Metabolic profiling of endocrine-disrupting compounds by on-line cytochrome p450 bioreaction coupled to on-line receptor affinity screening

We present a fully automated and hyphenated bioanalytical method for metabolic profiling of potentially harmful xenoestrogens. The system consists of an on-line cytochrome P450 bioreactor coupled to a reversed-phase, gradient high-performance liquid chromatograph. A C18 solid-phase extraction (SPE) unit is used as an interface between the P450 bioreactor and the HPLC column. The HPLC column is linked on-line to a high-resolution screening (HRS)-estrogen receptor alpha affinity detection (ERAD) assay. In effect, the P450 bioreactor produces metabolites that are subsequently trapped on-line by SPE and separated by HPLC. The separated metabolites are then screened on-line, at the moment of elution, for affinity toward estrogen receptor alpha (ERalpha) using the HRS-ERAD assay. The SPE method was optimized with methoxychlor (MXC) and its metabolites mono- and bis-OH-MXC. After optimization, the P450-bioreactor-SPE-HPLC system was made generally applicable to the biocatalysis and trapping of polar to highly apolar compounds. The precision of the P450-bioreactor-SPE-HPLC system is high (relative standard deviation相似文献   

On-line formation, separation, and estrogen receptor affinity screening of cytochrome P450-derived metabolites of selective estrogen receptor modulators.

We have developed a fully automated bioreactor coupled to an on-line receptor affinity detection system. This analytical system provides detailed information on pharmacologically active metabolites of selective estrogen receptor modulators (SERMs) generated by cytochromes P450 (P450s). We demonstrated this novel concept by investigating the metabolic activation of tamoxifen and raloxifene by P450-containing pig and rat liver microsomes. The high resolution screening (HRS) system is based on the coupling of a P450-bioreactor to an HPLC-based estrogen receptor alpha (ERalpha) affinity assay. P450-derived metabolites of the SERMs were generated in the bioreactor, subsequently trapped on-line with solid phase extraction, and finally separated with gradient HPLC. Upon elution, the metabolites were screened on affinity for ERalpha with an on-line HRS assay. With this HRS system, we were able to follow, in a time-dependent manner, the formation of ERalpha-binding metabolites of tamoxifen and raloxifene. By analyzing the bioaffinity chromatograms with liquid chromatography-tandem mass spectrometry, structural information of the pharmacologically active metabolites was obtained as well. For tamoxifen, 15 active and 6 nonactive metabolites were observed, of which 5 were of primary, 10 of secondary, and 6 of an as yet unknown order of metabolism. Raloxifene was biotransformed in three primary and three secondary metabolites. MS/MS analysis revealed that three of the observed active metabolites of raloxifene were not described before. The present automated on-line HRS system coupled to a P450-containing bioreactor and an ERalpha-affinity detector proved very efficient, sensitive, and selective in metabolic profiling of SERMs.     

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

AIMS: 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. METHODS: [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). RESULTS: 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. CONCLUSIONS: 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.     

The human intestinal cytochrome P450 "pie".

Cytochromes P450 (P450s) 3A, 2C, and 1A2 constitute the major "pieces" of the human liver P450 "pie" and account, on average, for 40, 25, and 18%, respectively, of total immunoquantified P450s (J Pharmacol Exp Ther 270:414-423, 1994). The P450 profile in the human small intestine has not been fully characterized. Therefore, microsomes prepared from mucosal scrapings from the duodenal/jejunal portion of 31 human donor small intestines were analyzed by Western blot using selective P450 antibodies. P450s 3A4, 2C9, 2C19, and 2J2 were detected in all individuals and ranged from 8.8 to 150, 2.9 to 27, <0.6 to 3.9, and <0.2 to 3.1 pmol/mg, respectively. CYP2D6 was detected in 29 individuals and ranged from <0.2 to 1.4 pmol/mg. CYP3A5 was detected readily in 11 individuals, with a range (average) of 4.9 to 25 (16) pmol/mg that represented from 3 to 50% of total CYP3A (CYP3A4 + CYP3A5) content. CYP1A1 was detected readily in three individuals, with a range (average) of 3.6 to 7.7 (5.6) pmol/mg. P450s 1A2, 2A6, 2B6, 2C8, and 2E1 were not or only faintly detected. As anticipated, average CYP3A content (50 pmol/mg) was the highest. Excluding CYP1A1, the remaining enzymes had the following rank order: 2C9 > 2C19 > 2J2 > 2D6 (8.4, 1.1, 0.9, and 0.5 pmol/mg, respectively). Analysis of a pooled preparation of the 31 donor specimens substantiated these results. In summary, as in the liver, large interindividual variation exists in the expression levels of individual P450s. On average, CYP3A and CYP2C9 represents the major pieces of the intestinal P450 pie, accounting for 80 and 15%, respectively, of total immunoquantified P450s.     

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 microM and 4.1 pmol min(-1) pmol(-1) P450, 44 microM and 10 pmol min(-1) pmol(-1) P450, and 34 microM 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.     

Metabolism of endosulfan-alpha by human liver microsomes and its utility as a simultaneous in vitro probe for CYP2B6 and CYP3A4.

Endosulfan-alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 microM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 microM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 microM, Vmax = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL(int) = 0.70 microl/min/pmol P450) than CYP3A4 (CL(int) = 0.09 microl/min/pmol P450). Using 16 individual human liver microsomes (HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r(2) = 0.79), whereas a moderate correlation with testosterone 6 beta-hydroxylase activity of CYP3A4 (r(2) = 0.54) was observed. Ticlopidine (5 microM), a potent CYP2B6 inhibitor, and ketoconazole (10 microM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha metabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan-alpha. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan-alpha is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities.     

Evaluation of Supermix as an in vitro model of human liver microsomal drug metabolism

SUPERMIX is a commercially available formulation of insect cell-expressed human drug-metabolizing cytochrome P450 (CYP) isoforms, mixed in proportions that are optimized to parallel their relative activities in human liver microsomes. We have evaluated the apparent functional affinity and capacity of individual CYP isoforms in SUPERMIX in comparison with microsomes from a panel of 12 human livers, using enzyme kinetic studies of isoform-selective index reactions. In addition, we have measured the concentration of NADPH cytochrome P450 oxidoreductase (OR) in SUPERMIX and compared it with the concentrations of this accessory electron transfer protein in human liver microsomes. No important differences were evident in the catalytic activities of CYPs 1A2, 2C8, 2C9, 2C19, 2D6 and 3A4 between SUPERMIX and human liver microsomes. However, SUPERMIX lacks CYP2B6 activity and did not hydroxylate the antidepressant bupropion, a clinically relevant substrate of this enzyme. In addition, the concentration of OR in SUPERMIX (1198 pmol mg protein(-1)) is 17-fold higher than the mean value in human liver microsomes (70 pmol mg protein(-1)). In conclusion, SUPERMIX lacks CYP2B6 activity and contains supraphysiological concentrations of the accessory electron transfer protein OR. These factors should be considered when this formulation is used as an in vitro model in human liver microsomal drug metabolism studies.     

CYP1A1 and CYP1B1, two hydrocarbon-inducible cytochromes P450, are constitutively expressed in neonate and adult goat liver, lung and kidney.

The ontogeny of cytochrome P-450 isozymes (P450) in goat liver, lung and kidney was studied using anion exchange HPLC separation of solublized microsomal proteins and Western immunoblotting. Comparison of the overall HPLC profile of goat P450 isozymes between liver, lung and kidney showed that while the P450's of goat liver were equally separated into five peaks of isozyme(s), only two peaks constitute the majority of P450 isozyme(s) in lung and kidney, thus demonstrating the tissue specific differences in P450 isozyme distribution in goats. Immunoblotting analysis using polyclonal antibodies against rat CYP1B1, and mouse CYP1B1, polyaromatic hydrocarbon-regulated P450's, revealed that goat orthologs of CYP1A1 and CYP1B1 are expressed constitutively in goats. The CYP1A1 was expressed in goat liver and lung as early as 1st day of age, and the levels of its expression in adult lung and liver were, respectively, 1.3 and 5.5 pmol per mg microsomal proteins. CYP1B1 was expressed in goat livers in substantial levels as of 1 week of age and increased thereafter to reach approximately 4.5 pmol per mg microsomal proteins in adult livers, while low level was detectable only in adult but not neonate lung tissues. Furthermore, polyclonal antibodies against rat CYP1A2 detected very high levels of CYP1A2 in livers of adult and 6 week old goats. The Ah receptor which controls the expression of CYP1A1/1A2 and CYP1B1, was detected in cytosolic fractions from these tissues as a 104 kDa and a minor level of the 106 kDa form. These are potentially very important findings in light of the role of CYP1A1/1A2 and CYP1B1 in activation of polyaromatic hydrocarbons, heterocyclic amines and nitroaromatic hydrocarbons to genotoxic metabolites, and the health consequences of these metabolites on humans, as consumers of goat milk and meat. Using polyclonal antibodies against rat hepatic CYP2B1 and CYP3A1, the goat CYP2B and CYP3A forms were not detectable in livers of goats at any age, but lungs of adult and 6 week old goats expressed these two CYPs in levels equivalent to the livers of phenobarbital-induced rats. On the other hand, anti-rat CYP2C6 antibodies specifically detected two goat ortholog forms which were expressed in all three tissues and exhibited age-dependent changes. In conclusion, results from both immunoblot and HPLC analyses confirmed that, as in other species, the expression of P450 isozymes in goat is under both developmental- and tissue-specific regulatory factors.     

Characterization of human cytochrome p450 enzymes involved in the metabolism of cilostazol.

Cilostazol (OPC-13013; 6-[4-(1-cyclohexl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone) is widely used as an antiplatelet vasodilator agent. In vitro, the hydroxylation of the quinone moiety of cilostazol to OPC-13326 [6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-4-hydroxy-2(1H)-quinolinone], is the predominant route, and the hydroxylation of the hexane moiety to OPC-13217 is the second most predominant route. This study was carried out to identify and kinetically characterize the human cytochrome P450 (P450) isozymes responsible for the formation of the two major metabolites of cilostazol, namely, OPC-13326 and OPC-13217 [3,4-dihydro-6-[4-[1-(cis-4-hydroxycyclohexyl)-1H-tetrazol-5-yl)butoxy]-2(1H)-quinolinone)]. In in vitro studies using 14 recombinant human P450 isozymes, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11, cilostazol was metabolized to OPC-13326 mainly by CYP3A4 (K(m) = 5.26 muM, intrinsic clearance (CL(int)) = 0.34 microl/pmol P450/min), CYP1B1 (K(m) = 11.2 microM, CL(int) = 0.03 microl/pmol P450/min), and CYP3A5 (K(m) = 2.89 microM, CL(int) = 0.05 microl/pmol P450/min) and to OPC-13217 mainly by CYP3A5 (K(m) = 1.60 microM, CL(int) = 0.57 microl/pmol P450/min), CYP2C19 (K(m) = 5.95 microM, CL(int) = 0.16 microl/pmol P450/min), CYP3A4 (K(m) = 5.35 microM, CL(int) = 0.10 microl/pmol P450/min), and CYP2C8 (K(m) = 33.8 microM, CL(int) = 0.009 microl/pmol P450/min). The present study showed that the two major metabolites of cilostazol in vitro, namely, OPC-13326 and OPC-13217, are mainly catalyzed by CYP3A4 and CYP3A5, respectively.     

Interaction of cisapride with the human cytochrome P450 system: metabolism and inhibition studies.

Using human liver microsomes (HLMs) and recombinant cytochrome P450s (CYP450s), we characterized the CYP450 isoforms involved in the primary metabolic pathways of cisapride and documented the ability of cisapride to inhibit the CYP450 system. In HLMs, cisapride was N-dealkylated to norcisapride (NORCIS) and hydroxylated to 3-fluoro-4-hydroxycisapride (3-F-4-OHCIS) and to 4-fluoro-2-hydroxycisapride (4-F-2-OHCIS). Formation of NORCIS, 3-F-4-OHCIS, and 4-F-2-OHCIS in HLMs exhibited Michaelis-Menten kinetics (K(m): 23.4 +/- 8.6, 32 +/- 11, and 31 +/- 23 microM; V(max): 155 +/- 91, 52 +/- 23, and 31 +/- 23 pmol/min/mg of protein, respectively). The average in vitro intrinsic clearance (V(max)/K(m)) revealed that the formation of NORCIS was 3.9- to 5. 9-fold higher than that of the two hydroxylated metabolites. Formation rate of NORCIS from 10 microM cisapride in 14 HLMs was highly variable (range, 4.9-133.6 pmol/min/mg of protein) and significantly correlated with the activities of CYP3A (r = 0.86, P =. 0001), CYP2C19, and 1A2. Of isoform-specific inhibitors, 1 microM ketoconazole and 50 microM troleandomycin were potent inhibitors of NORCIS formation from 10 microM cisapride (by 51 +/- 9 and 44 +/- 17%, respectively), whereas the effect of other inhibitors was minimal. Of 10 recombinant human CYP450s tested, CYP3A4 formed NORCIS from 10 microM cisapride at the highest rate (V = 0.56 +/- 0. 13 pmol/min/pmol of P450) followed by CYP2C8 (V = 0.29 +/- 0.08 pmol/min/pmol of P450) and CYP2B6 (0.15 +/- 0.04 pmol/min/pmol of P450). The formation of 3-F-4-OHCIS was mainly catalyzed by CYP2C8 (V = 0.71 +/- 0.24 pmol/min/pmol of P450) and that of 4-F-2-OHCIS by CYP3A4 (0.16 +/- 0.03 pmol/min/pmol of P450). Clearly, recombinant CYP2C8 participates in cisapride metabolism, but when the in vitro intrinsic clearances obtained were corrected for abundance of each CYP450 in the liver, CYP3A4 is the dominant isoform. Cisapride was a relatively potent inhibitor of CYP2D6, with no significant effect on other isoforms tested, but the K(i) value derived (14 +/- 16 microM) was much higher than the clinically expected concentration of cisapride (<1 microM). Our data suggest that CYP3A is the main isoform involved in the overall metabolic clearance of cisapride. Cisapride metabolism is likely to be subject to interindividual variability in CYP3A expression and to drug interactions involving this isoform.     

CYP1A1 and CYP1B1, Two Hydrocarbon-Inducible Cytochromes P450, are Constitutively Expressed in Neonate and Adult Goat Liver,Lung and Kidney *

Abstract: The ontogeny of cytochrome P-450 isozymes (P450) in goat liver, lung and kidney was studied using anion exchange HPLC separation of solublized microsomal proteins and Western immunoblotting. Comparison of the overall HPLC profile of goat P450 isozymes between liver, lung and kidney showed that while the P450's of goat liver were equally separated into five peaks of isozyme(s), only two peaks constitute the majority of P450 isozyme(s) in lung and kidney, thus demonstrating the tissue specific differences in P450 isozyme distribution in goats. Immunoblotting analysis using polyclonal antibodies against rat CYP1A1, and mouse CYP1B1, polyaromatic hydrocarbon-regulated P450's, revealed that goat orthologs of CYP1A1 and CYP1B1 are expressed constitutively in goats. The CYP1A1 was expressed in goat liver and lung as early as 1st day of age, and the levels of its expression in adult lung and liver were, respectively, 1.3 and 5.5 pmol per mg microsomal proteins. CYP1B1 was expressed in goat livers in substantial levels as of 1 week of age and increased thereafter to reach approximately 4.5 pmol per mg microsomal proteins in adult livers, while low level was detectable only in adult but not neonate lung tissues. Furthermore, polyclonal antibodies against rat CYP1A2 detected very high levels of CYP1A2 in livers of adult and 6 week old goats. The Ah receptor which controls the expression of CYP1A1/1A2 and CYP1B1, was detected in cytosolic fractions from these tissues as a 104 kDa and a minor level of the 106 kDa form. These are potentially very important findings in light of the role of CYP1A1/1A2 and CYP1B1 in activation of polyaromatic hydrocarbons, heterocyclic amines and nitroaromatic hydrocarbons to genotoxic metabolites, and the health consequences of these metabolites on humans, as consumers of goat milk and meat. Using polyclonal antibodies against rat hepatic CYP2B1 and CYP3A1, the goat CYP2B and CYP3A forms were not detectable in livers of goats at any age, but lungs of adult and 6 week old goats expressed these two CYPs in levels equivalent to the livers of phenobarbital-induced rats. On the other hand, anti-rat CYP2C6 antibodies specifically detected two goat ortholog forms which were expressed in all three tissues and exhibited age-dependent changes. In conclusion, results from both immunoblot and HPLC analyses confirmed that, as in other species, the expression of P450 isozymes in goat is under both developmental-and tissue-specific regulatory factors.     

Characterization of human cytochrome P450 enzymes involved in the in vitro metabolism of perospirone

In vitro studies were carried out to identify the major contribution of CYP2C8, CYP2D6 and CYP3A4 to the metabolism of perospirone (cis-N-[4-[4-(1,2-benzisothiazol-3-yl)-1-piperazinyl]butyl]cyclohexane-1,2-dicarboximide monohydrochloride dehydrate), a novel antipsychotic agent, using human liver microsomes and expressed P450 isoforms. Quinidine (a specific inhibitor of CYP2D6) did not markedly affect the metabolism of perospirone, whereas quercetin (an inhibitor of CYP2C8) and ketoconazole (an inhibitor of CYP3A4) caused a decrease in the metabolism with human liver microsomes in a concentration dependent fashion. With 10 microM quercetin, the metabolism of perospirone was inhibited by 60.0% and with 1 microM ketoconazole almost complete inhibition was apparent. Anti-CYP2C8 and anti-CYP2D6 antisera did not exert marked effects, whereas anti-CYP3A4 antiserum caused almost complete inhibition. With expressed P450s, K(m) and V(max) values were 1.09 microM and 1.93 pmol/min/pmol P450 for CYP2C8, 1.38 microM and 5.73 pmol/min/pmol P450 for CYP2D6, and 0.245 microM and 61.3 pmol/min/pmol P450 for CYP3A4, respectively. These results indicated that the metabolism of perospirone in human liver was mainly catalysed by CYP3A4, and to a lesser extent CYP2C8 and CYP2D6 were responsible because kinetic data (K(m) and V(max)) of CYP2C8 and CYP2D6 suggested catalytic potential.     

Diazinon is activated by CYP2C19 in human liver.

Phosphorothioate compounds are used throughout the world as agricultural and domestic pesticides. Here, the activation of the phosphorothioate diazinon to diazoxon in human liver is described. In an initial study using three human liver microsomal samples, K(m) for diazoxon formation varied markedly (31, 208, and 660 microM; V(max) 1125, 685, and 1028 pmol/min/mg protein, respectively), suggesting the involvement of more than one P450 enzyme. A wide variation in activity was found using 50 microM diazinon as substrate, (11-648 pmol/min/mg protein, n = 15), whereas, with 500 microM, variation was less (164-978 pmol/min/mg protein). Among eight P450-catalyzed reactions, the putative high-affinity component (50 microM diazinon) correlated with S-mephenytoin 4'-hydroxylase activity (r = 0.686, p < 0.01), suggesting the involvement of CYP2C19. The putative low-affinity component (500 microM diazinon) correlated with both S-mephenytoin 4'-hydroxylase (r = 0.714; p < 0.005) and high-affinity phenacetin O-deethylase activity (r = 0.625; p < 0.05). This activity was partially inhibited by furafylline, troleandomycin, and ketoconazole. These data suggest contributions from CYP2C19, CYP1A2, and CYP3A4. None of the inhibitors affected the high-affinity component. Of seven heterologously expressed human P450 enzymes, CYP2C19 activated diazinon (500 microM) at the fastest rate, followed by CYP3A4, CYP1A2, and CYP2C9. Both hepatic microsomal S-mephenytoin 4'-hydroxylase and high-affinity phenacetin O-deethylase activities were strongly inhibited by diazinon (IC50 < 2.5 microM), while no effect was seen on midazolam 1'-hydroxylase activity. These data indicate that CYP2C19 is the major enzyme involved in diazinon activation in human liver, while other enzymes including CYP1A2 may play a more minor role.     

Characterization of benidipine and its enantiomers' metabolism by human liver cytochrome P450 enzymes.

Benidipine is a dihydropyridine calcium antagonist that has been used clinically as an antihypertensive and antianginal agent. It is used clinically as a racemate, containing the (-)-alpha and (+)-alpha isomers of benidipine. This study was performed to elucidate the metabolism of benidipine and its enantiomers in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of benidipine. Human liver microsomal incubation of benidipine in the presence of NADPH resulted in the formation of two metabolites, N-desbenzylbenidipine and dehydrobenidipine. The intrinsic clearance (CL(int)) of the formation of N-desbenzylbenidipine and dehydrobenidipine metabolites from (-)-alpha isomer was similar to those from the (+)-alpha isomer (1.9 +/- 0.1 versus 2.3 +/- 2.3 microl/min/pmol P450 and 0.5 +/- 0.2 versus 0.6 +/- 0.6 microl/min/pmol P450, respectively). Correlation analysis between the known P450 enzyme activities and the rate of the formation of benidipine metabolites in the 15 HLMs showed that benidipine metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in liver microsomes and the incubation study of cDNA-expressed enzymes also showed that theN-debenzylation and dehydrogenation of benidipine are mainly mediated by CYP3A4 and CYP3A5. The total CL(int) values of CYP3A4-mediated metabolite formation from (-)-alpha isomer were similar to those from (+)-alpha isomer (17.7 versus 14.4 microl/min/pmol P450, respectively). The total CL(int) values of CYP3A5-mediated metabolite formation from (-)-alpha isomer were also similar to those from (+)-alpha isomer (8.3 versus 11.0 microl/min/pmol P450, respectively). These findings suggest that CYP3A4 and CYP3A5 isoforms are major enzymes contributing to the disposition of benidipine, but stereoselective disposition of benidipine in vivo may be influenced not by stereoselective metabolism but by other factors.     

CYP2A6 AND CYP2B6 are involved in nornicotine formation from nicotine in humans: interindividual differences in these contributions.

Nornicotine is an N-demethylated metabolite of nicotine. In the present study, human cytochrome P450 (P450) isoform(s) involved in nicotine N-demethylation were identified. The Eadie-Hofstee plot of nicotine N-demethylation in human liver microsomes was biphasic with high-affinity (apparent K(m) = 173 +/- 70 microM, V(max) = 57 +/- 17 pmol/min/mg) and low-affinity (apparent K(m) = 619 +/- 68 microM, V(max) = 137 +/- 6 pmol/min/mg) components. Among 13 recombinant human P450s expressed in baculovirus-infected insect cells (Supersomes), CYP2B6 exhibited the highest nicotine N-demethylase activity, followed by CYP2A6. The apparent K(m) values of CYP2A6 (49 +/- 12 microM) and CYP2B6 (550 +/- 46 microM) were close to those of high- and low-affinity components in human liver microsomes, respectively. The intrinsic clearances of CYP2A6 and CYP2B6 Supersomes were 5.1 and 12.5 nl/min/pmol P450, respectively. In addition, the intrinsic clearance of CYP2A13 expressed in Escherichia coli (44.9 nl/min/pmol P450) was higher than that of CYP2A6 expressed in E. coli (2.6 nl/min/pmol P450). Since CYP2A13 is hardly expressed in human livers, the contribution of CYP2A13 to the nicotine N-demethylation in human liver microsomes would be negligible. The nicotine N-demethylase activity in microsomes from 15 human livers at 20 microM nicotine was significantly correlated with the CYP2A6 contents (r = 0.578, p < 0.05), coumarin 7-hydroxylase activity (r = 0.802, p < 0.001), and S-mephenytoin N-demethylase activity (r = 0.694, p < 0.005). The nicotine N-demethylase activity at 100 microM nicotine was significantly correlated with the CYP2B6 contents (r = 0.677, p < 0.05) and S-mephenytoin N-demethylase activities (r = 0.740, p < 0.005). These results as well as the inhibition analyses suggested that CYP2A6 and CYP2B6 would significantly contribute to the nicotine N-demethylation at low and high substrate concentrations, respectively. The contributions of CYP2A6 and CYP2B6 would be dependent on the expression levels of these isoforms in any human liver.     

Human hepatic cytochrome p450-specific metabolism of parathion and chlorpyrifos.

Organophosphorus pesticides (OPs) remain a potential concern to human health because of their continuing worldwide use. Thiophosphorus OPs, once bioactivated by cytochromes P450 (P450s), form oxon metabolites, which are potent acetylcholinesterase inhibitors. This study investigated the rate of desulfation (activation) and dearylation (detoxification) of parathion and chlorpyrifos in human liver microsomes. In addition, recombinant human P450s were used to quantify, for the first time, the P450-specific kinetic variables (K(m) and V(max)) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. CYP1A2, 2B6, 2C9, 2C19, 3A4, 3A5, and 3A7 were found to be active to a widely varying degree in parathion metabolism, whereas all, with the exception of CYP2C9, were also found to be active in chlorpyrifos metabolism. CYP2B6 and CYP2C19 demonstrated low K(m) and high V(max) values for the metabolism of both model compounds, which supports their role as the primary enzymes that regulate metabolism at low-level human exposures to OPs. With K(m) and V(max) values of 0.61 microM, 4827 pmol/min/nmol P450 and 0.81 microM, 12,544 pmol/min/nmol for formation of paraoxon and chlorpyrifos-oxon, respectively, CYP2B6 favored the desulfation reaction. CYP2C19 activity favored dearylation with K(m) and V(max) values of 0.60 microM, 2338 pmol/min/nmol P450 and 1.63 microM, 13,128 pmol/min/nmol for formation of p-nitrophenol and 3,4,5-tricholorpyrindinol, respectively. P450-specific kinetic parameters for OP metabolism will be used with age-dependent hepatic P450 content to enhance PBPK/PD models so that OP exposures can be modeled to protect human health in different age groups.     

Disparity in holoprotein/apoprotein ratios of different standards used for immunoquantification of hepatic cytochrome P450 enzymes.

An analysis of reported hepatic abundances of CYP3A4 and 3A5 indicated that values determined by immunoquantification using commercially available, unpurified recombinant enzymes as standards are significantly lower than those determined using purified enzymes or human liver microsomes characterized with lysosomal peptides (CYP3A4: mean 45 versus 121 pmol/mg protein, p < 0.01; CYP3A5: mean 28 versus 83 pmol/mg protein, p < 0.05). When immunoquantifying cytochromes P450 (P450s), it is assumed that the holoprotein (holo)/apoprotein ratio is the same in the samples and the standard. Estimates of holo/apoprotein ratios from data reported for a range of P450s purified from human liver and non-commercial recombinant systems indicated less than complete and variable heme coupling dependent on enzyme and system.     

Kinetics and regulation of cytochrome P450-mediated etoposide metabolism.

Etoposide is a DNA topoisomerase II inhibitor widely used in the treatment of a variety of malignancies that is also associated with therapy-related leukemia. The cytochrome P450 (P450)-derived catechol and quinone metabolites of etoposide may be important in the damage to the MLL (mixed lineage leukemia) gene and other genes resulting in leukemia-associated chromosomal translocations. Kinetic analysis of catechol formation by recombinant P450s was determined using liquid chromatography/selected reaction monitoring/mass spectrometry. CYP3A4 was found to play a major role in etoposide metabolism (K(m) = 77.7 +/- 27.8 microM; V(max) = 314 +/- 84 pmol of catechol/min/nmol of P450). However, CYP3A5 (K(m) = 13. 9 +/- 3.1 microM; V(max) = 19.4 +/- 0.4 pmol of catechol/min/nmol of P450) may be involved in etoposide metabolism at therapeutic concentrations of free drug. Other P450s do not appear to be involved in etoposide catechol formation. Real-time polymerase chain reaction and Western blot analysis revealed significantly increased CYP3A4 mRNA and protein levels in hepatocytes treated with 10 microM rifampicin compared with untreated cells, but only modest effects of rifampicin on CYP3A5 induction. Etoposide (40, 5, 1, and 0.25 microM) caused a slight increase in CYP3A4 mRNA in three of five batches of hepatocytes but did not result in proportionately increased CYP3A4 protein levels. At high concentrations, etoposide induced only a modest increase in CYP3A5 mRNA and protein levels in four of five batches of hepatocytes. Alternatively, coadministration of other drugs with etoposide may account for the increase in etoposide catechol formation during therapy with etoposide.     

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: formation of the 4-hydroxy, 4'-hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen.

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 microM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 microM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 microM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 microM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.