oxide from GSK5182. The present data will be useful for understanding the pharmacokinetics and drug interactions of GSK5182 in vivo. Copyright © 2014 John Wiley & Sons, Ltd.