The use of liquid chromatography-atmospheric pressure chemical ionization mass spectrometry to explore the in vitro metabolism of cyanoalkyl piperidine derivatives

A LC/MS method using atmospheric pressure chemical ionization, positive ion mode and full scan to measure the in vitro metabolic stability of cyanoalkyl functionalized compounds with the human liver microsomes was employed. Percentage metabolism examined for the five cyanoalkyl piperidines revealed the optimal chain length and positioning of these functions to produce the most metabolically stable compound. The 4-cyanomethyl piperidine derivative was the most stable compound with 15% metabolism after 15 min incubation with human liver microsomes. In general, the major metabolites formed from the cyanoalkyl piperidine derivatives were due to oxidation of the cyanoalkyl chain or the piperidine fragment, resulting in a M+16 ion. However, the 2-cyanomethyl piperidine derivative exhibited an interesting biotransformation pathway with unusual metabolite peaks corresponding to M+5, M-11 and M+21 ions. Data-dependent MS/MS scanning was used to generate daughter ion spectra from the parent compound and its metabolite peaks. Based on the fragmentation analysis, a carboxylic acid, aldehyde and oxidative metabolite of the carboxylic acid structure have been proposed for M+5, M-11 and M+21 ions, respectively.     

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.     

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.     

Characterization of xylazine metabolism in rat liver microsomes using liquid chromatography–hybrid triple quadrupole–linear ion trap–mass spectrometry

ABSTRACT: Xylazine is an α₂‐adrenoceptor agonist and it is widely used in veterinary anesthesia in combination with ketamine. There is limited information on the metabolism of xylazine. A quantitative method for the determination of xylazine by HPLC‐ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray tandem mass spectrometry. The chromatographic separation was achieved using a Thermo Betasil Phenyl 100 × 2 mm column combined with an isocratic mobile phase composed of acetonitrile, methanol, water and formic acid (60:20:20:0.4) at a flow rate of 300 μL/min. The mass spectrometer was operating in selected reaction monitoring mode and the analytical range was set at 0.05–50 μm . The precision (%CV) and accuracy (%NOM) observed were 2.3–7.2 and 88.2–96.4%. In vitro metabolism studies were performed in rat liver microsomes and results showed moderate cytochrome P450 affinity (K_m = 10.1 μm ) and a low metabolic stability of xylazine with a half‐life of 4.1 min in rat liver microsomes. Five phase 1 metabolites were observed. The main metabolite observed was an oxidation of the thiazine moiety at m/z 235 and, to a lesser extent, we observed the formation of N‐(2,6‐dimethylphenyl)thiourea at m/z 181 and three distinctive hydroxylated metabolites at m/z 237. Further experiments with ketamine and ketoconazole strongly supported that the metabolism of xylazine to its main metabolite is mediated by CYP3A in rat liver microsomes. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of in vitro metabolites of trimethoprim and diaveridine in pig liver microsomes by liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry

Trimethoprim (TMP) and diaveridine (DVD) are used in combination with sulfonamides and sulfaquinoxlaine as an effective antibacterial agent and antiprotozoal agent, respectively, in humans and animals. To gain a better understanding of the metabolism of TMP and DVD in the food-producing animals, the metabolites incubated with liver microsomes of pigs were analyzed for the first time with high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. Seven TMP-related and six DVD-related metabolites were characterized based on the accurate MS2 spectra and known structure of the parent drug, respectively. The metabolites of TMP were identified as two O-demethylation metabolites, a di-O-demethylation metabolite, two N-oxides metabolites, a hydroxylated metabolite on the methylene carbon and a hydroxylated metabolite on the methyl group. DVD was also biotransformed to two O-demethylation metabolites, a di-O-demethylation metabolite, an N-oxide metabolite, a hydroxylation metabolite on the methylene carbon and a hydroxylation metabolite followed by O-demethylation. The results indicate that the two compounds have similar biotransformation pathways in pigs. O-Demethylation was the major metabolic route of TMP and DVD in the pig liver microsomes. The proposed metabolic pathways of TMP and DVD in liver microsomes will provide a basis for further studies of the in vivo metabolism of the two drugs in food-producing animals.     

Electrochemical Activation of the Natural Catalytic Cycle of Cytochrome P450s in Human Liver Microsomes

The natural catalytic cycle of cytochrome (cyt) P450 enzymes in human liver microsome (HLM) films was activated electrochemically via the electron transfer sequence electrode→cyt P450 reductase (CPR)→cyt P450. Cyclic voltammograms for HLM films had midpoint potentials of ?0.50 V vs. SCE at pH 7.4 characteristic of CPR, not cyt P450s. HLM and CPR microsomes without cyt P450s did not electrocatalytically reduce H₂O₂, and did not shift midpoint potential when CO was added, also indicating that the peaks do not correspond to iron heme cyt P450 enzymes. Electrochemical activation of the natural cyt P450 cycle for substrate conversion via CPR in HLM films was confirmed by catalytic electrolysis in an electrochemical microfluidic array designed to generate and detect reactive metabolites by measuring their reactivity with DNA.     

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.     

Electrochemistry/mass spectrometry as a tool in metabolism studies—A review

The combination of electrochemistry (EC) and mass spectrometry (MS) has become a more and more frequently used approach in metabolism studies in the last decade. This review provides insight into the importance of metabolism studies during the drug development process and gives a short overview about the conventionally used methods since electrochemistry is often intended to substitute or minimize animal-based studies. The optimization of the electrochemical conditions is of great importance for a successful comparison with in vitro approaches. The type of metabolism reactions, which can be simulated by EC, has been extended with new cell types and working electrodes. Although the mechanism differs from the enzyme-catalyzed turnover, electrochemistry can be used to simulate a significant number of the respective reactions.     

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.     

Potential Effects of Ibuprofen,Remdesivir and Omeprazole on Dexamethasone Metabolism in Control Sprague Dawley Male Rat Liver Microsomes (Drugs Often Used Together Alongside COVID-19 Treatment)

The role of individual cytochrome P450 (CYPs) responsible for the drug metabolism can be determined through their chemical inhibition. During the pandemic, dexamethasone and remdesivir with omeprazole were used for the treatment of COVID-19, while Ibuprofen was taken to treat the symptoms of fever and headache. This study aimed to examine the potency of ibuprofen remdesivir, and omeprazole as inhibitors of cytochrome P450s using rat liver microsomes in vitro. Dexamethasone a corticosteroid, sometimes used to reduce the body’s immune response in the treatment of COVID-19, was used as a probe substrate and the three inhibitors were added to the incubation system at different concentrations and analysed by a validated High Performance Liquid Chromatography (HPLC) method. The CYP3A2 isoenzyme is responsible for dexamethasone metabolism in vitro. The results showed that ibuprofen acts as a non-competitive inhibitor for CYP3A2 activity with K_i = 224.981 ± 1.854 µM and IC₅₀ = 230.552 ± 2.020 µM, although remdesivir showed a mixed inhibition pattern with a K_i = 22.504 ± 0.008 µM and IC₅₀ = 45.007 ± 0.016 µM. Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a K_i = 39.175 ± 0.230 µM and IC₅₀ = 78.351 ± 0.460 µM. These results suggest that the tested inhibitors would not exert a significant effect on the CYP3A2 isoenzyme responsible for the co-administered dexamethasone drug’s metabolism in vivo.