Recombinant Cytochrome P450 Research Articles

Utility of In Vitro Systems and Preclinical Data for the Prediction of Human Intestinal First-Pass Metabolism during Drug Discovery and Preclinical Development

A growing awareness of the risks associated with extensive intestinal metabolism has triggered an interest in developing robust methods for its quantitative assessment. This study explored the utility of intestinal S9 fractions, human liver microsomes, and recombinant cytochromes P450 to quantify CYP3A-mediated intestinal extraction in humans for a selection of marketed drugs that are predominantly metabolized by CYP3A4. A simple competing rates model is used to estimate the fraction of drug escaping gut wall metabolism (fg) from in vitro intrinsic clearance in humans. The fg values extrapolated from the three in vitro systems used in this study, together with literature-derived fg from human intestinal microsomes, were validated against fg extracted from human in vivo pharmacokinetic (PK) profiles using a generic whole-body physiologically-based pharmacokinetic (PBPK) model. The utility of the rat as a model for human CYP3A-mediated intestinal metabolism was also evaluated. Human fg from PBPK compares well with that from the grapefruit juice method, justifying its use for the evaluation of human in vitro systems. Predictive performance of all human in vitro systems was comparable [root mean square error (RMSE) = 0.22-0.27; n = 10]. Rat fg derived from in vivo PK profiles using PBPK has the lowest RMSE (0.19; n = 11) for the prediction of human fg for the selected compounds, most of which have a fraction absorbed close to 1. On the basis of these evaluations, the combined use of fg from human in vitro systems and rats is recommended for the estimation of CYP3A4-mediated intestinal metabolism in lead optimization and preclinical development phases.

In vitro inhibition of human CYP1A2, CYP2D6, and CYP3A4 by six herbs commonly used in pregnancy.

Black elderberry, cranberry, fennel, ginger, horsetail, and raspberry leaf, herbs frequently used in pregnancy, were investigated for their in vitro CYP1A2, 2D6, and 3A4 inhibitory potential. Aqueous or ethanolic extracts were made from commercially available herbal products, and incubations were performed with recombinant cDNA-expressed human CYP enzymes in the presence of positive inhibitory controls. Metabolite formation was determined by validated LCMS/MS or HPLC methodologies. IC50 inhibition constants were estimated from CYP activity inhibition plots using non-linear regression. The most potent inhibition was shown for fennel towards CYP2D6 and 3A4 with respective IC50 constants of 23 ± 2 and 40 ± 4 µg/ml, horsetail towards CYP1A2 with an IC50 constant of 27 ± 1 µg/ml, and raspberry leaf towards CYP1A2, 2D6, and 3A4 with IC50 constants of 44 ± 2, 47 ± 8, and 81 ± 11 µg/ml, respectively. Based on the recommended dosing of the different commercial herbal products, clinically relevant systemic CYP inhibitions could be possible for fennel, horsetail, and raspberry leaf. In addition, fennel and raspberry leaf might cause a clinically relevant inhibition of intestinal CYP3A4. The in vivo inhibitory potential of these herbs towards specific CYP enzymes should be further investigated.

Evaluations of In Vitro Metabolism, Drug–Drug Interactions Mediated by Reversible and Time-Dependent Inhibition of CYPs, and Plasma Protein Binding of MMB4 DMS

1,1'-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) dimethanesulfonate (DMS) is a bisquaternary pyridinium aldoxime that reactivates acetylcholinesterase inhibited by organophosphorus nerve agent. Drug metabolism and plasma protein binding for MMB4 DMS were examined using various techniques and a wide range of species. When (14)C-MMB4 DMS was incubated in liver microsomes, 4-pyridine aldoxime (4-PA) and an additional metabolite were detected in all species tested. Identity of the additional metabolite was postulated to be isonicotinic acid (INA) based on liquid chromatography with a tandem mass spectrometry analysis, which was confirmed by comparison with authentic INA. Formation of INA was dependent on species, with the highest level found in monkey liver microsomes. The MMB4 DMS exhibited reversible inhibition in a concentration-dependent manner toward cytochrome P450 1A2 (CYP1A2), CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in human liver microsomes showing the highest inhibition for CYP2D6. Human recombinant CYPs were used to evaluate inhibitory curves more adequately and determine detailed kinetic constants for reversible inhibition and potential time-dependent inhibition (TDI). The MMB4 DMS exhibited reversible inhibition toward human-recombinant CYP2D6 with an inhibition constant (K i) value of 66.6 µmol/L. Based on the k inact/K I values, MMB4 DMS was found to exhibit the most potent TDI toward CYP2D6. The MMB4 DMS at 5 different concentrations was incubated in plasma for 5 hours using an equilibrium dialysis device. For all species tested, there were no concentration-dependent changes in plasma protein binding, ranging from 10% to 17%. These results suggest that MMB4 was not extensively bound to plasma protein, and there were no overt species-related differences in the extent of MMB4 bound to plasma protein.

An LC–MS based study of the metabolic profile of primaquine, an 8-aminoquinoline antiparasitic drug, with an in vitro primary human hepatocyte culture model

The 8-aminoquinoline drug primaquine (PQ) is currently the only drug in use against the persistent malaria caused by the hypnozoite-forming strains P. vivax and P. ovale. However, despite decades of research, its complete metabolic profile is still poorly understood. In the present study, the metabolism of PQ was evaluated by incubating the drug with pooled human hepatocytes cultured in vitro as well as with recombinant cytochrome P450 (CYP) iso- enzymes, monoamine oxidases (MAO), and flavin-containing monooxygenases (FMO). Targeted LC-MS/MS analysis of hepatocyte incubations using chemical inhibitors indicated that PQ was predominantly metabolized by CYPs 3A4, 1A2 and 2D6, MAO-A, -B and FMO-3. Confirmation of these results was sought by incubation of PQ with the corresponding recombinant enzymes. Small amounts of carboxyprimaquine (CPQ), the major observed PQ metabolite in vivo, were detected in recombinant MAO-A incubations along with another peak at m/z 261, and no significant formation of CPQ with any other recombinant enzymes was observed. Incubations with all recombinant enzymes identified as potentially active towards PQ from the hepatocyte-based assay resulted in significant parent loss over the course of 1 h. These results suggest that several enzymes, including CYPs in combination with FMOs and MAOs, play a role in the overall metabolism of PQ and indicate a major role for MAO-A. Future studies to elucidate the potential role in cytotoxicity and/or efficacy of the PQ metabolite observed at m/z 261, as observed in MAO-A isoenzyme studies, are needed.

Metabolism and Disposition of the Metabotropic Glutamate Receptor 5 Antagonist (mGluR5) Mavoglurant (AFQ056) in Healthy Subjects

The disposition and biotransformation of (14)C-radiolabeled mavoglurant were investigated in four healthy male subjects after a single oral dose of 200 mg. Blood, plasma, urine, and feces collected over 7 days were analyzed for total radioactivity, mavoglurant was quantified in plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and metabolite profiles were generated in plasma and excreta by high-performance liquid chromatography (HPLC) and radioactivity detection. The chemical structures of mavoglurant metabolites were characterized by LC-MS/MS, wet-chemical and enzymatic methods, NMR spectroscopy, and comparison with reference compounds. Mavoglurant was safe and well tolerated in this study population. Mavoglurant absorption was ≥50% of dose reaching mean plasma Cmax values of 140 ng/ml (mavoglurant) and 855 ng-eq/ml (total radioactivity) at 2.5 and 3.6 hours, respectively. Thereafter, mavoglurant and total radioactivity concentrations declined with mean apparent half-lives of 12 and 18 hours, respectively. The elimination of mavoglurant occurred predominantly by oxidative metabolism involving primarily 1) oxidation of the tolyl-methyl group to a benzyl-alcohol metabolite (M7) and subsequently to a benzoic acid metabolite (M6), and 2) oxidation of the phenyl-ring leading to a hydroxylated metabolite (M3). The subjects were mainly exposed to mavoglurant and seven main metabolites, which combined accounted for 60% of (14)C-AUC0-72 h (area under the concentration-time curve from time 0 to infinity). The primary steps of mavoglurant metabolism observed in vivo could partially be reproduced in vitro in incubations with human liver microsomes and recombinant cytochrome P450 enzymes. After 7 days, the mean balance of total radioactivity excretion was almost complete (95.3% of dose) with 36.7% recovered in urine and 58.6% in feces.

Metabolic detoxification pathways for 5-methoxy-sterigmatocystin in primary tracheal epithelial cells

1. The health effects of inhaled mycotoxins remain poorly documented despite their presence in bioaerosols. 5-methoxy-sterigmatocystin is produced in association with sterigmatocystin by some Aspergillus spp., sometimes in larger amounts than sterigmatocystin. Whereas sterigmatocystin can be metabolized through cytochromes P450 (CYP), UDP-glucuronosyltransferases and sulfotransferases in airway epithelial cells, little is known about 5-methoxy-sterigmatocystin.2. The 5-methoxy-sterigmatocystin metabolites were analyzed using human recombinant CYP and porcine tracheal epithelial cell (PTEC) primary cultures at an air–liquid interface. The induction of xenobiotic-metabolizing enzymes was examined by real-time quantitative PCR for mRNA expression and 7-ethoxyresorufin O-deethylation activity.3. CYP1A1 metabolized 5-methoxy-sterigmatocystin into hydroxy-nor-methoxy-sterigmatocystin, nor-methoxy-sterigmatocystin and dihydroxy-methoxy-sterigmatocystin. CYP1A2 led to monohydroxy-methoxy-sterigmatocystin. In PTEC, 5-methoxy-sterigmatocystin metabolism resulted into a glucuroconjugate of 5-methoxy-sterigmatocystin, a sulfoconjugate and a glucuroconjugate of monohydroxy-methoxy-sterigmatocystin. The exposure of PTEC for 24 h to 1 µM 5-methoxy-sterigmatocystin induced a significant increase in the mRNA levels of CYP1A1, without significant induction of the 7-ethoxyresorufin O-deethylation activity.4. These data suggest that 5-methoxy-sterigmatocystin is mainly detoxified in airway cells through conjugation, as sterigmatocystin. However, while CYP produced a reactive metabolite of sterigmatocystin, no such metabolite was detected with 5-methoxy-sterigmatocystin. Nevertheless, 5-methoxy-sterigmatocystin increases the CYP1A1 mRNA levels. The long-term consequences remain unknown.

Ospemifene metabolism in humans in vitro and in vivo: metabolite identification, quantitation, and CYP assignment of major hydroxylations

The metabolism of ospemifene, a novel nonsteroidal selective estrogen receptor modulator, was investigated as part of its development. Metabolite identification, tentative quantitation, and CYP assignment of ospemifene were performed in human liver microsomes or homogenate incubations and in plasma samples from volunteer humans. The potential contributions of CYP enzymes were determined by recombinant human CYPs. Metabolite identification and tentative quantification were performed by liquid chromatography-mass spectrometry. The relative abundances of metabolites produced were dependent on ospemifene concentration and liver preparation, but the largest quantities of 4- and 4'-hydroxy-ospemifene (and their glucuronides in smaller quantities) were produced in human liver microsomes at low ospemifene concentrations. Other metabolites were detected in in vitro incubation with human liver including a direct glucuronide of ospemifene and some metabolites with only minor abundance. In human plasma samples, 4-hydroxy-ospemifene was the most abundant metabolite, representing about 25% of the abundance of the parent compound. All the other metabolites detected in plasma, including 4'-hydroxy-ospemifene, represented <7% of the abundance of ospemifene. Several CYP enzymes participated in 4-hydroxylation, including CYP2C9, CYP2C19, CYP2B6, and CYP3A4, whereas CYP3A enzymes were the only ones to catalyze 4'-hydroxylation. In vitro incubations with liver preparations provided a rather reliable starting point in the search for potential metabolites in clinical settings. The in vitro metabolite profile is informative for the in vivo metabolite profile, especially regarding the major hydroxylated metabolites. However, it is anticipated that extended in vivo exposures may result in an increased production of more distal metabolites from major metabolites.

Stereoselectivity in the cytochrome P450-dependent N-demethylation and flavin monooxygenase-dependent N-oxidation of N,N-dimethylamphetamine

N,N-dimethylamphetamine (DMA), a methamphetamine (MA) analog, is known as a weak central nervous system stimulant. As DMA possesses a chiral center, we investigated the enantioselective formation of N,N-dimethylamphetamine N-oxide (DMANO) and MA from DMA using human liver microsomes, recombinant cytochrome P450 (CYP) 2D6, and flavin monooxygenases (FMO) 1 and 3. d-DMA was preferentially metabolized to MA, whereas l-DMA was more rapidly transformed to DMANO in human liver microsomes. CYP2D6 showed a preference for catalyzing N-demethylation of d-DMA, and the intrinsic clearance (Clint) ratio of d-isomer to l-isomer was 1.41. FMO1 catalyzed the formation of slightly less d-DMANO than l-DMANO, and the Clint ratio of the D- to L-isomer was 0.78. The reverse was observed for the formation of DMANO by FMO3. However, given the minor contribution of FMO3 compared with FMO1, it would not affect the overall enantioselective formation of DMANO in human liver microsomes. Enantioselectivities in the formation of MA and DMANO in human liver microsomes were consistent with those of CYP2D6 and FMO1, respectively.

Identification of metabolites of dalfampridine (4-aminopyridine) in human subjects and reaction phenotyping of relevant cytochrome P450 pathways

Objectives:An extended release formulation of dalfampridine (4-aminopyridine; 4-AP), a potassium channel blocker is available in the USA to improve walking in patients with multiple sclerosis. This study investigated the human metabolites of 4-AP and the cytochrome P450 (CYP450) pathways responsible for 4-AP metabolism.Methods:Metabolites were identified, using thin layer chromatography, high performance liquid chromatography, and gas chromatography/mass spectroscopy, in plasma and urine samples obtained during an excretion balance study of four subjects who were administered a single oral 15-mg dose of 14C-4-AP. Samples were compared with authentic standards of 4-AP, 2-hydroxy-4AP, 3-hydroxy-4AP, and 4-AP-N-oxide. Reaction phenotyping was performed in vitro using human liver microsomes and recombinant CYP450 enzymes with incubation in the presence of direct and time-dependent inhibitors to determine the CYP450 pathways involved in metabolite formation.Results:While most (∼70%) of the radioactivity detected in plasma at each time point corresponded to unchanged 4-AP, two major metabolites were recovered. One metabolite co-localized with the authentic reference standard of 3-hydroxy-4-AP, and the other metabolite was identified as the sulfate conjugate of 3-hydroxy-4-AP. Two minor components were observed, one accounting for 2% of radioactivity and the other below the level of quantitation. Reaction phenotyping showed moderate correlations for conversion of 4-AP to 3-hydroxy-4AP with both CYP2E1 (r = 0.596; p < 0.001) and CYP2C8 (r = 0.608; p < 0.001). Use of a CYP2E1 metabolism-dependent inhibitor inhibited formation of 3-hydroxy-4-AP with and without pre-incubation (higher inhibition with pre-incubation), further supporting the likelihood of CYP2E1 as a metabolic pathway. The main limitation of this study was the inability to identify the CYP enzymes responsible for the 3-hydroxylation of 4-AP, although this conversion represents only a minor metabolic pathway.Conclusion:There is limited metabolism of 4-AP in humans. The two major metabolites were 3-hydroxy-4-AP and 3-hydroxy-4-AP sulfate, likely through CYP2E1 pathways; the possibility of other CYP enzymes playing a minor role in 4-AP metabolism could not be established unequivocally. Overall, these data suggest that there is a low risk for drug–drug interactions via an impact on 4-AP metabolism through cytochrome pathways.

Abstract 4552: Defining the metabolic pathways of 13-cis retinoic acid.

Abstract Introduction: Isotretinoin (13-cRA) induces differentiation of human neuroblastoma cells. Althoughhigh-dose, pulse 13-cis-RA treatment significantly improves overall survival of high-risk neuroblastoma patients, most recently together with ch14.18 antibody + cytokines, many children develop recurrent disease during or after 13-cis-RA treatment. Pharmacokinetic data from clinical trials showed a large variation in the levels of 13-cRA or a metabolite 4-oxo-13-cis-retinoic acid (4-oxo-13-cRA) with the metabolite being present up to 7-fold higher than 13-cRA in the plasma at steady-state (Br J Cancer 96:424-31, 2007). We sought to define the enzymes involved in metabolism of 13-cis-RA using liver microsomes. Methods: Phase I metabolism was assessed using pooled human liver (HLM) and intestinal (HIM) microsomes as well as supersomes expressing the recombinant human CYP isoenzymes (2B6, 2C9*1, 2C9*3, 3A5, 3A4, 2C19 and 2C8) for phase I metabolism. We tested inhibitors of CYP enzymes: fluconazole (50μM) (CYP2C9) and ketoconazole (50μM) (CYP3A4). For UGT (phase II) metabolism, we utilized UGT1A1, UGT1A3, UGT1A7, UGT1A9 and UGT1A8 isoenzymes. The generation of metabolites was analyzed by high performance liquid chromatography (HPLC). Results: We identified 4-oxo-13-cRA, monohydroxy-13-cRA, monohydroxy-4-oxo-13-cRA, and 13-cRA glucuronide and 4-oxo-13-cRA-glucuronide as metabolites in the plasma of a 13-cRA treated patient, of which 4-oxo-13-cRA was by far the most abundant. The enzyme kinetic studies revealed that the formation of 4-oxo-13-cRA by HLM (velocity: v=1.67) was much greater compared with HIM (v=0.08) at 100 μM 13-cRA. The formation of 4-oxo-13-cRA from 13-cRA by CYP3A4 isoenzyme was the highest, with CYP3A4 generating more than twice the 4-oxo-13ciRA compared with other isoenzymes tested at a given range of concentrations. In HLM, the CYP3A4 inhibitor ketoconazole (50μM) caused an 83% ± 0.0009 reduction in the formation of 4-oxo-13-cRA relative to no inhibitor controls, supporting that CYP3A4 is a key enzyme in the generation of 4-oxo-13-cRA. Other CYP isoenzymes (2B6, 2C9*1, 2C9*3, 3A5, 2C19 and 2C8) generated minimal amounts of 4-oxo-13-cRA. Glucuronidation of 13-cRA was &amp;gt;5 fold higher by UGT1A3 as compared to other UGT isoenzymes, while 4-oxo-13-cRA was glucuronized at similar rates by multiple isoenzymes of UGT (UGT1A1, UGT1A3, UGT1A7, UGT1A9, UGT1A8). Conclusions: CYP3A4 and UGT1A3 are the major enzymes metabolizing 13-cRA while the major metabolite, 4-oxo-13-cRA is a substrate for a broader spectrum of UGT enzymes. Future studies on the association between enzymatic polymorphisms and pharmacokinetics of 13-cRA may inform strategies to optimize dosing of 13-cRA in high-risk neuroblastoma patients. Citation Format: Poonam Sonawane, Hardeep Singh, C Patrick Reynolds, Min H. Kang. Defining the metabolic pathways of 13-cis retinoic acid. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4552. doi:10.1158/1538-7445.AM2013-4552

Biotransformation of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47) by Human Liver Microsomes: Identification of Cytochrome P450 2B6 as the Major Enzyme Involved

Polybrominated diphenyl ethers (PBDEs) were widely used flame retardants that have become persistent environmental pollutants. In the present study, we investigated the in vitro oxidative metabolism of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a major PBDE detected in human tissue and environmental samples. Biotransformation of BDE-47 by pooled and individual human liver microsomes and by human recombinant cytochrome P450 (P450) enzymes was assessed using a liquid chromatography/tandem mass spectrometry-based method. Of the nine hydroxylated metabolites of BDE-47 produced by human liver microsomes, seven metabolites were identified using authentic standards. A monohydroxy-tetrabrominated and a dihydroxy-tetrabrominated metabolite remain unidentified. Kinetic analysis of the rates of metabolite formation revealed that the major metabolites were 5-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (5-OH-BDE-47), 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE-47), and possibly the unidentified monohydroxy-tetrabrominated metabolite. Among the human recombinant P450 enzymes tested, P450 2B6 was the most active enzyme in the formation of the hydroxylated metabolites of BDE-47. Moreover, the formation of all metabolites of BDE-47 by pooled human liver microsomes was inhibited by a P450 2B6-specific antibody and was highly correlated with P450 2B6-mediated activity in single donor liver microsomes indicating that P450 2B6 was the major P450 responsible for the biotransformation of BDE-47. Additional experiments involving the incubation of liver microsomes with individual monohydroxy-tetrabrominated metabolites in place of BDE-47 demonstrated that 2,4-dibromophenol was a product of BDE-47 and several primary metabolites, but the dihydroxy-tetrabrominated metabolite was not formed by sequential hydroxylation of any of the monohydroxy-tetrabrominated metabolites tested. The present study provides a comprehensive characterization of the oxidative metabolism of BDE-47 by human liver microsomes and P450 2B6.

Hepatic biotransformation of alkylresorcinols is mediated via cytochrome P450 and β‐oxidation.

Alkylresorcinols (AR) are phenolic lipids present in the bran of whole grains, particularly in wheat and rye kernels. AR may serve as a biomarker of whole grain intake. While AR pharmacokinetics and principal end metabolites have been reported, the metabolic pathways contributing to their relatively rapid elimination from the circulation remain to be established. We investigated whether hepatic ω‐ and β‐oxidation mediate the catabolism of the AR homolog C19:0 to form 3,5‐dihydroxybenzoic acid and 3‐(3,5‐dihydroxyphenyl)‐1‐propanoic acid (DHPPA) using 3 in vitro platforms: human recombinant cytochrome P450 4F2 (CYP4F2), human liver S9 fraction (which includes microsomal and cytosolic enzymes), and HepG2 cells. After incubation with 10 μmol/L C19:0, one hydroxylated metabolite was formed by CYP4F2 and its production appeared to plateau after 1.0 h. After incubation of 10 μmol/L C19:0 with human S9 for up to 4 h, one hydroxylated and one carboxylated metabolite were identified, with their production increasing linearly with time. When HepG2 cells were treated with 5, 10, or 25 μmol/L C19:0 for 48 h, DHPPA was excreted into the media in a dose‐dependent manner. Our results are consistent with a metabolic pathway of absorbed AR to phenolic acids via CYP4F2‐mediated ω‐hydroxylation. Funded by the General Mills Inc. and USDA.

Regio‐ and stereo‐selective oxidation of fluorinated substrates by recombinant cytochrome P450 BM3 variants

We recently showed that, upon the addition of fluorine substituents on n‐octane, van der Waals interactions including dispersion between a ‐CF2‐ and an aromatic π system account for changes in the alkane hydroxylation mediated by variants of recombinant cytochrome P450 BM3. Here, for the first time, we demonstrate that P450 BM3 also recognizes fluorinated C12–C15 chain‐length fatty acids. Despite the fact that residues, Arg47 and Tyr52, exert significant controls for the sub‐terminal hydroxylation via hydrogen bonding interactions at carboxylate ends of these fatty acids, in any case, the fluorine substituent(s) at omega position plays an essential role in the regio‐selective hydroxylation occurred at omega‐3 position through unique van der Waals interactions. For instance, the catalytic turnover of fluorinated lauric acid substrates by recombinant cytochrome P450 BM3 exhibit higher activities than that of their parent substrate. In conclusion, our data suggest that hydrophobic residues in the heme pocket of P450 enzymes are involved in tuning the orientation of substrates and this further leads to the controlled oxidation of aliphatics mediated by high valent iron‐oxo species.This work was supported by Academia Sinica and grants from the National Science Council, R.O.C. (NSC 97–2113‐M‐001–006‐MY3 and 101–2113‐M‐001–007‐MY3).

In vitro intestinal and hepatic metabolism of Di(2-ethylhexyl) phthalate (DEHP) in human and rat

Species and organ differences in the intrinsic clearance and the enzymes involved in the metabolism of DEHP were examined in subcellular fractions of the intestine and liver as well as by recombinant cytochrome P450 (CYP) isoforms of human and rat. Estimated clearance (CLint) of DEHP via esterase-mediated pathway in human intestine was 2.4-fold greater than that in human liver while its value in rat intestine was 1.7-fold less than that in rat liver. Ranks of CLint for CYP-mediated oxidation/dealkylation of MEHP were human liver>rat liver>human intestine>rat intestine. Estimates of CLint for the production of mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethyl-5-oxohexyl) phthalate by human CYP2C9*1 were 4.2- and 2.6-fold greater than those by rat CYP2C6, respectively. Total CLint via hCYP2C9*3-mediated oxidation was 1.9- and 2.6-fold less than those by hCYP2C9*2 and 2C9*1, respectively. Estimated CLint for phthalic acid production by hCYP3A4 was 24.5μLnmolCYP−1min−1 while it was continuously produced by rCYP2C6 and 3A2 via passive mechanism. These species/organ differences in major metabolic pathway and CYP isoforms should be considered for appraisal of the potential adverse health effects of DEHP.

Metabolism of BYZX in Human Liver Microsomes and Cytosol: Identification of the Metabolites and Metabolic Pathways of BYZX

BYZX, [(E)-2-(4-((diethylamino)methyl)benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one], belongs to a series of novel acetylcholinesterase inhibitors and has been synthesized as a new chemical entity for the treatment of Alzheimer’s disease symptoms. When incubated with human liver microsomes (HLMs), BYZX was rapidly transformed into its metabolites M1, M2, and M3. The chemical structures of these metabolites were identified using liquid chromatography tandem mass spectrometry and nuclear magnetic resonance, which indicated that M1 was an N-desethylated and C = C hydrogenation metabolite of BYZX. M2 and M3 were 2 precursor metabolites, which resulted from the hydrogenation and desethylation of BYZX, respectively. Further studies with chemical inhibitors and human recombinant cytochrome P450s (CYPs), and correlation studies were performed. The results indicated that the N-desethylation of BYZX and M2 was mediated by CYP3A4 and CYP2C8. The reduced form of β-nicotinamide adenine dinucleotide 2′-phosphate was involved in the hydrogenation of BYZX and M3, and this reaction occurred in the HLMs and in the human liver cytosol. The hydrogenation reaction was not inhibited by any chemical inhibitors of CYPs, but it was significantly inhibited by some substrates of α,β-ketoalkene C = C reductases and their inhibitors such as benzylideneacetone, dicoumarol, and indomethacin. Our results suggest that α,β-ketoalkene C = C reductases may play a role in the hydrogenation reaction, but this issue requires further clarification.

Bioactivation of bisphenol A and its analogs (BPF, BPAF, BPZ and DMBPA) in human liver microsomes

Bisphenol A analogs are a class of chemicals known as diphenylmethanes, which contain two benzene rings separated by one central carbon atom, usually with a para-hydroxy group on both benzene rings. Bisphenol A (BPA) can induce an uterotrophic response in immature CD-1 mice and elicits estrogenic responses in many other experimental systems. Besides highlighting endocrine effects, a number of metabolic studies provide strong support for the idea that reactive species of BPA are formed in vitro and in vivo that can form covalent adducts with nucleophilic macromolecules and/or produce oxidative stress. We used a liquid chromatography with a triple quadrupole tandem mass spectrometry (LC–MS/MS) for the detection of metabolites and glutathione conjugates of BPA and its analogs (BPF, BPAF, BPZ and DMBPA) in human liver microsomes (HLM) or with recombinant CYP isozymes in the presence of NADPH and GSH as a trapping agent. We have confirmed that BPA and its structural analogs form hydroxylated metabolites and electrophilic species during bioactivation in HLM and CYP isozymes. These results provided important mechanistic insight into the metabolic fate of BPA structural analogs in vitro.

CYP450 1A2 and multiple UGT1A isoforms are responsible for jatrorrhizine metabolism in human liver microsomes

Jatrorrhizine, one of the protoberberine alkaloids derived from the plant Coptis chinensis, is expected to be developed as a new gastric prokinetic drug, but its metabolic characteristics in humans remain unknown. This study characterized the phase I and phase II metabolites, metabolic kinetics, and cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of jatrorrhizine in human liver microsomes (HLMs). Chemical inhibition in HLMs and metabolism by recombinant human CYP or UGT enzymes were employed to determine the key metabolic enzyme subtypes. In HLMs, demethyleneberberine (demethylated product) and jatrorrhizine glucuronide were identified as the phase I and phase II metabolites, respectively. The enzyme kinetics for both demethylation and glucuronidation were fitted to the Michaelis-Menten equation. Demethylation was inhibited significantly by furafylline and predominantly catalysed by recombinant CYP1A2, whereas glucuronidation was inhibited by silibinin, quercetin, as well as 1-naphthol and catalysed by recombinant UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9 and UGT1A10. These results showed that jatrorrhizine is metabolized by human CYP1A2 and multiple UGT1A isoforms.

In vitro Metabolism of Strychnine by Human Cytochrome P450 and Its Interaction with Glycyrrhetic Acid

ObjectiveTo investigate the metabolism of strychnine (STN) and the metabolic interaction between STN and glycyrrhetic acid (GA) in vitro. MethodsHuman liver microsomes (HLM) and human recombinant cytochrome P450 (CYP) isoforms were employed to study the metabolism of STN and the metabolic interaction of STN with GA in vitro. ResultsIn HLM, the Km, Vmax, and clearance of STN were 88.50 μmol/L, 0.88 nmol/(mg·min), and 9.93 mL/(mg·min), respectively. STN was metabolized mainly by CYP3A4. However, STN noncompetitively inhibited CYP3A4-catalyzed testosterone 6β-hydroxylation with IC50 value of 5.9 μmol/L and Ki value of 5.5 μmol/L. Moreover, GA competitively inhibited STN metabolism with IC50 value of 10.6 μmol/L and Ki value of 17.7 μmol/L. ConclusionAlthough STN is mainly metabolized by CYP3A4 in vitro, STN has noncompetitive inhibition on CYP3A4-catalyzed testosterone 6β-hydroxylation. Moreover, GA could competitively inhibit STN metabolism. The present work is helpful to elucidate the metabolic interaction between STN and GA.

Evaluation of the effect of compound aqueous solubility in cytochrome P450 inhibition assays

It is a common practice in drug discovery organizations to screen new chemical entities in order to predict future drug-drug interactions. For this purpose, there are two main assay strategies, one based on recombinant cytochrome P450 (rCYP) enzymes and fluorescent detection, and other on human liver microsomes (HLM) and liquid chromatography coupled to mass spectrometry. Many authors have reported a poor correlation between both technologies, giving rise to concerns about the usefulness of fluorometric methods for predicting drug-drug interactions. In this study, we investigated the role that compound aqueous kinetic solubility may play in this lack of correlation. We found that drug discovery compounds with unacceptable kinetic solubility, measured by a turbidimetric solutibility assay, tended to yield higher IC50 values in in vitro models based on human liver microsomes, whereas compounds with kinetic solubility values higher than 50 μM showed very similar IC50 values in both in vitro models. Our results show that the turbidimetric solubility assay is a useful tool to identify those discovery compounds that may require further investigation in order to avoid overlooking future drug-drug interactions.

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450.Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level.