Carbamazepine: a 'blind' assessment of CVP-associated metabolism and interactions in human liver-derived in vitro systems

1. The ability of various in vitro systems for CYP enzymes (computer modelling, human liver microsomes, precision-cut liver slices, hepatocytes in culture, recombinant enzymes) to predict various aspects of in vivo metabolism and kinetics of carbamazepine (CBZ) was investigated. 2. The study was part of the EUROCYP project that aimed to evaluate relevant human in vitro systems to study drug metabolism. 3. CBZ was given to the participating laboratories without disclosing its chemical nature. 4. The most important enzyme (CYP3A4) and metabolic route (10,11-epoxidation) were predicted by all the systems studied. 5. Minor enzymes and routes were predicted to a different extent by various systems. 6. Prediction of a clearance class, i.e. slow clearance, was correctly predicted by microsomes, slices, hepatocytes and recombinant enzymes (CYP3A4). 7. The 10,11-epoxidation of CBZ by the recombinant CYP3A4 was enhanced by the addition of exogenous cytochrome-b5, leading to a considerable over-prediction. 8. Induction potency of CBZ was predicted in cultured hepatocytes in which 7-ethoxycoumarin O-deethylase was used as an index activity. 9. It seems that for a principally CYP-metabolized substance such as CBZ, all liver-derived systems provide useful information for prediction of metabolic routes, rates and interactions.     

CYP3A4 and CYP3A7-mediated carbamazepine 10,11-epoxidation are activated by differential endogenous steroids.

Recently, we reported that several endogenous steroids affect CYP3A4-mediated drug metabolism, using human adult liver microsomes as an enzyme source. Especially, carbamazepine (CBZ) 10,11-epoxidation is activated by androstenedione (AND). In the present studies, we investigated the effects of endogenous steroids on the activity of CBZ 10,11-epoxidation by expressed CYP3A4 and CYP3A7. When expressed CYP3A4 was used as an enzyme source, the addition of AND to the reaction mixture also caused a drastic increase in the activity of CBZ 10,11-epoxidase, and resulted in a change in the kinetics from sigmoid to Michaelis-Menten type. On the other hand, expressed CYP3A7-mediated CBZ 10,11-epoxidation was activated by sulfate conjugate steroids, such as pregnenolone 3-sulfate, 17alpha-hydroxypregnenolone 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEA-S), whereas the unconjugated form corresponding to these three steroids did not activate the reaction. Especially, DHEA-S was found to be a potent activator of CBZ 10,11-epoxidation by expressed CYP3A7. The kinetic character of CBZ 10,11-epoxidation by CYP3A7 is Michaelis-Menten type regardless of the presence of DHEA-S. The presence of DHEA-S caused a decrease in K(m) and increase in V(max) for CYP3A7-mediated CBZ 10,11-epoxidation, whereas DHEA-S 16alpha-hydroxylation was not affected by the coexistence of CBZ. In conclusion, CYP3A4 and CYP3A7-mediated CBZ 10,11-epoxidations are activated by different types of endogenous steroids. This is the first report regarding CYP3A7 cooperativity.     

Assessment of the pulmonary CYP1A1 metabolism of mavoglurant (AFQ056) in rat

1.?AFQ056 phenotyping results indicate that CYP1A1 is responsible for the formation of the oxidative metabolite, M3. In line with the predominant assumption that CYP1A1 is mainly expressed in extrahepatic tissues, only traces of M3 were detected in hepatic systems. The aim of this study was to investigate the pulmonary CYP1A1 mediated metabolism of AFQ056 in rat.

2.?Western blot analysis confirmed that CYP1A1 is expressed in rat lung albeit at low levels. M3 formation was clearly observed in recombinant rat CYP1A1, lung microsomes and lung tissue slices and was strongly inhibited by ketoconazole in the incubations. As CYP3A4 and CYP2C9 metabolites were only observed at trace levels, we concluded that the reduced M3 formation was due to CYP1A1 inhibition.

3.?AFQ056 lung clearance (CL_lung) as estimated from in vitro data was predicted to be negligible (<1% pulmonary blood flow). This was confirmed by in vivo experiments where intravenous and intra-arterial dosing to rats failed to show significant pulmonary extraction.

4.?While rat lung may make a contribution to the formation of M3, it is unlikely to be the only organ involved in this process and further experiments are required to investigate the potential metabolic elimination routes for AFQ056.     

The metabolism of the 5HT3 antagonists ondansetron,alosetron and GR87442 I: A comparison of in vitro and in vivo metabolism and in vitro enzyme kinetics in rat,dog and human hepatocytes,microsomes and recombinant human enzymes

The metabolism of the structurally related 5HT₃ antagonists ondansetron, alosetron and GR87442 in the rat, dog and human was determined in hepatocytes, liver microsomes and human recombinant microsomes. The profiles of phase I metabolites were similar in human hepatocytes and microsomes. The metabolites of all three compounds produced in rat, dog and human microsomes and hepatocytes were similar to those seen in vivo, with the major routes of metabolism being N-dealkylation and/or hydroxylation. There was more extensive metabolic processing in hepatocytes than in microsomes; however, sequential metabolism was less extensive in vitro compared with in vivo. The pharmacokinetics of the three 5HT₃ antagonists investigated were dominated by CYP3A4 (and/or 2C9) compared with CYP1A2 in man, possibly determined by enzyme capacity rather than relative enzyme affinity. These data support the use of rat, dog and human hepatocytes for the prediction of in vivo metabolites of ondansetron, alosetron and GR87442.     

Effects of endogenous steroids on CYP3A4-mediated drug metabolism by human liver microsomes.

In the present study, we investigated the effects of 14 endogenous steroids on the CYP3A4-mediated drug metabolism by human liver microsomes in vitro. Nevirapine (NVP) 2-, 12-hydroxylations, carbamazepine (CBZ) 10,11-epoxidation, triazolam (TZM) 1'-, 4-hydroxylations, erythromycin (EM) N-demethylation, and 2-sulphamoylacetylphenol (SMAP) formation from zonisamide (ZNS) were investigated. The activities of the NVP 2-, 12-hydroxylations, the CBZ 10,11-epoxidation, and the TZM 4-hydroxylation were activated by endogenous androgens, such as androstenedione (AND), testosterone, and dehydroepiandrosterone. However, these androgens inhibited EM N-demethylation, TZM 1'-hydroxylation, and SMAP formation. To understand the mechanisms of these effects of androgens on CYP3A4 activities, we performed a kinetic analysis of the metabolism of CBZ and ZNS in the presence or absence of AND using the modified two-site equation model. The addition of AND to the reaction mixture caused a drastic increase in the activity of CBZ 10,11-epoxidase, especially at a low substrate concentration, and resulted in a change in the kinetics from the sigmoid to Michaelis-Menten type. On the other hand, the metabolism of ZNS was strongly inhibited by AND, although no allosteric change was observed in this case. These data demonstrate that endogenous steroids, especially androgens, strongly affect CYP3A4-mediated drug metabolism in vitro. The postulated mechanisms of the interactions between AND and CBZ or ZNS are discussed.     

Prediction of drug–drug interactions with carbamazepine-10,11-epoxide using a new in vitro assay for epoxide hydrolase inhibition

1.?Carbamazepine is an antiepileptic drug which is metabolized by CYP3A4 into carbamazepine-10,11-epoxide. This metabolite is then detoxified by epoxide hydrolase. As carbamazepine-10,11-epoxide has been associated with neurotoxicity, it is critical to identify whether a new antiepileptic drug has the potential to inhibit epoxide hydrolase and therefore increase carbamazepine-10,11-epoxide plasma levels.

2.?In this study, an in vitro assay was developed to evaluate epoxide hydrolase activity by using carbamazepine-10,11-epoxide as probe substrate. The ability of this assay to predict drug–drug interactions (DDI) at the epoxide hydrolase level was also investigated.

3.?To this aim, known inhibitors of epoxide hydrolase for which in vivo data are available were used. Firstly, carbamazepine-10,11-epoxide hydrolase activity was determined in liver microsomes, cytosol and hepatocytes. Thereafter, the IC₅₀ of epoxide hydrolase inhibitors (progabide, valproic acid, valpromide and valnoctamide) was determined in liver microsomes and hepatocytes. Finally, prediction of AUC increase was performed using the in vitro data generated.

4.?Interestingly, epoxide hydrolase activity was found to be much higher in human hepatocytes compared to liver microsomes/cytosol. Even though assessed on a limited number of compounds, this study demonstrated that the use of hepatocytes seems to be a more relevant model to assess and predict DDI at the epoxide hydrolase level.     

Metabolic activation of a pentafluorophenylethylamine derivative: Formation of glutathione conjugates in vitro in the rat

The aim was to investigate the metabolic activation potential of a pentafluorophenylethylamine derivative (compound I) in vitro in the rat and to identify the cytochrome P450 (CYP) enzymes that catalyse these metabolic activation processes. Reduced glutathione (GSH) was fortified in rat hepatocytes and liver microsomes to trap possible reactive intermediates. Four glutathione conjugates (M1–4) were identified by LC-MSⁿ following incubation of compound I in GSH-enriched rat hepatocytes and liver microsomes. Three of these conjugates (M2–4) have not been reported previously for pentafluorophenyl derivatives. Elemental composition analysis of these conjugates was obtained using high-resolution quadrupole time-of-flight mass spectrometry. The formation of GSH conjugate M1 was rationalized as a direct nucleophilic replacement of fluoride by glutathione, whereas the formation of the GSH conjugates M2–4 was proposed to occur by NADPH-dependent metabolic activation of the pentafluorophenyl ring via arene oxide, quinone and/or quinoneimine reactive intermediates. Formation of these conjugates was enhanced three- to five-fold in liver microsomes obtained from phenobarbital- and dexamethasone-treated rats. In incubations with pooled rat liver microsomes and recombinant rat CYP3A1 and CYP3A2, troleandomycin (TAO) reduced the formation of GSH conjugates M2–4 by 80–90%, but it had no effect on the formation of M1. Incubation of compound I with rat supersomes indicated that only CYP3A1 and CYP3A2 were capable of mediating these metabolic activation processes.     

In vitro–in vivo extrapolation of hepatic clearance: Biological tools,scaling factors,model assumptions and correct concentrations

Although the measurement of metabolite formation or substrate depletion in in vitro systems, from recombinant enzymes to tissue slices, is a relatively routine task, there are a number of more or less unresolved issues in the extrapolation of the enzymatic intrinsic clearance into hepatic metabolic clearance. Nominal concentrations of the drug added to the incubation system are not necessarily the concentration the transporter or the metabolizing enzyme sees. In addition, peculiarities of incubation set-ups should be assessed. Unbound drug fractions (concentrations) in the in vitro system itself should be measured or estimated for the appropriate assessment of enzymatic intrinsic clearance. In addition, blood and/or plasma concentrations to be encountered in the in vivo situation should be measured or estimated for the extrapolation. Extrapolation always means making a number of assumptions and the most important of these, such as scaling factors from recombinant enzymes, microsomes or hepatocytes to the mass unit of the liver, liver weight, blood flow, and distribution volume amongst others, and so on, should be explicitly stated and included in the extrapolation process. Despite all the above-mentioned reservations the in vitro–in vivo extrapolation of metabolic clearance seems to be a useful and mostly fairly precise tool for predicting the important pharmacokinetic processes of a drug.     

Preclinical Pharmacokinetics and In Vitro Metabolism of BMS-690514, a Potent Inhibitor of EGFR and VEGFR2

BMS-690514, a potent inhibitor of human epidermal growth factor receptor (HER) 1 (EGFR), 2, and 4, and vascular endothelial growth factor receptors (VEGFR) 1–3, is currently under investigation as an oral agent for the treatment of solid tumors. In vitro and in vivo studies were conducted to characterize the pharmacokinetics and metabolism. Through integration of in vitro and in vivo pharmacokinetic data and antitumor efficacy in nude mice, human pharmacokinetics and efficacious doses were projected for BMS-690514. The oral bioavailability of BMS-690514 was 78% in mice, ~ 100% in rats, 8% in monkeys, and 29% in dogs. The low oral bioavailability in monkeys could be attributed to high systemic clearance in that species, which was also consistent with predicted clearance using in vitro data from monkey liver microsomes. Permeability of BMS-690514 in Caco-2 cells was in the intermediate range with a moderate potential to be a P-gp substrate. Experiments using recombinant human CYP enzymes and human liver microsomes suggested that CYP2D6 and CYP3A4 are likely to play a key role in the metabolic clearance of BMS-690514; in addition, direct glucuronidation of BMS-690514 was also observed in human hepatocytes. BMS-690514 was able to cross the blood-brain barrier with a brain-to-plasma ratio of ~ 1. The preclinical ADME properties of BMS-690514 suggest good oral bioavailability in humans and metabolism by multiple pathways including oxidation and glucuronidation. Based on the efficacious AUC in nude mice and predicted human pharmacokinetics, the human efficacious QD dose is predicted to be in the range of 100-200 mg.     

Prediction of differences in in vivo oral clearance of N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100) between extensive and poor metabolizers from in vitro metabolic data in human liver microsomes lacking CYP2D6 activity and recombinant CYPs

1. It has previously been reported that N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) was predominantly metabolized by cytochrome P450 (CYP) 2D6 in human liver microsomes (HLM). In the present study, the contribution of CYP forms involved in the formation of the major metabolites of NE-100 in human liver lacking CYP2D6 activity (PM-HLM) has been predicted by use of in vitro kinetic data on recombinant CYPs microsomes (rCYPs).

2. In PM-HLM, NE-100 is predicted to be metabolized to N-despropyl-NE-100 (NE-098), p-hydroxy-NE-100 (NE-152) and m-hydroxyl-NE-100 (NE-163), but not to O-demethy-NE-100 (NE-125), which is a major metabolite in pooled human liver microsomes (EM-HLM). The relative activity factor approach assumed that NE-098 formation is predominantly catalysed by CYP3A4 and CYP2C9 and the NE-152?+?163mix (a mixture of two hydroxylated metabolites, NE-152 and NE-163) formation is only catalysed by CYP3A4.

3. The predicted contribution rates of CYP3A4 and CYP2C9 for NE-098 formation were 58.1 and 34.6%, respectively, in PM-HLM. These predicted results were strongly supported by kinetic and inhibition studies using PM-HLM. The intrinsic clearance of NE-100 predicted from rCYPs (the predicted CL_{int-HLM-total}) corresponded to those observed from EM- and PM-HLM (the observed CL_int-HLM).

4. The in vivo oral clearance (CL_oral) of NE-100 in extensive metabolizers and poor metabolizers of CYP2D6 was predicted to be 50?times higher in extensive metabolizers than poor metabolizers using in vitro–in vivo scaling method based on the dispersion model. These data suggest that polymorphism of CYP2D6 might greatly affect NE-100 metabolism in vivo.

     

A comprehensive assessment of repaglinide metabolic pathways: impact of choice of in vitro system and relative enzyme contribution to in vitro clearance

Repaglinide is presently recommended by the U.S. Food and Drug Administration as a clinical CYP2C8 probe, yet current in vitro and clinical data are inconsistent concerning the role of this enzyme in repaglinide elimination. The aim of the current study was to perform a comprehensive investigation of repaglinide metabolic pathways and assess their contribution to the overall clearance. Formation of four repaglinide metabolites was characterized using in vitro systems with differential complexity. Full kinetic profiles for the formation of M1, M2, M4, and repaglinide glucuronide were obtained in pooled cryopreserved human hepatocytes, human liver microsomes, human S9 fractions, and recombinant cytochrome P450 enzymes. Distinct differences in clearance ratios were observed between CYP3A4 and CYP2C8 for M1 and M4 formation, resulting in a 60-fold M1/M4 ratio in recombinant (r) CYP3A4, in contrast to 0.05 in rCYP2C8. Unbound K(m) values were within 2-fold for each metabolite across all in vitro systems investigated. A major system difference was seen in clearances for the formation of M2, which is suggested to be a main metabolite of repaglinide in vivo. An approximately 7-fold higher unbound intrinsic clearance was observed in hepatocytes and S9 fractions in comparison to microsomes; the involvement of aldehyde dehydrogenase in M2 formation was shown for the first time. This systematic analysis revealed a comparable in vitro contribution from CYP2C8 and CYP3A4 to the metabolism of repaglinide (<50%), whereas the contribution of glucuronidation ranged from 2 to 20%, depending on the in vitro system used. The repaglinide M4 metabolic pathway is proposed as a specific CYP2C8 probe for the assessment of drug-drug interactions.     

Identification and relative contributions of human cytochrome P450 isoforms involved in the metabolism of glibenclamide and lansoprazole: evaluation of an approach based on the in vitro substrate disappearance rate

1.?The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug.

2.?Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CL_int) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively.

3.?CL_int of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CL_int of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CL_int of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4.

4.?The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.     

Quantitative prediction of macrolide drug-drug interaction potential from in vitro studies using testosterone as the human cytochrome P4503A substrate

Objective Macrolide antibiotics are mechanism-based inactivators of CYP3A enzymes that exhibit varying degrees of inhibitory potency. Our aim was to predict quantitatively the drug-drug interaction (DDI) potential of five macrolides from in vitro studies using testosterone as the CYP3A substrate, and to compare the predictions generated from human liver microsomal and recombinant CYP3A4 data. Methods The in vitro kinetic constants of CYP3A inactivation (K _I and k _inact) were estimated by varying the time of pre-incubation and the concentration of troleandomycin, erythromycin, clarithromycin, roxithromycin or azithromycin. CYP3A activity was determined from the measurement of testosterone 6β-hydroxylation with human liver microsomes (HLM) and recombinant CYP3A4 as the enzyme sources. The mechanism-based pharmacokinetic model was fitted with inactivation data to predict the increase in oral area under the plasma concentration-time curve (AUC) for midazolam. Results All five macrolides inactivated testosterone 6β-hydroxylation by HLM and recombinant CYP3A4 with k _inact values in the range of 0.023 to 0.058 min⁻¹. The potency of inactivation (K _I) was higher using recombinant CYP3A4 as the enzyme source. The oral AUCs for midazolam were predicted from HLM data to increase 16.6, 5.3, 4.6, 1.6 and 1.2-fold due to the inhibition of metabolic clearance by troleandomycin, erythromycin, clarithromycin, roxithromycin and azithromycin, respectively. These results are within the range of the AUC ratios reported for clinical DDI studies. The predicted AUC increases generated using recombinant CYP3A4 overestimated the magnitude of the DDIs. Conclusions The DDI potential of five macrolide antibiotics was quantitatively predicted from in vitro studies using testosterone as the CYP3A substrate with HLM as the enzyme source.     

In vitro identification of metabolic pathways and cytochrome P450 enzymes involved in the metabolism of etoperidone

1. In vitro studies have been carried out to investigate the metabolic pathways and identify the hepatic cytochrome P450 (CYP) enzymes involved in etoperidone (Et) metabolism. 2. Ten in vitro metabolites were profiled, quantified and tentatively identified after incubation with human hepatic S9 fractions. Et was metabolized via three metabolic pathways: (A) alkyl hydroxylation to form OH-ethyl-Et (M1); (B) phenyl hydroxylation to form OH-phenyl-Et (M2); and (C) N-dealkylation to form 1-m-chlorophenylpiperazine (mCPP, M8) and triazole propyl aldehyde (M6). Six additional metabolites were formed by further metabolism of M1, M2, M6 and M8. 3. Kinetic studies revealed that all metabolic pathways were monophasic, and the pathway leading to the formation of OH-ethyl-Et was the most efficient at eliminating the drug. On incubation with microsomes expressing individual recombinant CYPs, formation rates of M1-3 and M8 were 10-100-fold greater for CYP3A4 than that for other CYP forms. The formation of these metabolites was markedly inhibited by the CYP3A4-specific inhibitor ketoconazole, whereas other CYP-specific inhibitors did not show significant effects. In addition, the production of M1-3 and M8 was strongly correlated with CYP3A4-mediated testosterone 6 β -hydroxylase activities in 13 different human liver microsome samples. 4. Dealkylation of the major metabolite M1 to form mCPP (M8) was also investigated using microsomes containing recombinant CYP enzymes. The rate of conversion of M1 to mCPP by CYP3A4 was 503.0 ± 3.1 pmole nmole^?1 min^?1. Metabolism of M1 to M8 by other CYP enzymes was insignificant. In addition, this metabolism in human liver microsomes was extensively inhibited by the CYP3A4 inhibitor ketoconazole, but not by other CYP-specific inhibitors. In addition, conversion of M1 to M8 was highly correlated with CYP3A4-mediated testosterone 6 β -hydroxylase activity. 5. The results strongly suggest that CYP3A4 is the predominant enzyme-metabolizing Et in humans.     

Approach to predict the contribution of cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage: The effect of the expression of cytochrome b5 with recombinant P450 enzymes

In order to evaluate the potential adverse effects due to genetic polymorphism and/or inter-individual variation, it is necessary to calculate the cytochrome P450 (CYP) contribution to the metabolism of new drugs. In the current study, the in vitro intrinsic clearance (CL_int) values of marker substrates and drugs were determined by measuring metabolite formation and substrate depletion, respectively. Recombinant CYP microsomes expressing CYP2C9, CYP2C19 and CYP3A4 with co-expressed cytochrome b₅ were used, but those expressing CYP1A2 and CYP2D6 did not have co-expressed cytochrome b₅. The following prediction methods were compared to determine the CL_int value using data from recombinant CYP enzymes: (1) relative CYP enzyme content in human liver microsomes; (2) relative activity factor (RAF) estimated from the V_max value; and (3) RAF estimated from the CL_int value. Estimating RAF from CL_int proved the most accurate prediction method among the three tested, and differences in the CYP3A4 marker reactions did not affect its accuracy. The substrate depletion method will be useful in the early drug-discovery stage when the main metabolite and/or metabolic pathway has not been identified. In addition, recombinant CYP microsomes co-expressed with cytochrome b₅ might be suitable for the prediction of the CL_int value.     

In vitro profiling of the metabolism and drug–drug interaction of tofogliflozin,a potent and highly specific sodium-glucose co-transporter 2 inhibitor,using human liver microsomes,human hepatocytes,and recombinant human CYP

Abstract

1. The metabolism and drug–drug interaction (DDI) risk of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, were evaluated by in vitro studies using human liver microsomes, human hepatocytes, and recombinant human CYPs.

2. The main metabolite of tofogliflozin was the carboxylated derivative (M1) in human hepatocytes, which was the same as in vivo. The metabolic pathway of tofogliflozin to M1 was considered to be as follows: first, tofogliflozin was catalyzed to the primary hydroxylated derivative (M4) by CYP2C18, CYP4A11 and CYP4F3B, then M4 was oxidized to M1.

3. Tofogliflozin had no induction potential on CYP1A2 and CYP3A4. Neither tofogliflozin nor M1 had inhibition potential on CYPs, with the exception of a weak CYP2C19 inhibition by M1.

4. Not only are multiple metabolic enzymes involved in the tofogliflozin metabolism, but the drug is also excreted into urine after oral administration, indicating that tofogliflozin is eliminated through multiple pathways. Thus, the exposure of tofogliflozin would not be significantly altered by DDI caused by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because tofogliflozin has no CYP induction or inhibition potency, and the main metabolite M1 has no clinically relevant CYP inhibition potency.     

Contributions of human cytochrome P450 enzymes to glyburide metabolism

Glyburide (GLB) is a widely used oral sulfonylurea for the treatment of gestational diabetes. The therapeutic use of GLB is often complicated by a substantial inter‐individual variability in the pharmacokinetics and pharmacodynamics of the drug in human populations, which might be caused by inter‐individual variations in factors such as GLB metabolism. Therefore, there has been a continued interest in identifying human cytochrome P450 (CYP) isoforms that play a major role in the metabolism of GLB. However, contrasting data are available in the present literature in this regard. The present study systematically investigated the contributions of various human CYP isoforms (CYP3A4, CYP3A5, CYP2C8, CYP2C9 and CYP2C19) to in vitro metabolism of GLB. GLB depletion and metabolite formation in human liver microsomes were most significantly inhibited by the CYP3A inhibitor ketoconazole compared with the inhibitors of other CYP isoforms. Furthermore, multiple correlation analysis between GLB depletion and individual CYP activities was performed, demonstrating a significant correlation between GLB depletion and the CYP3A probe activity in 16 individual human liver microsomal preparations, but not between GLB depletion and the CYP2C19, CYP2C8 or CYP2C9 probe activity. By using recombinant supersomes overexpressing individual human CYP isoforms, it was found that GLB could be depleted by all the enzymes tested; however, the intrinsic clearance (V_max/K_m) of CYP3A4 for GLB depletion was 4–17 times greater than that of other CYP isoforms. These results confirm that human CYP3A4 is the major enzyme involved in the in vitro metabolism of GLB. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of the human cytochrome P450s responsible for the in vitro metabolism of a leukotriene B4 receptor antagonist,CP-195,543

1.?The major human cytochrome P450 (CYP) form(s) responsible for the metabolism of CP-195,543, a potent leukotriene B4 antagonist, were investigated.

2.?Incubation of CP-195,543 with human liver microsomes resulted in the formation of three major metabolites, M1–3. M1 and M2 were diastereoisomers and formed by oxidation on the benzylic position. M3 was formed by aromatic oxidation of the benzyl group attached to the 3-position of the benzopyran ring.

3.?The results from experiments with recombinant CYPs, correlation studies and inhibition studies with form-selective inhibitors and a CYP3A antibody strongly suggest that the CYP3A4 plays a major role in the metabolism of CP-195,543. Recombinant CYP3A5 did not metabolize CP-195,543.

4.?The apparent K_m and V_max for the formation of M1–3 in human liver microsomes were determined as 36?μM and 4.1?pmol?min^?1?pmol^?1 P450, 44?μM and 10?pmol?min^?1?pmol^?1 P450, and 34?μM and 2.0?pmol?min^?1?pmol^?1 P450, respectively. The average in vitro intrinsic clearance for M2 was the highest both in human liver microsomes and recombinant CYP3A4 compared with M1 and M3. Intrinsic clearance for M2 in human liver microsomes and recombinant CYP3A4 was 0.231 and 0.736 ml?min^?1?pmol^?1 P450, respectively. The intrinsic clearances for M1 and M3 in human liver microsomes and CYP3A4 were 0.114 and 0.060 and 0.197 and 0.088 ml?min^?1?pmol^?1 P450, respectively. This suggests that benzylic oxidation is the predominant phase I metabolic pathway of CP-195,543 in man.     

Kinetics of diazepam metabolism in rat hepatic microsomes and hepatocytes and their use in predicting in vivo hepatic clearance

1. The rates of diazepam (DZ) metabolism to the primary metabolites 3-hydroxydi-azepam, 4'-hydroxydiazepam and nordiazepam were studied in vitro using rat hepatic microsomes and hepatocytes. 4'-hydroxydiazepam had the largest intrinsic clearance (V_max/K_m ratio, CL_int) in both microsomes and hepatocytes representing 49 and 70% of total metabolism respectively. Whereas the contribution of 3-hydroxydiazepam was similar in both systems (21–24%), the N-demethylation pathway was greater in microsomes (27%) than hepatocytes (9%).

2. The pharmacokinetics of DZ were determined in vivo using the intraportal route to avoid blood flow limitations due to the high clearance of DZ. No dose dependency was observed in either clearance or steady state volume of distribution, which were estimated to be 38 ml/min/SRW (where SRW is a standard rat weight of 250?g) and 1.3 L/SRW respectively. Blood binding of DZ was concentration independent, the unbound fraction being 0.22.

3. Scaling factors were used to relate the in vitro CL_int to the in vivo unbound clearance. Hepatocytes (123 ml/min/SRW) produced a more realistic prediction for the in vivo value (174 ml/min/SRW) than microsomes (41 ml/min/SRW). This situation is believed to arise from the quantitative differences in the three metabolic pathways in the two in vitro systems. It is speculated that end product inhibition is responsible for reduced total metabolism in microsomes whereas hepatocytes operate kinetically in a manner close to in vivo.     

Metabolite kinetics of ondansetron in rat. Comparison of hepatic microsomes,isolated hepatocytes and liver slices,with in vivo disposition

1. The kinetics of hydroxylation and N-demethylation of ondansetron have been determined in freshly isolated hepatocytes, hepatic microsomes and precision-cut liver slices from the male Sprague-Dawley rat. In vivo studies have also been carried out to characterize the pharmacokinetics of ondansetron and in vitro data have been assessed for their value as predictors of hepatic clearance.

2. In the three in vitro systems, the formation of hydroxylated and demethylated metabolites were characterized as a function of substrate concentration by a high-affinity, low-capacity site and a low-affinity, high-capacity site which was not saturated over the concentration range studied (2.5–500 μM). Slices gave consistently higher K_m's (20 and 30 μM for hydroxylation and demethylation respectively) than hepatocytes (3 and 13 μM respectively) and microsomes (2 and 5 μM respectively). The rank order of V_max and CL_int was the same for each system; hydroxylation rates exceeding demethylation rates. Although two hydroxylations (7- and 8-hydroxy metabolites) occurred exclusively in microsomes, these are believed to originate from a common precursor.

3. The high CL_int of ondansetron (150 ml/min/SRW, where SRW is a standard rat weight of 250 g) is well predicted by scaling either microsomal clearance for microsomal protein recovery or hepatocyte clearance for hepatocellularity (212 and 135 ml/min/SRW respectively). In contrast, the use of liver slice data scaled to a whole liver substantially underestimates CL_int (9 ml/min/SRW).