Panel Of Human Liver Microsomes Research Articles

In Vitro Hepatic Oxidative Biotransformation of Trimethoprim.

Trimethoprim (TMP) has been widely used since the 1960s, both alone and in combination with sulfamethoxazole. Unfortunately, information regarding the role that cytochrome P450 enzymes (P450s) play in the formation of TMP primary metabolites is scarce. Hence, we undertook in vitro studies to identify and more fully characterize the P450s that catalyze formation of six TMP primary metabolites: TMP 1-N-oxide (1-NO-TMP) and 3-N-oxide (3-NO-TMP), 3'- and 4'-desmethyl-TMP, a benzylic alcohol (Cα-OH-TMP), and an N-acetyl cysteine (NAC) adduct of TMP (Cα-NAC-TMP). Formation kinetics for each TMP metabolite in human liver microsomes (HLMs) were consistent with single-enzyme Michaelis-Menten kinetics, and Km values were markedly above (≥10-fold) the therapeutic concentrations of TMP (50 µM). The combined results from correlation studies between rates of metabolite formation and marker P450 activities in a panel of HLMs along with inhibition studies utilizing selective P450 inhibitors incubated with pooled HLMs suggested that 1-NO-TMP, Cα-NAC-TMP, and Cα-OH-TMP were predominantly formed by CYP3A4. In contrast, 3-NO-TMP was formed predominantly by CYP1A2 in HLMs and inhibited by α-naphthoflavone. 4'-Desmethyl-TMP, which is believed to be a reactive TMP metabolite precursor, was formed by several P450s, including CYP3A4, correlated with multiple P450 activities, but was inhibited primarily by ketoconazole (up to 50%), suggesting that CYP3A4 makes a major contribution to TMP 4'-demethylation. TMP 3'-demethylation was catalyzed by multiple P450s, including CYP2C9, correlated with CYP2C9 activity, and was inhibited by sulfaphenazole (up to 40%). Overall, CYP2C9 and CYP3A4 appear to be the most significant contributors to TMP primary metabolism.

Glucuronidation of a Sarpogrelate Active Metabolite Is Mediated by UDP-Glucuronosyltransferases 1A4, 1A9, and 2B4

Sarpogrelate is a selective serotonin 5-HT2A-receptor antagonist used to treat patients with peripheral arterial disease. This drug is rapidly hydrolyzed to its main metabolite (R,S)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2-propanol (M-1), which is mainly excreted as a glucuronide conjugate. Sarpogrelate was also directly glucuronidated to an O-acyl glucuronide and a N-glucuronide by UDP-glucuronosyltransferases (UGTs) in human liver microsomes (HLMs). Since M-1 is pharmacologically more active than sarpogrelate, we examined glucuronidation of this metabolite in HLMs and characterized the UGTs responsible for M-1 glucuronidation. Diastereomers of O-glucuronide (SMG1 and SMG3) and a N-glucuronide (SMG2) were identified by incubation of M-1 with HLMs in the presence of uridine 5'-diphosphoglucuronic acid (UDPGA), and their structures were confirmed by nuclear magnetic resonance and mass spectrometry analyses. Two O-glucuronides were identified as chiral isomers: SMG1 as R-isomer and SMG3 as S-isomer. Using recombinant UGT enzymes, we determined that SMG1 and SMG3 were predominantly catalyzed by UGT1A9 and UGT2B4, respectively, whereas SMG2 was generated by UGT1A4. In addition, significant correlations were noted between the SMG1 formation rate and propofol glucuronidation (a marker reaction of UGT1A9; r = 0.6269, P < 0.0031), and between the SMG2 formation rate and trifluoperazine glucuronidation (a marker reaction of UGT1A4; r = 0.6623, P < 0.0015) in a panel of HLMs. Inhibition of SMG1, SMG2, and SMG3 formation by niflumic acid, hecogenin, and fluconazole further substantiated the involvement of UGT1A9, UGT1A4, and UGT2B4, respectively. These findings collectively indicate that UGT1A4, UGT1A9, and UGT2B4 are the major UGT isoforms responsible for glucuronidation of M-1, an active metabolite of sarpogrelate.

An Inducible Cytochrome P450 3A4-Dependent Vitamin D Catabolic Pathway

Vitamin D(3) is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D(3) (25OHD(3)), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD(3) was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD(3) hydroxylation by human liver microsomes, with the formation of 4β,25-dihydroxyvitamin D(3) [4β,25(OH)(2)D(3)] dominating (V(max)/K(m) = 0.85 ml · min(-1) · nmol enzyme(-1)). 4β,25(OH)(2)D(3) was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D(3), and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4β,25(OH)(2)D(3) in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4β,25(OH)(2)D(3), but not CYP24A1-dependent 24R,25-dihydroxyvitamin D(3) formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD(3) metabolism may play an important role in the regulation of vitamin D(3) in vivo and in the etiology of drug-induced osteomalacia.

Repaglinide‐gemfibrozil drug interaction: inhibition of repaglinide glucuronidation as a potential additional contributing mechanism

To further explore the mechanism underlying the interaction between repaglinide and gemfibrozil, alone or in combination with itraconazole. Repaglinide metabolism was assessed in vitro (human liver subcellular fractions, fresh human hepatocytes, and recombinant enzymes) and the resulting incubates were analyzed, by liquid chromatography-mass spectrometry (LC-MS) and radioactivity counting, to identify and quantify the different metabolites therein. Chemical inhibitors, in addition to a trapping agent, were also employed to elucidate the importance of each metabolic pathway. Finally, a panel of human liver microsomes (genotyped for UGT1A1*28 allele status) was used to determine the importance of UGT1A1 in the direct glucuronidation of repaglinide. The results of the present study demonstrate that repaglinide can undergo direct glucuronidation, a pathway that can possibly contribute to the interaction with gemfibrozil. For example, [³H]-repaglinide formed glucuronide and oxidative metabolites (M2 and M4) when incubated with primary human hepatocytes. Gemfibrozil effectively inhibited (∼78%) both glucuronide and M4 formation, but had a minor effect on M2 formation. Concomitantly, the overall turnover of repaglinide was also inhibited (∼80%), and was completely abolished when gemfibrozil was co-incubated with itraconazole. These observations are in qualitative agreement with the in vivo findings. UGT1A1 plays a significant role in the glucuronidation of repaglinide. In addition, gemfibrozil and its glucuronide inhibit repaglinide glucuronidation and the inhibition by gemfibrozil glucuronide is time-dependent. Inhibition of UGT enzymes, especially UGT1A1, by gemfibrozil and its glucuronide is an additional mechanism to consider when rationalizing the interaction between repaglinide and gemfibrozil.

In vitro and in vivo oxidative metabolism and glucuronidation of anastrozole

Little information is available regarding the metabolic routes of anastrozole and the specific enzymes involved. We characterized anastrozole oxidative and conjugation metabolism in vitro and in vivo. A sensitive LC-MS/MS method was developed to measure anastrozole and its metabolites in vitro and in vivo. Anastrozole metabolism was characterized using human liver microsomes (HLMs), expressed cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs). Hydroxyanastrozole and anastrozole glucuronide were identified as the main oxidative and conjugated metabolites of anastrozole in vitro, respectively. Formation of hydroxyanastrozole from anastrozole was markedly inhibited by CYP3A selective chemical inhibitors (by >90%) and significantly correlated with CYP3A activity in a panel of HLMs (r= 0.96, P= 0.0005) and mainly catalyzed by expressed CYP3A4 and CYP3A5. The K(m) values obtained from HLMs were also close to those from CYP3A4 and CYP3A5. Formation of anastrozole glucuronide in a bank of HLMs was correlated strongly with imipramine N-glucuronide, a marker of UGT1A4 (r= 0.72, P < 0.0001), while expressed UGT1A4 catalyzed its formation at the highest rate. Hydroxyanastrozole (mainly as a glucuronide) and anastrozole were quantified in plasma of breast cancer patients taking anastrozole (1 mg day⁻¹); anastrozole glucuronide was less apparent. Anastrozole is oxidized to hydroxyanastrozole mainly by CYP3A4 (and to some extent by CYP3A5 and CYP2C8). Once formed, this metabolite undergoes glucuronidation. Variable activity of CYP3A4 (and probably UGT1A4), possibly due to genetic polymorphisms and drug interactions, may alter anastrozole disposition and its effects in vivo.

CYP4F Enzymes Are Responsible for the Elimination of Fingolimod (FTY720), a Novel Treatment of Relapsing Multiple Sclerosis

Fingolimod (FTY720, Gilenya, 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol) is a novel drug recently approved in the United States for the oral treatment of relapsing multiple sclerosis. The compound is eliminated predominantly by ω-hydroxylation, followed by further oxidation. The ω-hydroxylation was the major metabolic pathway in human liver microsomes (HLM). The enzyme kinetics in HLM were characterized by a Michaelis-Menten affinity constant (K(m)) of 183 μM and a maximum velocity (V(max)) of 1847 pmol/(min · mg). Rates of fingolimod metabolism by a panel of HLM from individual donors showed no correlation with marker activities of any of the major drug-metabolizing cytochrome P450 (P450) enzymes or of flavin-containing monooxygenase (FMO). Among 21 recombinant human P450 enzymes and FMO3, only CYP4F2 (and to some extent CYP4F3B) produced metabolite profiles similar to those in HLM. Ketoconazole, known to inhibit not only CYP3A but also CYP4F2, was an inhibitor of fingolimod metabolism in HLM with an inhibition constant (K(i)) of 0.74 μM (and by recombinant CYP4F2 with an IC(50) of 1.6 μM), whereas there was only a slight inhibition found with azamulin and none with troleandomycin. An antibody against CYP4F2 was able to inhibit the metabolism of fingolimod almost completely in HLM, whereas antibodies specific to CYP2D6, CYP2E1, and CYP3A4 did not show significant inhibition. Combining the results of these four enzyme phenotyping approaches, we demonstrated that CYP4F2 and possibly other enzymes of the CYP4F subfamily (e.g., CYP4F3B) are the major enzymes responsible for the ω-hydroxylation of fingolimod, the main elimination pathway of the drug in vivo.

Human Cytochrome P450 1A2 Involvement in the Formation of Reactive Metabolites from a Species-Specific Hepatotoxic Pyrazolopyrimidine Derivative, 5-n-Butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine

5-n-Butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine) (OT-7100) is a pyrazolopyrimidine derivative with potential analgesic effects. Exclusively limited elevations in serum levels of aspirate- and alanine-aminotransferase were abnormally observed in a clinical study, in contrast to no toxicological potential to experimental animals. The aim of this study was to clarify the mechanism responsible for species-specific hepatotoxicity of this model compound. OT-7100 was primarily metabolized to a carboxylic acid derivative and an amino derivative (5-n-butyl-pyrazolo[1,5-a]pyrimidine, M-5) by hydrolysis in humans and rats. In human liver, pyrazolo[1,5-a]pyrimidine derivative M-5 was further metabolized to mainly M-23OH (a C-3-position hydroxyl derivative, 3-hydroxy-5-n-butyl-pyrazolo[1,5-a]pyrimidine). Studies with recombinant cytochrome P450s (P450s), correlation analysis using a panel of human liver microsomes as well as immunoinhibition with anti-P450 antibodies collectively suggested that human liver microsomal P450 1A2 preferentially metabolized M-5 to predominantly M-23OH. Human liver microsomes were capable of activating M-5 to a covalently bound metabolite faster than rat liver microsomes: reduced glutathione prevented the bindings. A cysteine adduct derivative of M-23OH at the C-6-position was structurally confirmed. On the contrary, rat liver microsomal P450 1A2 could metabolize M-5 to equally M-23OH, M-22OH (a C-6-position hydroxyl derivative, 6-hydroxy-5-n-butyl-pyrazolo[1,5-a]pyrimidine), or an unknown metabolite. These results suggest that differences in the regiospecific metabolic function of human and rat P450 1A2 would be responsible for the human-specific metabolic activation of the primary metabolite of OT-7100 to a proximate form. It is presumed that hepatotoxicity associated with OT-7100 could be likely related to the formation of a human-specific reactive metabolite from M-23OH. OT-7100 activation by inducible P450 1A2 may therefore exhibit marked individual differences.

Pathways of Carbamazepine Bioactivation in Vitro. III. The Role of Human Cytochrome P450 Enzymes in the Formation of 2,3-Dihydroxycarbamazepine

Conversion of the carbamazepine metabolite 3-hydroxycarbamazepine (3-OHCBZ) to the catechol 2,3-dihydroxycarbamazepine (2,3-diOHCBZ) followed by subsequent oxidation to a reactive o-quinone species has been proposed as a possible bioactivation pathway in the pathogenesis of carbamazepine-induced hypersensitivity. Initial in vitro phenotyping studies implicated CYP3A4 as a primary catalyst of 2,3-diOHCBZ formation: 2-hydroxylation of 3-OHCBZ correlated significantly (r(2) > or = 0.929, P < 0.001) with CYP3A4/5 activities in a panel of human liver microsomes (n = 14) and was markedly impaired by CYP3A inhibitors (>80%) but not by inhibitors of other cytochrome P450 enzymes (< or = 20%). However, in the presence of troleandomycin, the rate of 2,3-diOHCBZ formation correlated significantly with CYP2C19 activity (r(2) = 0.893, P < 0.001) in the panel of human liver microsomes. Studies with a panel of cDNA-expressed enzymes revealed that CYP2C19 and CYP3A4 were high (S50 = 30 microM) and low (S50 = 203 microM) affinity enzymes, respectively, for 2,3-diOHCBZ formation and suggested that CYP3A4, but not CYP2C19, might be inactivated by a metabolite formed from 3-OHCBZ. Subsequent experiments demonstrated that preincubation of 3-OHCBZ with human liver microsomes or recombinant CYP3A4 led to decreased CYP3A4 activity, which was both preincubation time- and concentration-dependent, but not inhibited by inclusion of glutathione or N-acetylcysteine. CYP3A4, CYP3A5, CYP3A7, CYP2C19, and CYP1A2 converted [14C]3-OHCBZ into protein-reactive metabolites, but CYP3A4 was the most catalytically active enzyme. The results of this study suggest that CYP3A4-dependent secondary oxidation of 3-OHCBZ represents a potential carbamazepine bioactivation pathway via formation of reactive metabolites capable of inactivating CYP3A4, potentially generating a neoantigen that may play a role in the etiology of carbamazepine-induced idiosyncratic toxicity.

Glucuronidation of the soyabean isoflavones genistein and daidzein by human liver is related to levels of UGT1A1 and UGT1A9 activity and alters isoflavone response in the MCF-7 human breast cancer cell line

The soyabean isoflavones genistein and daidzein, which may protect against some cancers, cardiovascular disease and bone mineral loss, undergo substantial Phase 2 metabolism, predominantly glucuronidation. We observed a correlation between rates of metabolism of marker substrates of specific UGTs and rates of glucuronidation of genistein and daidzein in vitro by a panel of human liver microsomes, demonstrating that UGT1A1 and UGT1A9, but not UGT1A4, make a major contribution to the metabolism of these isoflavones by human liver. These findings were substantiated by observations that recombinant human UGT1A1 and UGT1A9, but not UGT1A4, catalysed the production of the major glucuronides of both genistein and daidzein in vitro. Recombinant human UGT1A8 also metabolised both genistein and daidzein, whereas UGT1A6 was specific to genistein and UGTs 2B7 and 2B15 were inactive, or only marginally active, with either isoflavone as substrate. The intestinal isoform UGT1A10 metabolised either both isoflavones or genistein only, depending on the commercial supplier of the recombinant enzyme, possibly as a result of a difference in amino acid sequence, which we were unable to confirm. Daidzein (16 μM) increased cell death in the MCF-7 human breast cancer cell line and this effect was reversed by glucuronidation. In view of a well-characterised functional polymorphism in UGT1A1, these observations may have implications for inter-individual variability in the potential health-beneficial effects of isoflavone consumption.

Impact of CYP2B6 Polymorphism on Hepatic Efavirenz Metabolism in Vitro

To determine the influence of cytochrome P450 2B6 (CYP2B6) genotype on the rate of oxidative efavirenz metabolism in human liver microsomes. Formation rates of 8-hydroxyefavirenz, 7-hydroxyefavirenz and 8,14-dihydroxyefavirenz were determined in vitro with efavirenz as a substrate (10 microM) in a large panel of human liver microsomes (n = 87) that were genotyped for variants of the CYP2B6 gene and phenotyped for CYP2B6 protein expression and bupropion hydroxylation. Efavirenz 8-hydroxylation, the major route of efavirenz clearance, was detected in all samples, exhibiting an overall interindividual variability of 44.7-fold; 8,14-dihydroxyefavirenz and 7-hydroxyefavirenz were also detected in most samples. The formation rate of 8-hydroxyefavirenz correlated significantly with CYP2B6 protein (Spearman's r(S) = 0.54; p < 0.0001) and bupropion hydroxylase activity (r(S) = 0.73; p < 0.0001). Compared with the *1/*1 genotype, efavirenz 8-hydroxylation was significantly lower in samples with *1/*6 and *6/*6 genotype, which also had significantly decreased CYP2B6 protein (Mann-Whitney test, p < 0.05). A decrease in CYP2B6 protein was also observed in samples with *1/*5 and *5/*6 genotypes, but this did not result in significant reduction of efavirenz metabolism, probably due to differences in specific activity of the protein variants. Lower CYP2B6 protein and activity, as well as efavirenz 8-hydroxylation was also found in several samples with rarer genotypes. We found no effect of gender and age on any of the phenotypes tested, but prior exposure to carbamazepine markedly increased CYP2B6 protein expression and activity as well as efavirenz 8-hydroxylation. We have shown that CYP2B6 genetic polymorphism markedly influences the metabolism of efavirenz in human liver microsomes. Importantly, the CYP2B6*6 allele harboring the SNPs c.516G>T [Q172H] and c.785A>G [K262R] was significantly associated with a pronounced decrease in CYP2B6 expression and activity, as well as a low rate of efavirenz 8-hydroxylation. These results represent a first step towards elucidating the mechanism by which this allele identifies patients exhibiting very high efavirenz plasma concentrations.

Ethanol oxidation into acetaldehyde by 16 recombinant human cytochrome P450 isoforms: Role of CYP2C isoforms in human liver microsomes

The involvement of cytochromes P450 (CYPs) in the oxidation of ethanol into acetaldehyde was investigated by using 16 recombinant human CYP isoforms. Apparent K m and V m were determined for CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9*1, CYP2C9*2, CYP2C9*3, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP4A11. All of the tested CYPs, except CYP2A6 and CYP2C18, metabolized ethanol into significant amounts of acetaldehyde and displayed K m values around 10 mM. The significant correlation found between ethanol oxidation and CYP2E1, CYP3A4 and CYP1A2 catalytic activities in a panel of human liver microsomes confirmed the strong implication of these CYPs in ethanol metabolism. The contribution of CYP2C isoforms which are the most abundant in the liver after CYP3A4, was studied using selective inhibitors either with recombinant CYP2C isoforms or in human liver microsomes. Tienilic acid (100 μM) and ticlopidine (20 μM), mechanism-based inhibitors of CYP2C9 and CYP2C19, respectively, decreased ethanol oxidation by 8 ± 1.2% and 7.6 ± 1.6% in human liver microsomal samples while selective inhibitors of CYP2E1 (DEDTC 100 μM), CYP3A4 (TAO 50 μM) and CYP1A2 (furafylline 25 μM) decreased it by 11.9 ± 2.1%, 19.8 ± 1.9% and 16.3 ± 3.9%, respectively. As ethanol can be metabolized by most of CYPs, it helps to explain or predict alcohol–xenobiotics interactions which are of high importance in medical prescription.

BIOTRANSFORMATION OF FLUTICASONE: IN VITRO CHARACTERIZATION

Fluticasone propionate (FTP) is a synthetic trifluorinated glucocorticoid with potent anti-inflammatory action that is commonly used in patients with asthma. After oral or intranasal administration, FTP undergoes rapid hepatic biotransformation; the principal metabolite formed is a 17beta-carboxylic acid derivative (M1). M1 formation has been attributed largely to cytochrome P450 3A4 (CYP3A4); however, there are no published data that confirm this assertion. Hence, in vitro studies were conducted to determine the role that human P450s play in the metabolism of FTP. Consistent with in vivo data, human liver microsomes catalyzed the formation of a single metabolite (M1) at substrate concentrations <or=10 microM (mean plasma Cmax = 1 nM). Under these conditions, the kinetics of M1 formation in human liver microsomes were consistent with those of a single enzyme (Km congruent with 5 microM). Formation of M1 correlated significantly (r > 0.95) with CYP3A4/5 activities in a panel of human liver microsomes (n = 14) and was markedly impaired by the CYP3A inhibitor ketoconazole (>94%) but not by inhibitors of other P450 enzymes (<or=10%). Studies with a panel of cDNA-expressed enzymes revealed that M1 formation was catalyzed primarily by CYP3A enzymes at FTP concentrations <or=1 microM. M1 formation was catalyzed by P450s 3A4, 3A5, and 3A7; in vitro intrinsic clearance values (Vmax/Km) were comparable for all three CYP3A enzymes. These results suggest that at pharmacologically relevant concentrations, biotransformation of FTP to M1 is mediated predominantly by CYP3A enzymes in the liver.

Pathways of Carbamazepine Bioactivation In Vitro II. The Role of Human Cytochrome P450 Enzymes in the Formation of 2-Hydroxyiminostilbene

Conversion of the carbamazepine metabolite, 2-hydroxycarbamazepine, to the potentially reactive species, carbamazepine iminoquinone (CBZ-IQ), has been proposed as a possible bioactivation pathway in the pathogenesis of carbamazepine-induced hypersensitivity. Generation of CBZ-IQ has been proposed to proceed through the intermediate, 2-hydroxyiminostilbene (2-OHIS); however, data suggested that 2-hydroxycarbamazepine is oxidized by cytochromes P450 (P450s) directly to CBZ-IQ, followed by NADPH-mediated reduction to 2-OHIS. In vitro studies were conducted to identify the P450s responsible for converting 2-hydroxycarbamazepine to 2-OHIS and to determine functional consequences of this bioactivation pathway. Formation of 2-OHIS in human liver microsomes (HLMs) was consistent with monophasic, Michaelis-Menten kinetics. The sample-to-sample variation in the rate of 2-OHIS formation correlated significantly (r2 > or = 0.706) with CYP3A4/5 and CYP2B6 activities in a panel of HLMs (n = 10). Studies with a panel of cDNA-expressed enzymes revealed that CYP3A4 preferentially catalyzed 2-OHIS formation; CYP3A4 formed 2-OHIS at a rate >10 times that of other enzymes capable of forming 2-OHIS (CYP1A1, CYP2C19, and CYP3A7). Inhibitors of CYP3A enzymes markedly impaired 2-OHIS formation in HLMs, whereas inhibitors of other P450s resulted in < or = 20% inhibition. Although CYP3A4 was primarily responsible for converting 2-hydroxycarbamazepine to 2-OHIS, neither 2-hydroxycarbamazepine, 2-OHIS, nor CBZ-IQ caused time-dependent inactivation of CYP3A activity. No thiol adducts were formed directly from 2-hydroxycarbamazepine. However, glutathione- and N-acetylcysteine-conjugates were formed with 2-OHIS or CBZ-IQ as substrates. Thus, CYP3A4-dependent secondary oxidation of 2-hydroxycarbamazepine represents a potential carbamazepine bioactivation pathway leading to the formation of thiol-reactive metabolites, intermediates that may play a role in the etiology of idiosyncratic toxicity attributed to carbamazepine.

CYTOCHROME P450 3A-MEDIATED METABOLISM OF BUSPIRONE IN HUMAN LIVER MICROSOMES

This study was carried out to determine the metabolic pathways of buspirone and cytochrome P450 (P450) isoform(s) responsible for buspirone metabolism in human liver microsomes (HLMs). Buspirone mainly underwent N-dealkylation to 1-pyrimidinylpiperazine (1-PP), N-oxidation on the piperazine ring to buspirone N-oxide (Bu N-oxide), and hydroxylation to 3'-hydroxybuspirone (3'-OH-Bu), 5-hydroxybuspirone (5-OH-Bu), and 6'-hydroxybuspirone (6'-OH-Bu) in HLMs. The apparent K(m) values for buspirone metabolite formation in pooled HLMs were 8.7 (1-PP), 34.0 (Bu N-oxide), 4.3 (3'-OH-Bu), 11.4/514 (5-OH-Bu), and 8.8 microM (6'-OH-Bu). CYP3A inhibitor ketoconazole (1 microM) completely inhibited the formation of all major metabolites in HLMs (0-16% of control), whereas the chemical inhibitor selective to other P450 isoforms had little or no inhibitory effect. Recombinant CYP3A4, CYP3A5, and CYP2D6 exhibited buspirone oxidation activities among nine P450 isoforms tested. The overall metabolism rate of 5 microM buspirone by CYP3A4 was 18-fold greater than that by CYP2D6 and 35-fold greater than that by CYP3A5. In a panel of HLMs from 16 donors, buspirone metabolism correlated well CYP3A activity (r2 = 0.85-0.96, rho < 0.0005), but not the activities of other P450 isoforms. The metabolism rates of buspirone in CYP2D6 poor-metabolizer genotype HLMs were comparable to those in pooled HLMs. Taken together, these data suggest that CYP3A, mostly likely CYP3A4, is primarily responsible for the metabolism of buspirone in HLMs.

Antitumor activity of methoxymorpholinyl doxorubicin: potentiation by cytochrome P450 3A metabolism.

Methoxymorpholinyl doxorubicin (MMDX) is a novel liver cytochrome P450 (P450)-activated anticancer prodrug whose toxicity toward cultured tumor cells can be potentiated up to 100-fold by incubation with liver microsomes and NADPH. In the present study, a panel of human liver microsomes activated MMDX with potentiation ratios directly correlated to the CYP3A-dependent testosterone 6beta-hydroxylase activity of each liver sample. Microsome-activated MMDX exhibited nanomolar IC(50) values in growth-inhibition assays of human tumor cell lines representing multiple tissues of origin: lung (A549 cells), brain (U251 cells), colon (LS180 cells), and breast (MCF-7 cells). Analysis of individual cDNA-expressed CYP3A enzymes revealed that rat CYP3A1 and human CYP3A4 activated MMDX more efficiently than rat CYP3A2 and that human P450s 3A5 and 3A7 displayed little or no activity. MMDX cytotoxicity was substantially increased in Chinese hamster ovary cells after stable expression of CYP3A4 in combination with P450 reductase. CYP3A activation of MMDX abolished the parent drug's residual cross-resistance in a doxorubicin-resistant MCF-7 cell line that overexpresses P-glycoprotein. CYP3A-activated MMDX displayed a comparatively high intrinsic stability, with a t(1/2) of approximately 5.5 h at 37 degrees C. MMDX was rapidly activated by CYP3A at low ( approximately 1-5 nM) prodrug concentrations, with 100% tumor cell kill obtained after as short as a 2-h exposure to the activated metabolite. These findings demonstrate that MMDX can be activated by CYP3A metabolism to a potent, long-lived, and cell-permeable cytotoxic metabolite and suggest that this anthracycline prodrug may be used in combination with CYP3A4 in a P450 prodrug activation-based gene therapy for cancer treatment.

Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes.

The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent Km and Vmax values were 21.1 microM and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 microM and 412.0 pmol/min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (rs=0.829; P<0.01), CYP2B6-catalzyed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (rs=0.691; P<0.05), and CYP2C8-catalyzed paclitaxel 6alpha-hydroxylation (rs=0.797; P<0.05). CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.

Characterization of human cytochrome P450 enzymes catalyzing domperidone N-dealkylation and hydroxylation in vitro.

To confirm the identity of the major metabolites of domperidone and to characterize the cytochrome P450s (CYPs) involved in their formation. Human liver microsomes (HLMs) were used to characterize the kinetics of domperidone metabolism and liquid chromatography-mass spectrometry to identify the products. Isoform-specific chemical inhibitors, correlation analysis and expressed human CYP genes were used to identify the CYPs involved in domperidone oxidation. In HLMs, domperidone underwent hydroxylation to form 5-hydroxydomperidone (MIII) and N-dealkylation to form 2,3-dihydro-2-oxo-1H-benzimidazole-1-propionic acid (MI) and 5-chloro-4-piperidinyl-1,3-dihydro-benzimidazol-2-one (MII). The formation of all three metabolites (n = 4 HLMs) followed apparent Michaelis-Menten kinetics. The mean Km values for MI, MII and MIII formation were 12.4, 11.9, and 12.6 micro m, respectively. In a panel of HLMs (n = 10), the rate of domperidone (5 microm and 50 microm) metabolism correlated with the activity of CYP3A (r > 0.94; P < 0.0001). Only ketoconazole (1 microm) (by 87%) and troleandomycin (50 microm) (by 64%) inhibited domperidone (5 microm) metabolism in HLMs. Domperidone (5 and 50 microm) hydroxylation and N-dealkylation was catalyzed by expressed CYP3A4 at a higher rate than the other CYPs. CYP1A2, 2B6, 2C8 and 2D6 also hydroxylated domperidone CYP3A-catalyzed N-dealkylation and aromatic hydroxylation are the major routes for domperidone metabolism. The drug would be expected to demonstrate highly variable bioavailability due to hepatic, and possibly intestinal first-pass metabolism after oral administration. Increased risk of adverse effects might be anticipated during concomitant administration with CYP3A inhibitors, as well as decreased efficacy with inducers of this enzyme.

Role of CYP3A enzymes in fluticasone biotransformation

Fluticasone propionate (FLU) is a synthetic, trifluorinated glucocorticoid with potent anti-inflammatory action that is commonly used in patients with asthma. After oral or intranasal administration, FLU undergoes rapid hepatic inactivation to a single metabolite, a 17β-carboxylic acid derivative (M1). M1 formation has been attributed largely to cytochrome P450 (CYP) 3A4, however, there are no published data that confirm this assertion. Hence, in vitro studies were conducted to determine the role that human CYPs play in the metabolism of FLU. Consistent with in vivo data, human liver microsomes (HLM) catalyzed the formation of a single metabolite (M1) at low substrate concentrations (<1 μM; mean plasma Cmax = 1 nM). At FLU concentrations <1 μM, the kinetics of M1 formation in HLM were consistent with those of a single enzyme (Km ≅800 nM). M1 formation correlated significantly (r>0.95) with CYP3A4/5 activities in a panel of HLM (n=14) and was markedly impaired by the CYP3A inhibitor, ketoconazole, (>94%), but not by inhibitors of other CYPs (<10%). Studies with a panel of cDNA-expressed enzymes revealed that M1 formation was catalyzed solely by CYP3A enzymes at FLU concentrations <1 μM. M1 formation was catalyzed by CYPs 3A4, 3A5 and 3A7, however CYP3A5 was ~4-fold less active than CYP3A4 or 3A7. These results suggest that at pharmacologically relevant concentrations of FLU, biotransformation of FLU to M1 is mediated predominantly by CYPs 3A4 and 3A7. Clinical Pharmacology & Therapeutics (2004) 75, P84–P84; doi: 10.1016/j.clpt.2003.11.320

CYP2C8 and CYP3A4 are the principal enzymes involved in the human in vitro biotransformation of the insulin secretagogue repaglinide.

To identify the principal human cytochrome P450 (CYP) enzyme(s) responsible for the human in vitro biotransformation of repaglinide. Previous experiments have identified CYP3A4 as being mainly responsible for the in vitro metabolism of repaglinide, but the results of clinical investigations have suggested that more than one enzyme may be involved in repaglinide biotransformation. [14C]-Repaglinide was incubated with recombinant CYP and with human liver microsomes (HLM) from individual donors in the presence of inhibitory antibodies specific for individual CYP enzymes. Metabolites, measured by high-performance liquid chromatography (HPLC) with on-line radiochemical detection, were identified by liquid chromatography-mass spectrophotometry (LC-MS) and LC-MS coupled on-line to a nuclear magnetic resonance spectrometer (LC-MS-NMR). CYP3A4 and CYP2C8 were found to be responsible for the conversion of repaglinide into its two primary metabolites, M4 (resulting from hydroxylation on the piperidine ring system) and M1 (an aromatic amine). Specific inhibitory monoclonal antibodies against CYP3A4 and CYP2C8 significantly inhibited (> 71%) formation of M4 and M1 in HLM. In a panel of HLM from 12 individual donors formation of M4 and M1 varied from approximately 160-880 pmol min-1 mg-1 protein and from 100-1110 pmol min-1 mg-1 protein, respectively. The major metabolite generated by CYP2C8 was found to be M4. The rate of formation of this metabolite in HLM correlated significantly with paclitaxel 6alpha-hydroxylation (rs = 0.80; P = 0.0029). Two other minor metabolites were also detected. One of them was M1 and the other was repaglinide hydroxylated on the isopropyl moiety (M0-OH). The rate of formation of M4 in CYP2C8 Supersomes was 2.5 pmol min-1 pmol-1 CYP enzyme and only about 0.1 pmol min-1 pmol-1 CYP enzyme in CYP3A4 Supersomes. The major metabolite generated by CYP3A4 was M1. The rate of formation of this metabolite in HLM correlated significantly with testosterone 6beta-hydroxylation (rs = 0.90; P = 0.0002). Three other metabolites were identified, namely, M0-OH, M2 (a dicarboxylic acid formed by oxidative opening of the piperidine ring) and M5. The rate of M1 formation in CYP3A4 Supersomes was 1.6 pmol min-1 pmol-1 CYP enzyme but in CYP2C8 Supersomes it was only approximately 0.4 pmol min-1 pmol-1 CYP enzyme. The results confirm an important role for both CYP3A4 and CYP2C8 in the human in vitro biotransformation of repaglinide. This dual CYP biotransformation may have consequences for the clinical pharmacokinetics and drug-drug interactions involving repaglinide if one CYP pathway has sufficient capacity to compensate if the other is inhibited.

Pathways of carbamazepine bioactivation in vitro I. Characterization of human cytochromes P450 responsible for the formation of 2- and 3-hydroxylated metabolites.

In vitro studies were conducted to identify the cytochromes P450 (P450s) involved in the formation of 2- and 3-hydroxycarbamazepine, metabolites that may serve as precursors in the formation of protein-reactive metabolites. Human liver microsomes (HLMs) converted carbamazepine (30-300 microM) to 3-hydroxycarbamazepine at rates >25 times those of 2-hydroxycarbamazepine. Both the 2- and 3-hydroxylation of carbamazepine appeared to conform to monophasic Michaelis-Menten kinetics in HLMs (apparent K(m) values, approximately 1640 and approximately 217 microM; apparent V(max) values, approximately 5.71 and approximately 46.9 pmol/mg of protein/min, respectively). Rates of carbamazepine 2- and 3-hydroxylation correlated strongly with CYP2B6 activity (r >or= 0.757) in a panel of HLMs (n = 8). Carbamazepine 3-hydroxylation also correlated significantly with CYP2C8 activity at a carbamazepine concentration of 30 microM. Formation of 2- and 3-hydroxycarbamazepine did not correlate significantly with any other P450 activities. The chemical inhibitors ketoconazole (CYP3A) and 7-EFC (CYP2B6) inhibited both 2- and 3-hydroxycarbamazepine formation whereas 4-methylpyrazole (CYP2E1) markedly decreased 2-hydroxycarbamazepine formation. Several recombinant P450s catalyzed carbamazepine 2- and 3-hydroxylation, but after adjustment for relative hepatic abundance, CYP3A4 and CYP2B6 appeared to be the major catalysts of carbamazepine 3-hydroxylase activity, and at least five P450s were significant contributors to 2-hydroxycarbamazepine formation; CYP2E1 made the greatest contribution to the Cl(int) of carbamazepine 2-hydroxylation (approximately 30%), but P450s CYP1A2, 2A6, 2B6, and 3A4 also made significant contributions (approximately 13-18%). These results suggest that CYP2B6 and CYP3A4 are largely responsible for the formation of 3-hyrdoxycarbamazepine, whereas multiple P450s (CYP1A2, 2A6, 2B6, 2E1, and 3A4) contributed to 2-hydroxycarbamazepine formation.