Recombinant CYP2D6 Research Articles

Identification of the human Cytochrome P450 enzymes (P450s) responsible for metabolizing infigratinib to its pharmacologically active Metabolites, BHS697, and CQM157, and assessment of their in vitro inhibition of P450s and UDP-glucuronosyltransferases (UGTs)

Infigratinib, an oral FGFR inhibitor for advanced cholangiocarcinoma, yielded two active metabolites, BHS697 and CQM157, with similar receptor affinity. Our study characterized P450s that are responsible for the metabolism of infigratinib to its two major active metabolites, BHS697 and CQM157. In vitro inhibition of P450s and UGTs by infigratinib, BHS697 or CQM157 was further investigated. The unbound apparent Km values for metabolism of infigratinib to BHS697 by HLM, human recombinant CYP2C8, CYP2C19, CYP2D6 and CYP3A4 enzymes are 4.47, 0.65, 2.50, 30.6 and 2.08 μM, while Vmax values are 90.0 pmol/min/mg protein, 0.13, 0.027, 0.81, and 0.56 pmol/min/pmol protein, respectively. The unbound apparent Km value for metabolism of infigratinib to CQM157 by HLM is 0.049 μM, while the Vmax value is 0.32 pmol/min/mg protein respectively. In HLM, infigratinib displayed moderate inhibition of CYP3A4 and CYP2C19 and weak or negligible inhibition of other P450 isoforms. BHS697 exhibited weak inhibition of CYP2B6, CYP2C9, CYP2C19 and CYP3A4, and no inhibition of CYP2C8 and CYP2D6. CQM157 moderately inhibited CYP2C9 and CYP3A4, and weakly or negligibly inhibited other P450 isoforms. Regarding UGTs, infigratinib moderately inhibited UGT1A4 and weakly inhibited UGT1A1, respectively. BHS697 weakly inhibited UGT1A1. In contrast, CQM157 moderately inhibited both UGT1A1 and UGT1A4. Our findings provide novel insights into the metabolism of and potential DDIs implicating infigratinib.

In vitro and in vivo metabolism of psilocybin's active metabolite psilocin.

In vivo, psilocybin is rapidly dephosphorylated to psilocin which induces psychedelic effects by interacting with the 5-HT2A receptor. Psilocin primarily undergoes glucuronidation or conversion to 4-hydroxyindole-3-acetic acid (4-HIAA). Herein, we investigated psilocybin's metabolic pathways in vitro and in vivo, conducting a thorough analysis of the enzymes involved. Metabolism studies were performed using human liver microsomes (HLM), cytochrome P450 (CYP) enzymes, monoamine oxidase (MAO), and UDP-glucuronosyltransferase (UGT). In vivo, metabolism was examined using male C57BL/6J mice and human plasma samples. Approximately 29% of psilocin was metabolized by HLM, while recombinant CYP2D6 and CYP3A4 enzymes metabolized nearly 100% and 40% of psilocin, respectively. Notably, 4-HIAA and 4-hydroxytryptophol (4-HTP) were detected with HLM but not with recombinant CYPs. MAO-A transformed psilocin into minimal amounts of 4-HIAA and 4-HTP. 4-HTP was only present in vitro. Neither 4-HIAA nor 4-HTP showed relevant interactions at assessed 5-HT receptors. In contrast to in vivo data, UGT1A10 did not extensively metabolize psilocin in vitro. Furthermore, two putative metabolites were observed. N-methyl-4-hydroxytryptamine (norpsilocin) was identified in vitro (CYP2D6) and in mice, while an oxidized metabolite was detected in vitro (CYP2D6) and in humans. However, the CYP2D6 genotype did not influence psilocin plasma concentrations in the investigated study population. In conclusion, MAO-A, CYP2D6, and CYP3A4 are involved in psilocin's metabolism. The discovery of putative norpsilocin in mice and oxidized psilocin in humans further unravels psilocin's metabolism. Despite limitations in replicating phase II metabolism in vitro, these findings hold significance for studying drug-drug interactions and advancing research on psilocybin as a therapeutic agent.

Effect of apatinib on the pharmacokinetics of tramadol and O-desmethyltramadol in rats.

Since the combination of anticancer drugs and opioids is very common, apatinib and tramadol are likely to be used in combination clinically. This study evaluated the effects of apatinib on the pharmacokinetics of tramadol and its main metabolite O-desmethyltramadol in Sprague-Dawley (SD) rats and the inhibitory effects of apatinib on tramadol in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP2D6.1. The samples were determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The in vivo results showed that compared with the control group, apatinib increased the AUC(0-t), AUC(0-∞) and Cmax values of tramadol and O-desmethyltramadol, and decreased the values of VZ/F and CLz/F. In addition, the MRT(0-t), MRT(0-∞) values of O-desmethyltramadol were increased. In vitro, apatinib inhibited the metabolism of tramadol by a mixed way with IC50 of 1.927 µM in RLMs, 2.039 µM in HLMs and 15.32 µM in CYP2D6.1. In summary, according to our findings, apatinib has a strong in vitro inhibitory effect on tramadol, and apatinib can increase the analgesic effect of tramadol and O-desmethyltramadol in rats.

Inhibitory effects of herbal medicines with claimed anticancer indications on cytochrome P450—An evaluation of drug-herb interactions risk

Herbs have the potential to interact with conventional medicines or each other upon concurrent administration. This can be at the level of absorption, distribution, metabolism and excretion (ADME). With an estimation that 80% of the population in poor regions of the world utilise herbal medicines for primary healthcare, there is a likelihood that some people will take both herbal and conventional medicines. This could present a risk for herb-drug interactions (HDI) at the various levels of the ADME process. Most common HDI have been observed at metabolism level involving inhibition or induction of the major drug metabolising liver enzymes called cytochrome P450s. In this study, we investigated the inhibitory effects of Parinari curatellifolia (MC), Fluggea virosa (ST), Garcinia livingstonei (MT), Pterocarpus angolensis (M), Tapinanthus quequensis (MF) and Cussonia arborea (GM) on recombinant CYP1A2, CYP2D6, CYP2C9 and CYP2C19. Plate based fluorescent assays were used to follow enzyme activity. Inhibition was determined at 5 mg/ml and 100 mg/ml of herbal extract, with IC50 determined for herbs that showed potent inhibition. A total of 10 out of 13 extracts showed significant inhibitory effects. Pterocarpus angolensis(bark), Fluggea virossa (leaf), Cussonia arborea(bark), Parinari curatellifollia (root) had very potent effects on CYP1A2 as indicated by an IC50 of <9.9 µg/ml. Based on the worst-case-scenario of complete absorption and exposure, these herbs are predicted to result in HDI if co-administered with medicines which depend on the affected CYPs for metabolism and elimination. Significance of the main findingsThere is a potential threat of drug toxicities due to drug-herb interactions for patients undergoing chemotherapy or taking other medications where co-morbidities exist during chemotherapy. Drugs that are substrates of CYP1A2 should be administered with caution for patients who are taking the herbs which have been studied in this research as risk for drug toxicities may be high. The findings act as a guide to medical doctors and patients in evaluating potential for herb-drug interactions during prescribing medications which are substrates for the studied CYPs.

Hydroxychloroquine is Metabolized by Cytochrome P450 2D6, 3A4, and 2C8, and Inhibits Cytochrome P450 2D6, while its Metabolites also Inhibit Cytochrome P450 3A in vitro.

This study aimed to explore the cytochrome P450 (CYP) metabolic and inhibitory profile of hydroxychloroquine (HCQ). Hydroxychloroquine metabolism was studied using human liver microsomes (HLMs) and recombinant CYP enzymes. The inhibitory effects of HCQ and its metabolites on nine CYPs were also determined in HLMs, using an automated substrate cocktail method. Our metabolism data indicated that CYP3A4, CYP2D6, and CYP2C8 are the key enzymes involved in HCQ metabolism. All three CYPs formed the primary metabolites desethylchloroquine (DCQ) and desethylhydroxychloroquine (DHCQ) to various degrees. Although the intrinsic clearance (CLint) value of HCQ depletion by recombinant CYP2D6 was > 10-fold higher than that by CYP3A4 (0.87 versus 0.075 µl/min/pmol), scaling of recombinant CYP CLint to HLM level resulted in almost equal HLM CLint values for CYP2D6 and CYP3A4 (11 and 14 µl/min/mg, respectively). The scaled HLM CLint of CYP2C8 was 5.7 µl/min/mg. Data from HLM experiments with CYP-selective inhibitors also suggested relatively equal roles for CYP2D6 and CYP3A4 in HCQ metabolism, with a smaller contribution by CYP2C8. In CYP inhibition experiments, HCQ, DCQ, DHCQ, and the secondary metabolite didesethylchloroquine were direct CYP2D6 inhibitors, with 50% inhibitory concentration (IC50) values between 18 and 135 µM. HCQ did not inhibit other CYPs. Furthermore, all metabolites were time-dependent CYP3A inhibitors (IC50 shift 2.2-3.4). To conclude, HCQ is metabolized by CYP3A4, CYP2D6, and CYP2C8 in vitro. HCQ and its metabolites are reversible CYP2D6 inhibitors, and HCQ metabolites are time-dependent CYP3A inhibitors. These data can be used to improve physiologically-based pharmacokinetic models and update drug-drug interaction risk estimations for HCQ. SIGNIFICANCE STATEMENT: While CYP2D6, CYP3A4, and CYP2C8 have been shown to mediate chloroquine biotransformation, it appears that the role of CYP enzymes in hydroxychloroquine (HCQ) metabolism has not been studied. In addition, little is known about the CYP inhibitory effects of HCQ. Here, we demonstrate that CYP2D6, CYP3A4, and CYP2C8 are the key enzymes involved in HCQ metabolism. Furthermore, our findings show that HCQ and its metabolites are inhibitors of CYP2D6, which likely explains the previously observed interaction between HCQ and metoprolol.

Inhibitory effects of fluoxetine and duloxetine on the pharmacokinetics of metoprolol in vivo and in vitro.

Depression is common among people with cardiovascular diseases. Therefore, the combined use of antidepressants and cardiovascular drugs is very common, which increases the possibility of drug interaction. Simultaneously compare the effects of duloxetine and fluoxetine on metoprolol metabolism, and provide evidence-based guidance for medication safety. Sprague-Dawley rats were randomly divided into three groups: group A (10.3mg/kg metoprolol alone), group B (10.3mg/kg metoprolol + 6.2mg/kg fluoxetine), and group C (10.3mg/kg metoprolol + 6.2mg/kg duloxetine). Tail vein blood was collected and subjected to the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection. Moreover, in vitro inhibition of fluoxetine and duloxetine were assessed by incubating liver microsomes and CYP2D6.1 with metoprolol. In in vivo study, the administration of fluoxetine or duloxetine significantly increased the AUC(0-𝑡) and AUC(0-∞) of metoprolol (P < 0.05). Differences between fluoxetine and duloxetine in plasma concentration were also investigated, and their pharmacokinetic parameters such as AUC(0-𝑡) and AUC(0-∞) were significantly distinct (P < 0.05). In vitro, fluoxetine and duloxetine inhibited the metabolism of metoprolol via mixed competitive mechanism of cytochrome P450. IC50 values of fluoxetine and duloxetine were 12.86 and 2.51 μM, respectively. Moreover, the metabolism rate of metoprolol was inhibited to 19.62% and 17.14% in recombinant human CYP2D6.1 by fluoxetine and duloxetine, respectively. Duloxetine showed a more significant inhibitory potential compared to fluoxetine in vitro, but the main pharmacokinetic parameters of fluoxetine and duloxetine revealed differences in inhibiting metoprolol metabolism in vivo.

Spectroscopic observations of β-eudesmol binding to human cytochrome P450 isoforms 3A4 and 1A2, but not to isoforms 2C9, 2C19, and 2D6

β-Eudesmol (BEU) is a sesquiterpenoid component of Atractylodes lancea with cytotoxic activity against cholangiocarcinoma. Its lipophilic nature makes BEU a likely substrate of human cytochrome P450 (P450) enzymes. Using ligand-binding difference spectroscopy, the affinities of this compound to recombinant CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were investigated in Escherichia coli membrane preparations. CYP3A4 showed a type I spectral change, with a binding constant Ks of 77 ± 23 (mean ± SD) μM at 0.5 μM P450 (Ks/[P450] ≈ 155). The reference substrate testosterone (TES) and the inhibitor fluconazole bound to the enzyme with apparent affinities of 86 ± 4 μM (type I) and 21 μM (type II), respectively. BEU was bound by CYP3A4 in a non-cooperative manner (Hill coefficient n ≈ 0.8). CYP1A2 showed reverse type I difference spectra with either BEU or caffeine (CAF). The CYP1A2 affinity for BEU was higher (0.23 mM) than for CAF (0.37 mM) but lower than for phenacetin (0.11 mM, type I). BEU did not bind significantly to CYP2C9, CYP2C19, and CYP2D6. Confirmation of metabolic activity and studies on the involvement of other human P450 isoforms are required. Double-beam spectrometry is needed to validate Ks measurements made with a microplate reader.

Alisertib shows negligible potential for perpetrating pharmacokinetic drug-drug interactions on ABCB1, ABCG2 and cytochromes P450, but acts as dual-activity resistance modulator through the inhibition of ABCC1 transporter

Alisertib (MLN8237), a novel Aurora A kinase inhibitor, is currently being clinically tested in late-phase trials for the therapy of various malignancies. In the present work, we describe alisertib's potential to perpetrate pharmacokinetic drug-drug interactions (DDIs) and/or to act as an antagonist of multidrug resistance (MDR). In accumulation assays, alisertib potently inhibited ABCC1 transporter, but not ABCB1 or ABCG2. The results of molecular modeling suggested a bifunctional mechanism for interaction on ABCC1. In addition, alisertib was characterized as a low- to moderate-affinity inhibitor of recombinant CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 isoenzymes, but without potential clinical relevance. Drug combination studies revealed the capability of alisertib to synergistically antagonize ABCC1-mediated resistance to daunorubicin. Although alisertib exhibited substrate characteristics toward ABCB1 transporter in monolayer transport assays, comparative proliferation studies showed lack of its MDR-victim behavior in cells overexpressing ABCB1 as well as ABCG2 and ABCC1. Lastly, alisertib did not affect the expression of ABCC1, ABCG2, ABCB1 transporters and CYP1A2, CYP3A4, CYP2B6 isozymes on mRNA level in various systemic and tumoral models. In conclusion, our study suggests that alisertib is a drug candidate with negligible potential for perpetrating systemic pharmacokinetic DDIs on ABCB1, ABCG2 and cytochromes P450. In addition, we introduce alisertib as an effective dual-activity chemosensitizer whose MDR-antagonistic capacities are not impaired by efflux or effect on MDR phenotype. Our in vitro findings provide important pieces of information for clinicians when introducing alisertib into the clinical area.

Pirfenidone 5-hydroxylation is mainly catalysed by CYP1A2 and partly catalysed by CYP2C19 and CYP2D6 in the human liver

Pirfenidone is a first-line drug for the treatment of idiopathic pulmonary fibrosis. The primary metabolic pathways of pirfenidone in humans are 5-hydroxylation and subsequent oxidation to 5-carboxylpirfenidone. The aims of this study were to determine the cytochrome P450 isoforms responsible for pirfenidone 5-hydroxylation and to evaluate their contributions in human liver microsomes (HLM). Among the recombinant P450 isoforms, CYP1A2, CYP2D6, CYP2C19, CYP2A6, and CYP2B6 were shown to catalyse the 5-hydroxylation of pirfenidone. Pirfenidone 5-hydroxylase activity by HLM was inhibited by α-naphthoflavone (by 45%), 8-methoxypsolaren (by 84%), tranylcypromine (by 53%), and quinidine (by 15%), which are CYP1A2, CYP1A2/CYP2A6/CYP2C19, CYP2A6/CYP2C19, and CYP2D6 inhibitors, respectively. In 17 individual HLM donors, pirfenidone 5-hydroxylase activity was significantly correlated with phenacetin O-deethylase (r = 0.89, P < 0.001) and S-mephenytoin 4’-hydroxylase activities (r = 0.51, P < 0.05), which are CYP1A2 and CYP2C19 marker activities, respectively. By using the relative activity factors, the contributions of CYP1A2, CYP2C19, and CYP2D6 to pirfenidone 5-hydroxylation in the human liver were 72.8%, 11.8%, and 8.9%, respectively. In conclusion, we clearly demonstrated the predominant P450 involved in pirfenidone 5-hydroxylation in the human liver is CYP1A2, with CYP2C19 and CYP2D6 playing a minor role.

In vitro investigation of metabolic fate of \u03b1-mangostin and gartanin via skin permeation by LC-MS/MS and in silico evaluation of the metabolites by ADMET predictor\u2122

BackgroundMangosteen, Garciniam angostana L., is a juicy fruit commonly found in Thailand. The rinds of Garciniam angostana L.have been used as a traditional medicine for the treatment of trauma, diarrhea and skin infection. It is also used in dermatological product such as in cosmetics. The mangosteen pericarp can be used to extract valuable bioactive xanthone compounds such as α-mangostin and gartanin. This study is aimed to predict the metabolism of α-mangostin and gartanin using in silico and in vitro skin permeation strategies.MethodsBased on their 2D molecular structures, metabolites of those compounds were predicted in silico using ADMET Predictor™. The Km and Vmax, for 5 important recombinant CYP isozymes 1A2, 2C9, 2C19, 2D6 and 3A4 were predicted. Moreover, the in vitro investigation of metabolites produced during skin permeation using human epidermal keratinocyte cells, neonatal (HEKn cells) was performed by LC-MS/MS.ResultsIt was found that the results derived from in silico were in excellent alignment with those obtained from in vitro studies for both compounds. The prediction referred that gartanin and α-mangostin were the substrate of CYP1A2, 2C9, 2C19 and 3A. In the investigation of α-mangostin metabolites by LC-MS/MS system, the MW of the parent compound was increased from 411.200 to 459.185 Da. Therefore, α-mangostin might be metabolized via tri-oxidation process. The increased molecular weight of parent compound (397.200 to 477.157 Da) illustrated that gartanin might be conjugated to sulfated derivatives.ConclusionsIn all the studies, α-mangostin and gartanin were predicted to be.metabolized via phase I and phase II metabolism (sulfation), respectively.

The Respective Roles of CYP3A4 and CYP2D6 in the Metabolism of Pimozide to Established and Novel Metabolites.

Pimozide is a dopamine receptor antagonist indicated for the treatment of Tourette syndrome. Prior in vitro studies characterized N-dealkylation of pimozide to 1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one (DHPBI) via CYP3A4 and, to a lesser extent, CYP1A2 as the only notable routes of pimozide biotransformation. However, drug-drug interactions between pimozide and CYP2D6 inhibitors and CYP2D6 genotype-dependent effects have since been observed. To reconcile these incongruities between the prior in vitro and in vivo studies, we characterized two novel pimozide metabolites: 5-hydroxypimozide and 6-hydroxypimozide. Notably, 5-hydroxypimozide was the major metabolite produced by recombinant CYP2D6 (Km ∼82 nM, V max ∼0.78 pmol/min per picomoles), and DHPBI was the major metabolite produced by recombinant CYP3A4 (apparent Km ∼1300 nM, V max ∼2.6 pmol/min per picomoles). Kinetics in pooled human liver microsomes (HLMs) for the 5-hydroxylation (Km ∼2200 nM, V max ∼59 pmol/min per milligram) and N-dealkylation (Km ∼3900 nM, V max ∼600 pmol/min per milligram) reactions were also determined. Collectively, formation of DHPBI, 5-hydroxypimozide, and 6-hydroxypimozide accounted for 90% of pimozide depleted in incubations of NADPH-supplemented pooled HLMs. Studies conducted in HLMs isolated from individual donors with specific cytochrome P450 isoform protein abundances determined via mass spectrometry revealed that 5-hydroxypimozide (r 2 = 0.94) and 6-hydroxypimozide (r 2 = 0.86) formation rates were correlated with CYP2D6 abundance, whereas the DHPBI formation rate (r 2 = 0.98) was correlated with CYP3A4 abundance. Furthermore, the HLMs differed with respect to their capacity to form 5-hydroxypimozide relative to DHPBI. Collectively, these data confirm a role for CYP2D6 in pimozide clearance via 5-hydroxylation and provide an explanation for a lack of involvement when only DHPBI formation was monitored in prior in vitro studies. SIGNIFICANCE STATEMENT: Current CYP2D6 genotype-guided dosing information in the pimozide label is discordant with available knowledge regarding the primary biotransformation pathways. Herein, we characterize the CYP2D6-dependent biotransformation of pimozide to previously unidentified metabolites. In human liver microsomes, formation rates for the novel metabolites and a previously identified metabolite were determined to be a function of CYP2D6 and CYP3A4 content, respectively. These findings provide a mechanistic basis for observations of CYP2D6 genotype-dependent pimozide clearance in vivo.

Cannabinoid metabolites as potential inhibitors of major CYP450 enzymes. Implication for cannabis‐drug interactions.

The recent legalization of marijuana in many parts of the United States and other countries has led to an increased need for a more comprehensive understanding of the metabolism of marijuana constituents, specifically in regard to potential drug‐drug interactions (DDIs) with cannabinoids and their numerous metabolites. To understand the DDI potential of cannabis, we are evaluating cannabis constituents and cannabinoid metabolites as possible inhibitors of phase I metabolizing enzymes. We hypothesize that inhibition of major drug metabolizing enzymes by cannabinoids metabolites [(−)‐trans‐Δ‐9‐ tetrahydrocannabinol (THC), 11‐hydroxy Δ9‐tetra hydrocannabinol(11‐OH‐THC), 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannbinol (THC‐COOH), THC‐COO‐glucuronide, cannabidiol (CBD) and cannabinol (CBN] can lead to an increase in hepatic and systemic exposure to certain drugs. In vitro assays demonstrated that THC, its major metabolites 11‐OH‐THC, THC‐COOH and THC‐COO‐glucuronide, CBD and CBN inhibit several major CYP enzymes. THC (IC50= 10 ± 4.7 μM), 11‐OH‐THC (IC50= 2.8 ± 1.4 μM), THC‐COO‐glucuronide (IC50= 7.6 ± 4.8 μM), and CBD (2.8 ± 1.2 μM) all inhibited the dextromethorphan‐O‐demethylase activity of recombinant CYP2D6. THC (IC50= 8.3 ± 3.2 μM), THC‐COO‐glucuronide (IC50= 7.2 ± 4.1 μM), CBD (IC50= 8.1 ± 4.3 μM) and CBN (IC50= 5.5 ± 3.1 μM) all inhibited the omeprazole‐5‐hydroxylase activity of recombinant CYP2C19, while 11‐OH‐THC (IC50= 5.02 ± 2.3 μM), THC‐COO‐glucuronide (IC50= 5.2 μM ± 3.5 μM), CBD (IC50= 0.8 ± 0.5 μM), and CBN (IC50= 1.5 ± 0.9 μM) all inhibited the bupropion hydroxylation activity of recombinant CYP2B6. In addition, CBD and CBN inhibited the chlorzoxazone‐6‐hydroxylase activity of recombinant CYP2E1 (IC50 were 0.9 μM ± 0.5 μM and 0.6 μM ± 0.4 μM, respectively). These data indicate that cannabinoids and their major metabolites are able to inhibit the activities of multiple CYP enzymes, suggesting a strong possibility for DDI with many exogenous compounds including a variety of drugs and carcinogens.Support or Funding InformationThis investigation was supported in part by funds provided by the State of Washington Initiative Measure No. 502

Metabolism of Benzalkonium Chlorides by Human Hepatic Cytochromes P450.

Benzalkonium chlorides (BACs) are widely used as disinfectants in cleaning products, medical products, and the food processing industry. Despite a wide range of reported toxicities, limited studies have been conducted on the metabolism of these compounds in animal models and none in human-derived cells or tissues. In this work, we report on the metabolism of BACs in human liver microsomes (HLM) and by recombinant human hepatic cytochrome P450 (CYP) enzymes. BAC metabolism in HLM was NADPH-dependent and displayed apparent half-lives that increased with BAC alkyl chain length (C10 < C12 < C14 < C16), suggesting enhanced metabolic stability of the more lipophilic, longer chain BACs. Metabolites of d7-benzyl labeled BAC substrates retained all deuteriums and there was no evidence of N-dealkylation. Tandem mass spectrometry fragmentation of BAC metabolites confirmed that oxidation occurs on the alkyl chain region. Major metabolites of C10-BAC were identified as ω-hydroxy-, (ω-1)-hydroxy-, (ω, ω-1)-diol-, (ω-1)-ketone-, and ω-carboxylic acid-C10-BAC by liquid chromatography-mass spectrometry comparison with synthetic standards. In a screen of hepatic CYP isoforms, recombinant CYP2D6, CYP4F2, and CYP4F12 consumed substantial quantities of BAC substrates and produced the major microsomal metabolites. The use of potent pan-CYP4 inhibitor HET0016, the specific CYP2D6 inhibitor quinidine, or both confirmed major contributions of CYP4- and CYP2D6-mediated metabolism in the microsomal disappearance of BACs. Kinetic characterization of C10-BAC metabolite formation in HLM demonstrated robust Michaelis-Menten kinetic parameters for ω-hydroxylation (Vmax = 380 pmol/min/mg, Km = 0.69 μM) and (ω-1)-hydroxylation (Vmax = 126 pmol/min/mg, Km = 0.13 μM) reactions. This work illustrates important roles for CYP4-mediated ω-hydroxylation and CYP2D6/CYP4-mediated (ω-1)-hydroxylation during the hepatic elimination of BACs, an environmental contaminant of emerging concern. Furthermore, we demonstrate that CYP-mediated oxidation of C10-BAC mitigates the potent inhibition of cholesterol biosynthesis exhibited by this short-chain BAC.

P.3.10 The role of CYP2D6 gene in treatment resistant schizophrenia and the effectiveness of treatment resistant schizophrenia

l-Corydalmine (l-CDL) was under development as an oral analgesic agent, exhibiting potent analgesic activity in preclinical models. The objective of this study was to compare metabolic profiles of l-CDL in liver microsomes from mouse, rat, monkey, dog and human. Six metabolites (M1–M6) were identified using LC-Q/TOF in liver microsomes from the five species. The metabolism of l-CDL included O-demethylation (M1–3) and hydroxylation (M4–6). The desmethyl metabolites were the major ones among the five species, which accounted for more than 84%. Data from chemical inhibition in human liver microsomes (HLM) and human recombinant CYP450s demonstrated that CYP2D6 exhibited strong catalytic activity towards M1 and M2 formations, while CYP2C9 and CYP2C19 also catalyzed M2 formation. Formations of M3 and hydroxyl metabolites (M4 and M5) were mainly catalyzed by CYP3A4. Further studies showed that M1 and M2 were main metabolites in HLM. The kinetics of M1 and M2 formations in HLM and recombinant CYP450s were also investigated. The results showed that M1 and M2 formations in HLM and recombinant CYP2D6 characterized biphasic kinetics, whereas sigmoid Vmax model was better used to fit M2 formation by recombinant CYP2C9 and CYP2C19. The contributions of CYP2D6 to M1 and M2 formations in HLM were estimated to be 75.3% and 50.7%, respectively. However, the contributions of CYP2C9 and CYP2C19 to M2 formation were only 5.0% and 4.1%, respectively. All these data indicated that M1 and M2 were main metabolites in HLM, and CYP2D6 was the primary enzyme responsible for their formations.

Effect of an anxiolytic botanical containing Souroubea sympetala and Platanus occidentalis on in-vitro diazepam human cytochrome P450-mediated metabolism

A novel anxiolytic natural health product (NHP) containing Souroubea sympetala and Platanus occidentalis is available for the companion animal market and is currently being developed for clinical evaluation. Addressing the risk of potential NHP-drug interactions, this study investigated S.sympetala and P.occidentalis plant extracts, and their identified bioactive compounds, for effects on the activity of cytochrome P450 (CYP) isozymes and the metabolism of the conventional anti-anxiety medication diazepam. Souroubea sympetala and P.occidentalis extracts, a 1:1 blend of the two extracts, and five triterpenes were tested for inhibitory effects on human recombinant CYP3A4, CYP2D6, CYP2C9 and CYP2C19 activity using a fluorometric plate assay. Direct effects on the metabolism of diazepam were evaluated using human liver microsomes with drug and metabolite quantification by ultra-high-pressure liquid chromatography and mass spectroscopy. The active substances betulinic acid (BA) and ursolic acid (UA) strongly inhibited CYP3A4 activity while UA and lupeol moderately inhibited CYP2C19. All extracts exhibited strong activity against the tested isozymes at 50-100μg/ml. BA and all plant extracts blocked the formation of major diazepam metabolites. Betulinic acid, UA and both the extracts and blended product are expected to affect the metabolism of diazepam when given in high dose.

Decreasing the CYP2D6 contribution to metabolism of a CK1ε inhibitor

Our internal casein kinase 1ε lead inhibitor, compound 1 was partially cleared by the polymorphic cytochrome P450 2D6. CYP2D6 involvement in metabolism implies more extensive clinical trials. We therefore wanted to reduce the contribution to clearance by this enzyme. We utilized metabolism reports for compound 1 performed in recombinant CYP2D6 together with structure-metabolism variation in structures of closely related analogs in order to see if we could incorporate similar substitution patterns in our lead compound. In addition, we utilized a previously established docking method using a modified CYP2D6 crystal structure to see if the metabolism patterns in CYP2D6 could be reproduced to afford the metabolites in the metabolism reports as well as those for the compounds used in the structure-metabolism relationship. All three of these steps, the metabolism report, the establishment of structure-metabolism relationships and the docking, lead to compound 10 where CYP2D6 was not involved in the clearance pathways.

Evaluation of Carbazeran 4-Oxidation and O 6-Benzylguanine 8-Oxidation as Catalytic Markers of Human Aldehyde Oxidase: Impact of Cytosolic Contamination of Liver Microsomes.

The present study investigated the contribution of microsomal cytochrome P450 and cytosolic aldehyde oxidase-1 (AOX-1) to carbazeran 4-oxidation and O 6-benzylguanine 8-oxidation in human liver microsomal, cytosolic, and S9 fractions. Incubations containing carbazeran and human liver microsomes with or without exogenously added NADPH yielded comparable levels of 4-oxo-carbazeran. O 6-Benzylguanine 8-oxidation occurred in microsomal incubations, and the extent was increased by NADPH. Human recombinant CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 did not catalyze carbazeran 4-oxidation, whereas CYP1A2 was highly active in O 6-benzylguanine 8-oxidation. 1-Aminobenzotriazole, a pan-cytochrome P450 inhibitor, decreased O 6-benzylguanine 8-oxidation, but not carbazeran 4-oxidation, in microsomal incubations, whereas 1-aminobenzotriazole and furafylline (a CYP1A2-selective inhibitor) did not inhibit carbazeran 4-oxidation or O 6-benzylguanine 8-oxidation in human liver S9 fraction. Carbazeran 4-oxidation in incubations containing human liver microsomes (from multiple donors and commercial suppliers) was attributed to microsomal preparations contaminated with AOX-1, as suggested by liver microsomal experiments indicating a decrease in carbazeran 4-oxidation by an AOX-1 inhibitor (hydralazine), and to detection of AOX-1 protein (at one-third the level of that in liver cytosol). Cytosolic contamination of liver microsomes was further demonstrated by the formation of dehydroepiandrosterone sulfate (catalyzed by cytosolic sulfotransferases) in liver microsomal incubations containing dehydroepiandrosterone. In conclusion, carbazeran 4-oxidation and O 6-benzylguanine 8-oxidation are enzyme-selective catalytic markers of human AOX-1, as shown in human liver S9 fraction expressing cytochrome P450 and AOX-1. This study highlights the negative impact of cytosolic contamination of liver microsomes on the interpretation of reaction phenotyping data collected in an in vitro study performed in microsomal fractions.

Effect of 95% Ethanol Khat Extract and Cathinone on in vitro Human Recombinant Cytochrome P450 (CYP) 2C9, CYP2D6, and CYP3A4 Activity.

A significant number of people worldwide consume khat on daily basis. Long term of khat chewing has shown negative impact on several organ systems. It is likely that these people are co-administered khat preparations and conventional medication, which may lead to khat-drug interactions. This study aimed to reveal the inhibitory potencies of khat ethanol extract (KEE) and its major active ingredient (cathinone) on human cytochrome P450 (CYP) 2C9, CYP2D6, and CYP3A4 enzymes activities, which are collectivelyresponsible for metabolizing 70-80% clinically used drugs. In vitro fluorescence-based enzyme assays were developed and the CYP enzyme activities were quantified in the presence and absence of KEE and cathinone employing Vivid® CYP450 Screening Kits. KEE inhibited human CYP2C9, CYP2D6, and CYP3A4 enzyme activities with IC50 of 42, 62, and 18μg/ml. On the other hand, cathinone showed negligible inhibitory effect on these CYPs. Further experiments with KEE revealed that KEE inhibited CYP2C9 via non-competitive or mixed mode with Ki of 14.7μg/ml, CYP2D6 through competitive or mixed mode with Ki of 17.6μg/ml, CYP3A4 by mixed inhibition mode with Ki of 12.1μg/ml. Khat-drug interactions are possible due to administration of clinical drugs metabolized by CYP2C9/CYP2D6/CYP3A4 together with khat chewing. Further in vivo studies are required to confirm our findings and identify the causative constituents of these inhibitory effects.

Celecoxib is a substrate of CYP2D6: Impact on celecoxib metabolism in individuals with CYP2C9*3 variants

Celecoxib was characterized as a substrate of human cytochrome P450 (CYP) 2D6 in vitro. In recombinant CYP2D6, celecoxib hydroxylation showed atypical substrate inhibition kinetics with apparent Km, Ki, and Vmax of 67.2 μM, 12.6 μM, and 1.33 μM/min, respectively. In human liver microsomes (HLMs), a concentration-dependent inhibition of celecoxib hydroxylation by quinidine was observed after CYP2C9 and CYP3A4 were inhibited. In individual HLMs with variable CYP2D6 activities, a significant correlation was observed between celecoxib hydroxylation and CYP2D6-selective dextromethorphan O-demethylation when CYP2C9 and CYP3A4 activities were suppressed (r = 0.97, P < 0.0001). Molecular modeling showed two predominant docking modes of celecoxib with CYP2D6, resulting in either a substrate or an inhibitor. A second allosteric binding antechamber, which stabilized the inhibition mode, was revealed. Modeling results were consistent with the observed substrate inhibition kinetics. Using HLMs from individual donors, the relative contribution of CYP2D6 to celecoxib metabolism was found to be highly variable and dependent on CYP2C9 genotypes, ranging from no contribution in extensive metabolizers with CYP2C9*1*1 genotype to approximately 30% in slow metabolizers with allelic variants CYP2C9*1*3 and CYP2C9*3*3. These results demonstrate that celecoxib may become a potential victim of CYP2D6-associated drug-drug interactions, particularly in individuals with reduced CYP2C9 activity.

Evaluation of Carbazeran as an Enzyme‐Selective Substrate of Human Aldehyde Oxidase: Evidence for Regiospecific Oxidation of Carbazeran by CYP1A2, CYP2D6, CYP3A4, and CYP3A5

Aldehyde oxidase‐1 (AOX‐1) is a cytosolic xenobiotic‐metabolizing enzyme that has gained increasing interest because of its emerging role in drug metabolism and toxicity. It is a catalyst of carbazeran 4‐oxidation, resulting in the formation of 4‐oxo‐carbazeran. However, cytochrome P450 is also a catalyst of oxidative reactions. Therefore, we tested the hypothesis that cytochrome P450 and AOX‐1 catalyze carbazeran 4‐oxidation in the endoplasmic reticulum and cytosol, respectively. Incubations containing human liver microsomes with or without NADPH yielded 4‐oxo‐carbazeran, but to a lesser extent than that in incubations containing human liver cytosol. Cytochrome P450 inhibitors (1‐aminobenzotriazole and ketoconazole) did not decrease the extent of carbazeran 4‐oxidation in liver microsomes, whereas an AOX‐1 inhibitor (hydralazine) decreased it in liver cytosol. Human recombinant CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 did not catalyze carbazeran 4‐oxidation, in contrast to the catalysis by recombinant AOX‐1. The extent of carbazeran 4‐oxidation in incubations containing liver microsomes was attributed to the presence of AOX‐1 in the microsomal preparations, as indicated by the decrease in carbazeran 4‐oxidation by an AOX‐1 inhibitor (hydralazine) and the detection of AOX‐1 protein in liver microsomes (at a level that was up to one‐third of that in liver cytosol). Cytosolic sulfotransferases‐catalyzed dehydroepiandrosterone sulfation increased with increasing amount of microsomes, indicating that the two lots of hepatic microsomes are contaminated with not only AOX‐1, but also other cytosolic enzymes. An isomer of 4‐oxo‐carbazeran, putatively identified as 5‐hydroxy‐carbazeran or 8‐hydroxy‐carbazeran, was detected in incubations containing carbazeran and CYP1A2, CYP2D6, CYP3A4, or CYP3A5. The 5‐hydroxy‐carbazeran or 8‐hydroxy‐carbazeran peak was detected in human liver microsomal incubation and the peak area increased with increasing incubation time. The 5‐hydroxy‐carbazeran or 8‐hydroxy‐carbazeran peak was decreased by a pan‐CYP inhibitor (1‐aminobenzotriazole), but not by an AOX‐1 inhibitor (hydralazine). The addition of 1‐aminobenzotriazole increased the 4‐oxo‐carbazeran formation, indicating a shunting of carbazeran metabolism to AOX‐1 in the presence of the CYP inhibitor. In conclusion, carbazeran is a dual substrate of AOX‐1 and cytochrome P450. However, AOX‐1, but not cytochrome P450, catalyzes carbazeran 4‐oxidation.Support or Funding InformationSingapore Ministry of Education Academic Research Fund Tier 1 [Grant R‐148‐000‐218‐112 to AJL]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.