CYP1A2 is the major isoform responsible for paeonol O-demethylation in human liver microsomes

1. Paeonol, the primary active component of a traditional Chinese medicine Moutan Cortex, has a wide range of pharmacological activities. In the present study, the metabolism of paeonol by cytochrome P450s (CYPs) was investigated in human liver microsomes.

2. One O-demethylated metabolite was detected in reaction catalysed by human liver microsomes, and was identified as resacetophenone by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound.

3. The study with a chemical selective inhibitor, cDNA-expressed human CYPs, a correlation assay, and a kinetics study demonstrated that CYP1A2 was the major isoform responsible for the paeonol O-demethylation in human liver microsomes.

     

UDP-glucuronosyltransferase 1A1 is the principal enzyme responsible for puerarin metabolism in human liver microsomes

Puerarin has multiple pharmacological effects and is widely prescribed for patients with cardiovascular diseases, including hypertension, cerebral ischemia, myocardial ischemia, diabetes mellitus, and arteriosclerosis. While puerarin is a useful therapeutic agent, its mechanisms of action have not been well defined. Understanding puerarin metabolism, in particular its interactions with metabolizing enzymes, will contribute to our understanding of its toxic and therapeutic effects and may help to elucidate potential negative drug–drug interactions. In this study, the major metabolite of puerarin was obtained from the urine of rats administered puerarin, by a semi-preparative high-performance liquid chromatography method. The major metabolite was identified as puerarin-7-O-glucuronide. In vitro, we used a UDP-glucuronosyltransferase (UGT) reaction screening method with 12 recombinant human UGTs to demonstrate that formation of puerarin-7-O-glucuronide was catalyzed by UGT1A1, 1A9, 1A10, 1A3, 1A6, 1A7, and 1A8. UGT1A1, 1A9, and 1A10 significantly catalyzed puerarin-7-O-glucuronide formation, and the activity of UGT1A1 was significantly higher than those of 1A9 and 1A10. The V _max of UGT1A1 was two- to threefold higher than the levels of UGT1A9 or 1A10, with a lower K _m value and a higher V _max/K _m value. The kinetics of puerarin-7-O-glucuronide formation catalyzed by UGT1A1 were similar to those of the pooled human liver microsomes (HLMs), with V _max values of 186.3 and 149.2?pmol/min/mg protein, and K _m values of 811.3 and 838.9?μM, respectively. Furthermore, bilirubin and β-estradiol, probe substrates for UGT1A1, significantly inhibited the formation of puerarin-7-O-glucuronide in HLMs.     

Udp-glucuronosyltransferase 2b7 is the major enzyme responsible for gemcabene glucuronidation in human liver microsomes.

The predominant metabolic pathway of gemcabene in humans is glucuronidation. The principal human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of gemcabene were determined in this study. Glucuronidation of gemcabene was catalyzed by recombinant UGT1A3, recombinant UGT2B7, and recombinant UGT2B17, as well as by human liver microsomes (HLM). Gemcabene glucuronidation in recombinant UGTs and HLM followed non-Michaelis-Menten kinetics consistent with homotropic activation, but pharmacokinetics in humans were linear over the dose range tested (total plasma C(max), 0.06-0.88 mM). Gemcabene showed similar affinity (S(50)) for recombinant UGTs (0.92-1.45 mM) and HLM (1.37 mM). S-Flurbiprofen was identified as a more selective inhibitor of recombinant UGT2B7-catalyzed gemcabene glucuronidation (>23-fold lower IC(50)) when compared with recombinant UGT1A3- or recombinant UGT2B17-catalyzed gemcabene glucuronidation. The IC(50) for S-flurbiprofen inhibition of gemcabene glucuronidation was similar in HLM (60.6 microM) compared with recombinant UGT2B7 (27.4 microM), consistent with a major role for UGT2B7 in gemcabene glucuronidation in HLM. In addition, 5,6,7,3',4',5'-hexamethoxyflavone inhibited recombinant UGT1A3 and recombinant UGT2B17-catalyzed gemcabene glucuronidation (with 4-fold greater potency for recombinant UGT1A3) but did not inhibit gemcabene glucuronidation in HLM, suggesting that UGT1A3 and UGT2B17 do not contribute significantly to gemcabene glucuronidation. Reaction rates for gemcabene glucuronidation from a human liver bank correlated well (r(2)=0.722, P<0.0001; n=24) with rates of glucuronidation of the UGT2B7 probe substrate 3'-azido-3'-deoxythymidine. In conclusion, using the three independent experimental approaches typically used for cytochrome P450 reaction phenotyping, UGT2B7 is the major enzyme contributing to gemcabene glucuronidation in human liver microsomes.     

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

Jatrorrhizine, one of the protoberberine alkaloids derived from the plant Coptis chinensis, is expected to be developed as a new gastric prokinetic drug, but its metabolic characteristics in humans remain unknown. This study characterized the phase I and phase II metabolites, metabolic kinetics, and cytochrome P450 (CYP) and UDP‐glucuronosyltransferase (UGT) enzymes responsible for the metabolism of jatrorrhizine in human liver microsomes (HLMs). Chemical inhibition in HLMs and metabolism by recombinant human CYP or UGT enzymes were employed to determine the key metabolic enzyme subtypes. In HLMs, demethyleneberberine (demethylated product) and jatrorrhizine glucuronide were identified as the phase I and phase II metabolites, respectively. The enzyme kinetics for both demethylation and glucuronidation were fitted to the Michaelis–Menten equation. Demethylation was inhibited significantly by furafylline and predominantly catalysed by recombinant CYP1A2, whereas glucuronidation was inhibited by silibinin, quercetin, as well as 1‐naphthol and catalysed by recombinant UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9 and UGT1A10. These results showed that jatrorrhizine is metabolized by human CYP1A2 and multiple UGT1A isoforms. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification of cytochrome P4503A4 as the major enzyme responsible for the metabolism of ivermectin by human liver microsomes

1. Ivermectin was extensively metabolized by human liver microsomes to at least 10 metabolites. The structure of many of them (mostly hydroxylated and demethylated) was determined by ¹H-NMR and LC MS. 2. To determine which human cytochrome P450 isoform(s) is responsible for the metabolism of ivermectin, chemical inhibitors including sulphaphenazole, quinidine, furafylline, troleandomycin (TAO) and diethyldithiocarbamate (DDC) were used to evaluate their effect on ivermectin metabolism. TAO, a specific inhibitor of cytochrome P4503A4, was the most potent inhibitor, inhibiting the total metabolism as well as formation of each metabolite. Metabolismwas also inhibited byananti-humancytochrome 3A4 antibody by 90%. 3. When ivermectin was incubated with microsomes from cells expressing CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 or 3A4 at 4?mg/ml protein concentrations, metabolic activity was only detected with the microsomes containing CYP3A4. The metabolic profile from cDNA-expressed CYP3A4 microsomes was qualitatively similar to that from human liver microsomes. 4. Thus, cytochrome P4503A4 is the predominant isoform responsible for the metabolism of ivermectin by human liver microsomes.     

Identification of CYP3A4 as the predominant isoform responsible for the metabolism of ambroxol in human liver microsomes

1. In humans, ambroxol is metabolized to dibromoanthranilic acid (DBAA) and 6,8-dibromo-3-(trans-4-hydroxycyclohexyl)-1,2,3,4-tetrahydroquinazoli ne (DHTQ). The formation of DHTQ proceeds non-enzymatically, whereas that of DBAA requires NADPH. Studies have been performed to identify the CYP isozyme(s) involved in the formation of DBAA using human liver microsomes and microsomes expressing recombinant human CYP isozymes (1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 4A11). 2. The apparent Vmax and Km for the formation of DBAA were 472+/-192 pmol/ min/mg protein and 248+/-40.6 microM respectively (mean +/- S.D., n = 3). 3. Of the recombinant CYP examined, only CYP3A4 metabolized ambroxol to DBAA. The apparent Vmax and Km were 1.42 pmol/min/pmol P450 and 287 microM respectively. 4. Among the CYP inhibitors examined (furafylline, sulphaphenazole, quinidine, diethyldithiocarbamic acid, ketoconazole), only ketoconazole inhibited the production of DBAA (> 80%) at 1 microM and anti-CYP3A antiserum almost completely inhibited the formation of DBAA. 5. These results suggest that CYP3A4 is predominantly involved in the metabolism of ambroxol to DBAA in humans.     

Identification of CYP3A4 as the predominant isoform responsible for the metabolism of ambroxol in human liver microsomes

1. In humans, ambroxol is metabolized to dibromoanthranilic acid (DBAA) and 6,8- dibromo-3-(trans-4-hydroxycyclohexyl)-1,2,3,4-tetrahydroquinazoline (DHTQ). The formation of DHTQ proceeds non-enzymatically, whereas that of DBAA requires NADPH. Studies have been performed to identify the CYP isozyme(s) involved in the formation of DBAA using human liver microsomes and microsomes expressing recombinant human CYP isozymes (1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 4A11). 2. The apparent V_max and K_m for the formation of DBAA were 472 +/- 192 pmol/min/mg protein and 248 +/- 40.6 muM respectively (mean +/- S.D., n = 3). 3. Of the recombinant CYP examined, only CYP3A4 metabolized ambroxol to DBAA. The apparent V_max and K_m were 1.42 pmol/min/pmol P450 and 287 muM respectively. 4. Among the CYP inhibitors examined (furafylline, sulphaphenazole, quinidine, diethyldithiocarbamic acid, ketoconazole), only ketoconazole inhibited the production of DBAA (&gt;80%) at 1 muM and anti-CYP3A antiserum almost completely inhibited the formation of DBAA. 5. These results suggest that CYP3A4 is predominantly involved in the metabolism of ambroxol to DBAA in humans.     

Identification of enzymes responsible for rifalazil metabolism in human liver microsomes

1. The major metabolites of rifalazil in human are 25-deacetyl-rifalazil and 32-hydroxy-rifalazil. Biotransformation to these metabolites in pooled human liver microsomes, cytosol and supernatant 9000g (S9) fractions was studied, and the enzymes responsible for rifalazil metabolism were identified using inhibitors of esterases and cytochromes P450 (CYP). 2. The 25-deacetylation and 32-hydroxylation of rifalazil occurred in incubations with microsomes or S9 but not with cytosol, indicating that both the enzymes responsible for rifalazil metabolism were microsomal. Km and Vmax of the rifalazil-25-deacetylation in microsomes were 6.5 microM and 11.9 pmol/min/mg with NADPH, and 2.6 microM and 6.0 pmol/min/mg without NADPH, indicating that, although rifalazil-25-deacetylation did not require NADPH, NADPH activated it. Rifalazil-32-hydroxylation was NADPH dependent, and its Km and Vmax were 3.3 microM and 11.0 pmol/min/mg respectively. 3. Rifalazil-25-deacetylation in microsomes was completely inhibited by diisopropyl fluorophosphate, diethyl p-nitrophenyl phosphate and eserine, but not by p-chloromercuribenzoate or 5,5'-dithio-bis(2-nitrobenzoic acid), indicating that the enzyme responsible for the rifalazil-25-deacetylation is a B-esterase. 4. Rifalazil-32-hydroxylation in microsomes was completely inhibited by CYP3A4-specific inhibitors (fluconazole, ketoconazole, miconazole, troleandomycin) and drugs metabolized by CYP3A4 such as cyclosporin A and clarithromycin, indicating that the enzyme responsible for the rifalazil-32-hydroxylation is CYP3A4.     

Identification of enzymes responsible for rifalazil metabolism in human liver microsomes

1. The major metabolites of rifalazil in human are 25-deacetyl-rifalazil and 32- hydroxy-rifalazil. Biotransformation to these metabolites in pooled human liver microsomes, cytosol and supernatant 9000g (S9) fractions was studied, and the enzymes responsible for rifalazil metabolism were identified using inhibitors of esterases and cytochromes P450 (CYP). 2. The 25-deacetylation and 32-hydroxylation of rifalazil occurred in incubations with microsomes or S9 but not with cytosol, indicating that both the enzymes responsible for rifalazil metabolism were microsomal. K_m and V_max     

Identification of CYP3A4 and CYP2C8 as the major cytochrome P450 s responsible for morphine N-demethylation in human liver microsomes

1. The aim was to identify the cytochrome P450 (CYP) enzymes responsible for the N-demethylation of morphine in vitro. 2. In human liver microsomes, normorphine formation followed Michaelis-Menten kinetics with mean Km and Vmax of 12.4 +/- 2.2 mM and 1546 +/- 121 pmol min(-1) mg(-1), respectively. In microsomes from a panel of 14 human livers phenotyped for 10 CYP enzymes, morphine N-demethylation correlated with testosterone 6beta-hydroxylation (r=0.91, p<0.001) and paclitaxel 6-alpha hydroxylation (r=0.72, p<0.001), two specific markers of CYP3A4 and CYP2C8, respectively. Normorphine formation decreased when incubated in the presence of troleandomycin or quercetin (by 46 and 33-36%, respectively), which further corroborates the contribution of CYP3A4 and CYP2C8. 3. Among eight recombinant human CYP enzymes tested, CYP3A4 and CYP2C8 exhibited the highest intrinsic clearance. More than 90% of morphine N-demethylation could be accounted for via the action of both CYP3A4 and CYP2C8. 4. The in vitro findings suggest that hepatic CYP3A4, and to a lesser extent CYP2C8, play an important role in the metabolism of morphine into normorphine.     

Territrems B and C metabolism in human liver microsomes: major role of CYP3A4 and CYP3A5

Human liver microsomes, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, and control and CYP3A4 cDNA-transfected V79 Chinese hamster cells were tested for their ability to metabolize territrem B (TRB) and territrem C (TRC). Two TRB metabolites, designated MB(2) and MB(4), and one TRC metabolite, designated MC, were formed by all of these preparations. Of the nine supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5), only those expressing CYP3A4 or CYP3A5 metabolized TRB and TRC. MB(2), MB(4), and MC were formed by CYP3A4 cDNA-transfected V79MZ Chinese hamster cells, but not by non-transfected cells. In order to investigate which CYP450 isoforms were responsible for MB(2), MB(4) and MC formation in human liver microsomal preparations, six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketoconazole, and diethyldithiocarbamate) and antibodies against CYP3A4 were used. MB(2), MB(4), and MC formation was markedly inhibited by ketoconazole, but less affected by quinidine and sulfaphenazole. Anti-CYP3A4 antibody markedly inhibited MB(2), MB(4), and MC formation and also 6 beta-hydroxytestosterone formation from testosterone. The CYP3A-dependent reaction of testosterone 6 beta-hydroxylation showed a high correlation with 4 beta-C hydroxylation of TRB (r(2)=0.97, P<0.0001), O-demethylation of TRB (r(2)=0.95, P<0.0001), and 4 beta-C hydroxylation of TRC (r(2)=0.99, P<0.0001). Immunoblotting and RT-PCR showed that CYP3A4 and CYP3A5 were expressed in all four human liver microsomal preparations tested (HLM1-HLM4). The amount of MB(2), MB(4), and MC formed using different HLM preparations was related to the 6 beta-testosterone hydroxylase activity of the preparations. However, the extent of MB(2), MB(4), and MC formation was not related to the age or gender of the person from whom the microsomal sample was prepared. It was therefore suggest that CYP3A4 and CYP3A5 are the major enzymes responsible for TRB and TRC metabolism by human liver microsomes.     

Identification of CYP3A4 as the primary cytochrome P450 responsible for the metabolism of tandospirone by human liver microsomes.

The present study was carried out to characterize the human P450 isoforms involved in the metabolism of tandospirone, an anxiolytic agent known for its superior efficacy and safety. Among 11 yeast-expressed recombinant P450 isoforms tested, CYP2D6 and CYP3A4 exhibited the highest tandospirone metabolic activity. Although there was no qualitative difference between the two isoforms, a quantitative difference in metabolite profiling was found i.e., M4 (hydroxylation of the pyrimidine ring) was the major metabolite formed with CYP2D6 while M2 (hydroxylation of the norbornan ring) and 1-PP (oxidative cleavage of the butyl chain) predominated with CYP3A4. The metabolite profile on incubation with CYP3A4 was qualitatively and quantitatively similar to that obtained with human liver microsomes. In vitro intrinsic clearance (CLint) values derived from kinetic analysis using both P450 isoforms were similar (2.2 and 1.6 ml/min/nmol P450), but the hepatic content of CYP3A4 was found to be more abundant than that of CYP2D6. The in vitro metabolism of tandospirone by human liver microsomes was markedly inhibited by ketoconazole (a CYP3A4 inhibitor) but not by quinidine (a CYP2D6 inhibitor). These results indicate that the metabolism of tandospirone by human liver microsomes primarily involves CYP3A4, and to a lesser extent CYP2D6.     

Identification of CYP3A4 as the primary cytochrome P450 responsible for the metabolism of tandospirone by human liver microsomes.

The present study was carried out to characterize the human P450 isoforms involved in the metabolism of tandospirone, an anxiolytic agent known for its superior efficacy and safety. Among 11 yeast-expressed recombinant P450 isoforms tested, CYP2D6 and CYP3A4 exhibited the highest tandospirone metabolic activity. Although there was no qualitative difference between the two isoforms, a quantitative difference in metabolite profiling was found i.e., M4 (hydroxylation of the pyrimidine ring) was the major metabolite formed with CYP2D6 while M2 (hydroxylation of the norbornan ring) and 1-PP (oxidative cleavage of the butyl chain) predominated with CYP3A4. The metabolite profile on incubation with CYP3A4 was qualitatively and quantitatively similar to that obtained with human liver microsomes. In vitro intrinsic clearance (CLint) values derived from kinetic analysis using both P450 isoforms were similar (2.2 and 1.6 ml/min/nmol P450), but the hepatic content of CYP3A4 was found to be more abundant than that of CYP2D6. The in vitro metabolism of tandospirone by human liver microsomes was markedly inhibited by ketoconazole (a CYP3A4 inhibitor) but not by quinidine (a CYP2D6 inhibitor). These results indicate that the metabolism of tandospirone by human liver microsomes primarily involves CYP3A4, and to a lesser extent CYP2D6.     

CYP3A4 mediated in vitro metabolism of vinflunine in human liver microsomes

AIM: To study the metabolism of vinflunine and the effects of selective cytochrome P-450 (CYP450) inhibitors on the metabolism of vinflunine in human liver microsomes. METHODS: Individual selective CYP450 inhibitors were used to investigate their effects on the metabolism of vinflunine and the principal CYP450 isoform involved in the formation of metabolites M(1) and M(2) in human liver microsomes. RESULTS: Vinflunine was rapidly metabolized to 2 metabolites: M(1) and M(2) in human liver microsomes. M(1) and M(2) were tentatively presumed to be the N-oxide metabolite or hydroxylated metabolite and epoxide metabolite of vinflunine, respectively. Ketoconazole uncompetitively inhibited the formation of M(1), and competitively inhibited the formation of M(2), while alpha-naphthoflavone, sulfaphenazole, diethyl dithiocarbamate, tranylcypromine and quinidine had little or no inhibitory effect on the formation of M(1) and M(2). CONCLUSION: Vinflunine is rapidly metabolized in human liver microsomes, and CYP3A4 is the major human CYP450 involved in the metabolism of vinflunine.     

CYP3A4 is mainly responsibile for the metabolism of a new vinca alkaloid, vinorelbine, in human liver microsomes.

The metabolism of vinorelbine, a new anticancer agent belonging to the vinca alkaloid family, was investigated in human liver microsomes. Vinorelbine biotransformation consisted of one saturable and one nonsaturable process, and the K(m) and V(max) values for the saturable process were 1.90 microM and 25.3 pmol/min/mg of protein, respectively. Several studies, including metabolism by cytochrome P450 (CYP) enzymes in a cDNA expression system and inhibition by specific antibodies and chemical inhibitors, showed that the main CYP enzyme involved in vinorelbine metabolism was CYP3A4. Also, the effects of vinorelbine on each of the CYP activities in human liver microsomes were investigated. High concentrations (100 microM) of vinorelbine inhibited CYP3A4 activity (testosterone 6beta-hydroxylation activity) by 45.2%. However, the inhibitory effects of vinorelbine on the other CYP activities were minimal. The 50% inhibitory concentration (IC(50)) of vinorelbine for testosterone 6beta-hydroxylase was estimated to be 155 microM. The plasma concentration in patients is expected to be much lower than this value. These results indicate that vinorelbine metabolism is expected to be modulated by the drugs that are able to inhibit or induce CYP3A activity.     

Involvement of CYP2E1 as A low-affinity enzyme in phenacetin O-deethylation in human liver microsomes.

Phenacetin O-deethylation (POD) exhibits biphasic kinetics in human liver microsomes. Although cytochrome P-450 (CYP) 1A2 is responsible for the high-affinity component of POD, the enzyme(s) that catalyzes the low-affinity reaction is still unknown. We examined the roles of human CYPs in POD by using human liver microsomes and recombinant CYPs from baculovirus-infected insect cells. Of the recombinant CYPs studied, CYP1A2 showed the highest POD activity. CYP1A1, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 also showed POD activity at 500 microM phenacetin. K(M) values of recombinant CYP1A2 and CYP2E1 (28 +/- 2 microM and 785 +/- 125 microM, respectively) were similar to those of the high- and low-affinity components of POD in pooled human liver microsomes (15 +/- 5 and 894 +/- 189 microM, respectively). Fluvoxamine (10 microM) and anti-CYP1A2 antibodies potently inhibited POD activity at 500 microM phenacetin in pooled human liver microsomes to 22.8 and 34.2% of controls, respectively. CYP2E1 inhibitors diethyldithiocarbamate and aniline also reduced POD activity. The combination of fluvoxamine (10 microM) and aniline (1 mM) further inhibited the residual POD activity not inhibited by fluvoxamine alone. Microsomal POD activity in 12 human livers in the absence of fluvoxamine was correlated with immunoquantified CYP1A2 levels (r = 0.961, p <.001) and, in the presence of 10 microM fluvoxamine, was correlated with immunoquantified CYP2E1 levels (r = 0.589, p <.01) or chlorzoxazone 6-hydroxylase activity (r = 0.823, p <.001). These results suggest that CYP2E1 is responsible for the low-affinity component of POD in human liver microsomes.     

Identification of CYP isozymes involved in benzbromarone metabolism in human liver microsomes

Benzbromarone (BBR) is metabolized to 1′‐hydroxy BBR and 6‐hydroxy BBR in the liver. 6‐Hydroxy BBR is further metabolized to 5,6‐dihydroxy BBR. The aim of this study was to identify the CYP isozymes involved in the metabolism of BBR to 1′‐hydroxy BBR and 6‐hydroxy BBR and in the metabolism of 6‐hydroxy BBR to 5,6‐dihydroxy BBR in human liver microsomes. Among 11 recombinant P450 isozymes examined, CYP3A4 showed the highest formation rate of 1′‐hydroxy BBR. The formation rate of 1′‐hydroxy BBR significantly correlated with testosterone 6β‐hydroxylation activity in a panel of 12 human liver microsomes. The formation of 1′‐hydroxy BBR was completely inhibited by ketoconazole in pooled human liver microsomes. On the other hand, the highest formation rate of 6‐hydroxy BBR was found in recombinant CYP2C9. The highest correlation was observed between the formation rate of 6‐hydroxy BBR and diclofenac 4′‐hydroxylation activity in 12 human liver microsomes. The formation of 6‐hydroxy BBR was inhibited by tienilic acid in pooled human liver microsomes. The formation of 5,6‐dihydroxy BBR from 6‐hydroxy BBR was catalysed by recombinant CYP2C9 and CYP1A2. The formation rate of 5,6‐dihydroxy BBR was significantly correlated with diclofenac 4′‐hydroxylation activity and phenacetin O‐deethylation activity in 12 human liver microsomes. The formation of 5,6‐dihydroxy BBR was inhibited with either tienilic acid or α‐naphthoflavone in human liver microsomes. These results suggest that (i) the formation of 1′‐hydroxy BBR and 6‐hydroxy BBR is mainly catalysed by CYP3A4 and CYP2C9, respectively, and (ii) the formation of 5,6‐dihydroxy BBR is catalysed by CYP2C9 and CYP1A2 in human liver microsomes. Copyright © 2012 John Wiley & Sons, Ltd.     

CYP3A4 is a major isoform responsible for oxidation of 7-hydroxy-Delta(8)-tetrahydrocannabinol to 7-oxo-delta(8)-tetrahydrocannabinol in human liver microsomes.

The human liver enzyme microsomal alcohol oxygenase was able to oxidize both 7alpha- and 7beta-hydroxy-Delta(8)-tetrahydrocannabinol (7alpha- and 7beta-hydroxy-Delta(8)-THC) to 7-oxo-Delta(8)-THC. The oxidative activity was determined by using a panel of 12 individual cDNA-expressed human cytochrome P450s (CYPs) (1A1, 1A2, 2A6, 2B6, 2C8, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Met, 2D6-Val, 2E1 and 3A4). Among the CYP isoforms examined, CYP3A4 showed the highest activity for both of substrates. The metabolism of 7alpha- and 7beta-hydroxy-Delta(8)-THC to 7-oxo-Delta(8)-THC was also detected for CYPs 1A1 (4.8% of CYP3A4), 1A2 (4.7%), 2A6 (2.3%), 2C8 (16.6%), and 2C9-Cys (5.4%), and CYPs 1A1 (0.4%), 2C8 (1.3%), 2C9-Arg (4.3%), and 2C9-Cys (0.9%), respectively. The 7alpha- and 7beta-hydroxy-Delta(8)-THC microsomal alcohol oxygenase activities in human liver were significantly inhibited by addition of 100 microM troleandomycin, 1 microM ketoconazole, and anti-CYP3A antibody, although these activities were not inhibited by 1 microM 7, 8-benzoflavone and 50 microM sulfaphenazole. When the substrates were incubated with the CYP3A4-expressed microsomes under oxygen-18 gas phase, atmospheric oxygen was incorporated into 35% of 7-oxo-Delta(8)-THC formed from 7alpha-OH-Delta(8)-THC, but only 12% of 7-oxo-Delta(8)-THC formed from 7beta-OH-Delta(8)-THC. These results indicate that CYP3A4 is a major isoform responsible for the oxidation of 7alpha- and 7beta-hydroxy-Delta(8)-THC to 7-oxo-Delta(8)-THC in liver microsomes of humans, although the oxidation mechanisms for 7alpha- and 7beta-hydroxy-Delta(8)-THC might be different.     

Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms.

1. The primary metabolism of diazepam was studied in human liver microsomes in order to investigate the kinetics and to identify the cytochrome P450 (CYP) isoforms responsible for the formation of the main diazepam metabolites, temazepam and N-desmethyldiazepam. 2. The formation kinetics of both metabolites were atypical and consistent with the occurrence of substrate activation. A sigmoid Vmax model equivalent to the Hill equation was used to fit the data. The degree of sigmoidicity was greater for temazepam formation than for N-desmethyldiazepam formation, so that the ratio of desmethyldiazepam:temazepam formation increased as the substrate (diazepam) concentration decreased. 3. alpha-Naphthoflavone activated both reactions but with a greater effect on temazepam formation than on N-desmethyldiazepam formation. In the presence of 25 microM alpha-naphthoflavone the kinetics for both pathways were approximated by Michaelis-Menten kinetics. 4. Studies with a series of CYP isoform selective inhibitors and with an inhibitory anti-CYP2C antibody indicated that temazepam formation was carried out mainly by CYP3A isoforms, whereas the formation of N-desmethyldiazepam was mediated by both CYP3A isoforms and S-mephenytoin hydroxylase.