Model for the drug–drug interaction responsible for CYP3A enzyme inhibition. II: establishment and evaluation of dexamethasone-pretreated female rats

1.?Cytochrome P450 (CYP) 3A catalysis of testosterone 6β-hydroxylation in female rat liver microsomes was significantly induced, then reached a plateau level after pretreatment with 80?mg?kg^?1?day^?1 dexamethasone (DEX) for 3 days.

2.?Midazolam was mainly metabolized by CYP3A in DEX-treated female rat liver microsomes from an immuno-inhibition study, and the apparent K_m was 1.8?μM, similar to that in human microsomes.

3.?Ketoconazole and erythromycin, typical CYP3A inhibitors, demonstrated extensive inhibition of midazolam metabolism in DEX-treated female rat liver microsomes, and the apparent K_i values were 0.088 and 91.2?μM, respectively. The values were similar to those in humans, suggesting that DEX-treated female rat liver microsomes have properties similar to those of humans.

4.?After oral administration of midazolam, the plasma midazolam concentration in DEX-treated female rats significantly decreased compared with control female rats. The area under the plasma concentration curve (AUC) and elimination half-life were one-11th and one-20th of those of control female rats, respectively.

5.?Using DEX-treated female rats, the effect of CYP3A inhibitors on midazolam pharmacokinetics was evaluated. The AUC and maximum concentration in plasma (C_max) increased when ketoconazole was co-administered with midazolam.

6.?It was shown that the drug–drug interaction that occurs in vitro is also observed in vivo after oral administration of midazolam. In conclusion, the DEX-treated female rat could be a useful model for evaluating drug–drug interactions based on CYP3A enzyme inhibition.     

Model for the drug–drug interaction responsible for CYP3A enzyme inhibition. I: evaluation of cynomolgus monkeys as surrogates for humans

1.?Anti-human cytochrome P450 (CYP) 3A4 antiserum completely inhibited midazolam metabolism in monkey liver microsomes, suggesting that midazolam was mainly metabolized by CYP3A enzyme(s) in monkey liver microsomes.

2.?Midazolam metabolism was also inhibited in vitro by typical chemical inhibitors of CYP3A, such as ketoconazole, erythromycin and diltiazem, and the apparent K_i values for ketoconazole, erythromycin and diltiazem were 0.127, 94.2 and 29.6?μM, respectively.

3.?CYP3A inhibitors increased plasma midazolam concentrations when midazolam and CYP3A inhibitors were co-administered orally. However, the pharmacokinetic parameters of midazolam were not changed by treatment with CYP3A inhibitors when midazolam was given intravenously. This suggests that CYP3A inhibitors modified the first-pass metabolism in the liver and/or intestine, but not systemic metabolism.

4.?The drug–drug interaction responsible for CYP3A enzyme(s) inhibition was observed when midazolam and inhibitors were co-administrated orally. Therefore, it was concluded that monkeys given midazolam orally could be useful models for predicting drug–drug interactions in man based on CYP3A enzyme inhibition.     

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

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

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

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

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

In vitro identification of human cytochrome P450 isoforms involved in the metabolism of Geissoschizine methyl ether,an active component of the traditional Japanese medicine Yokukansan

1.?Yokukansan (YKS) is a traditional Japanese medicine also called kampo, which has been used to treat neurosis, insomnia, and night crying and peevishness in children. Geissoschizine methyl ether (GM), a major indole alkaloid found in Uncaria hook, has been identified as a major active component of YKS with psychotropic effects. Recently, GM was reported to have a partial agonistic effect on serotonin 5-HT_1A receptors. However, there is little published information on GM metabolism in humans, although several studies reported the blood kinetics of GM in rats and humans. In this study, we investigated the GM metabolic pathways and metabolizing enzymes in humans.

2.?Using recombinant human cytochrome P450 (CYP) isoforms and polyclonal antibodies to CYP isoforms, we found that GM was metabolized into hydroxylated, dehydrogenated, hydroxylated+dehydrogenated, demethylated and water adduct forms by some CYP isoforms.

3.?The relative activity factors in human liver microsomes were calculated to determine the relative contributions of individual CYP isoforms to GM metabolism in human liver microsomes (HLMs). We identified CYP3A4 as the CYP isoform primarily responsible for GM metabolism in human liver microsomes.

4.?These findings provide an important basis for understanding the pharmacokinetics and pharmacodynamics of GM and YKS.     

Predictive value of animal models for human cytochrome P450 (CYP)-mediated metabolism: A comparative study in vitro

One major challenge in drug development is defining of the optimal animal species to serve as a model of metabolism in man. The study compared the hepatic drug metabolism characteristics of humans and six widely used experimental animal species. Classical in vitro model enzyme assays with known human cytochrome P450 (CYP) enzyme selectivity were employed and optimized to target human hepatic CYP forms. The profile of CYP activities best resembling the human was seen in mouse followed by monkey, minipig, and dog liver microsomes, with rats displaying the most divergent. The widest interindividual variability was found in CYP3A-mediated midazolam α-hydroxylase, and omeprazole sulphoxidase activities in human and monkey liver microsomes. These data demonstrate that if hepatic xenobiotic-metabolizing characteristics were to be the sole reason for the selection of animal species for toxicity studies, then the rat might not be the most appropriate model to mimic human CYP activity patterns.     

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor

1.?The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied.

2.?The cytochrome P450s (CYPs) responsible for metabolism of IN-1130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomes?, were identified using specific CYP inhibitors. The order of disappearance of IN-1130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog.

3.?Five distinct metabolites (M1–M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomes? examined, CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19 were involved in the metabolism of IN-1130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1130 (M1–M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites.

4.?Hence, it is concluded that metabolism of IN-1130 is mediated by CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19.     

Oxidative metabolism of koumine is mainly catalyzed by microsomal CYP3A4/3A5

1.?Gelsemium elegans Benth (Loganiaceae) is a toxic plant that can be used for committing suicide besides alleviating pains. Its anti-inflammatory and analgesic effect mainly come from its active ingredient, namely koumine. Koumine, an indole alkaloid, possesses widely pharmacological effects especially inhibition of neuropathic pain.

2.?This study aimed to investigate the metabolic profile of koumine using human liver microsomes (HLMs), selective chemical inhibitors and recombinant human CYP isoforms. Ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) was used to detect and identify metabolites.

3.?Four major metabolites of koumine were found after incubation with HLMs or individual CYP isoforms. The metabolic pathways of koumine included demethylation, dehydrogenation, oxidation and demethyl-dehydrogenation. Chemical inhibition study showed that the inhibitor of CYP3A4/3A5 significantly decreased (93%) the formation of koumine metabolites. Further, CYP3A4/3A5 was shown as the most efficient isoform in biotransformation of koumine, among a series of CYP isoforms tested.

4.?In conclusion, koumine was metabolized into four oxidative metabolites in HLMs. And CYP3A4/3A5 was probably the main contributor to the hepatic oxidative metabolism of koumine.     

Effect of ursodeoxycholic acid on the pharmacokinetics of midazolam and CYP3A in the liver and intestine of rats

1. The elimination half-life of midazolam administered intravenously (5 mg kg^?1) or orally (15 mg kg^?1) was significantly decreased by 70% and 73%, respectively, 24 h after a single oral administration of ursodeoxycholic acid (UDCA, 300 mg kg^?1) in rats. In the liver there was a significant enhancement of the hydroxylation of midazolam in the microsomes and expression of cytochrome P450 (CYP) 3A1 messenger RNA (mRNA) and CYP3A2 mRNA.

2. The C_max and area under the curve (AUC)_0–∞ of midazolam were significantly (1.8–2.3 fold) increased by the single oral treatment with UDCA (100 and 300 mg kg^?1). Thus, the oral bioavailability, estimated from the AUC_0–∞, of midazolam administered intravenously and orally was significantly (1.8- and 2.3-fold, respectively) increased by the treatment with UDCA.

3. Repeated administration of UDCA (300 mg kg^?1 day^?1) for 7 days did not alter the pharmacokinetics of midazolam administered intravenously or orally, and the expression of mRNA for CYP3As in the rat liver.

4. The study has shown that a single administration of UDCA in rats induces significant hepatic CYP3A activity and increases significantly the oral bioavailability of midazolam. Such effects on the pharmacokinetics of midazolam were little observed on the repeated administration of UDCA.

     

In vitro characterization of 4′-(p-toluenesulfonylamide)-4-hydroxychalcone using human liver microsomes and recombinant cytochrome P450s

1.?4′-(p-Toluenesulfonylamide)-4-hydroxychalcone (TSAHC) is a synthetic sulfonylamino chalcone compound possessing anti-cancer properties. The aim of this study was to elucidate the metabolism of TSAHC in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of TSAHC.

2.?TSAHC was incubated with HLMs or recombinant P450 isoforms (rP450) in the presence of an nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system. The metabolites were identified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). P450 isoforms, responsible for TSAHC metabolite formation, were characterized by chemical inhibition and correlation studies in HLMs and enzyme kinetic studies with a panel of rP450 isoforms.

3.?Two hydroxyl metabolites, that is M1 and M2, were produced from the human liver microsomal incubations (K_m and V_max values were 2.46?µM and 85.1?pmol/min/mg protein for M1 and 9.98?µM and 32.1?pmol/min/mg protein for M2, respectively). The specific P450 isoforms responsible for two hydroxy-TSAHC formations were identified using a combination of chemical inhibition, correlation analysis and metabolism by expressed recombinant P450 isoforms. The known P450 enzyme activities and the rate of TSAHC metabolite formation in the 15 HLMs showed that TSAHC metabolism is correlated with CYP2C and CYP3A activity. The P450 isoform-selective inhibition study in HLMs and the incubation study of cDNA-expressed enzymes also showed that two hydroxyl metabolites M1 and M2 biotransformed from TSAHC are mainly mediated by CYP2C and CYP3A, respectively. These findings suggest that CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 isoforms are major enzymes contributing to TSAHC metabolism.     

Comparison of predicted intrinsic hepatic clearance of 30 pharmaceuticals in canine and feline liver microsomes

1.?Known cytochrome P450 (CYP) substrates in humans are used in veterinary medicine, with limited knowledge of the similarity or variation in CYP metabolism. Comparison of canine and feline CYP metabolism via liver microsomes report that human CYP probes and inhibitors demonstrate differing rates of intrinsic clearance (CL_int).

2.?The purpose of this study was to utilize a high-throughput liver microsome substrate depletion assay, combined with microsomal and plasma protein binding to compare the predicted hepatic clearance (CL_hep) of thirty therapeutic agents used off-label in canines and felines, using both the well-stirred and parallel tube models.

3.?In canine liver microsomes, 3/30 substrates did not have quantifiable CL_int, while midazolam and amitriptyline CL_int was too rapid for accurate determination. A CL_hep was calculated for 29/30 substrates in feline microsomes. Overall, canine CL_hep was faster compared to the feline, with fold differences ranging from 2–20-fold.

4.?A comparison between the well-stirred and parallel tube model indicates that the parallel tube model reports a slighter higher CL_hep in both species.

5.?The differences in CYP metabolism between canine and feline highlight the need for additional research into CYP expression and specificity.     

Time-dependent changes in hepatic and intestinal induction of cytochrome P450 3A after administration of dexamethasone to rats

Abstract

1.?We investigated the effects of the dose of and the number of times an inducer was administered and the duration of induction of hepatic and intestinal cytochrome P450 3A (CYP3A) in rats using dexamethasone 21-phosphate (DEX-P) and midazolam (MDZ) as an inducer and a substrate to CYP3A, respectively.

2.?The number of times DEX-P was administered was not a significant factor in the induction of either hepatic or intestinal CYP3A; however, administration of DEX-P multiple times markedly decreased the bioavailability of DEX-P by self-induction of CYP3A.

3.?CYP3A induction in the liver increased depending on the dose of DEX-P, whereas that in intestine showed a mild increase, but the induction level was almost constant regardless of the dose of DEX-P.

4.?Administration of a single dose of DEX-P showed a temporal increase in CYP3A activity in both tissues and the induction ratios reached maximum values at 12?h after DEX-P administration. On the other hand, a mild increase of CYP3A activity, which lasted for at least 48?h, was observed in both tissues after administration of multiple doses.

5.?Some physiological compounds such as cytokines might be involved in decreasing the CYP3A activity to maintain homeostasis of the body.     

Homology modelling of CYP3A4 from the CYP2C5 crystallographic template: analysis of typical CYP3A4 substrate interactions

1.?The results of homology modelling of cytochrome P4503A4 (CYP3A4), which is a human enzyme of major importance for the Phase 1 metabolism of drug substrates, from the CYP2C5 crystal structure is reported.

2.?The overall homology between the two protein sequences was generally good (46%) with 24%?of amino acid residues being identical and a 22%?similarity between matched pairs in the CYP3A4 and CYP2C5 aligned sequences, thus indicating that CYP2C5 represents a viable template for modelling CYP3A4 by homology.

3.?The CYP3A4 model appears to show consistency with the reported findings from the extensive site-directed mutagenesis studies already published.

4.?Typical CYP3A4 substrates, such as midazolam, testosterone, nifedipine and verapamil, are shown to fit the putative active site of the enzyme structure in a manner consistent with their known positions of metabolism.     

In vitro metabolism of the anti-inflammatory clerodane diterpenoid polyandric acid A and its hydrolysis product by human liver microsomes and recombinant cytochrome P450 and UDP-glucuronosyltransferase enzymes

1.?The metabolism of the anti-inflammatory diterpenoid polyandric acid A (PAA), a constituent of the Australian Aboriginal medicinal plant Dodonaea polyandra, and its de-esterified alcohol metabolite, hydrolysed polyandric acid A (PAAH) was studied in vitro using human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzymes.

2.?Hydrolysis of PAA to yield PAAH occurred upon incubation with HLM. Further incubations of PAAH with HLM in the presence of UGT and CYP cofactors resulted in significant depletion, with UGT-mediated depletion as the major pathway.

3.?Reaction phenotyping utilising selective enzyme inhibitors and recombinant human UGT and CYP enzymes revealed UGT2B7 and UGT1A1, and CYP2C9 and CYP3A4 as the major enzymes involved in the metabolism of PAAH.

4.?Analysis of incubations of PAAH with UDP-glucuronic acid-supplemented HLM and recombinant enzymes by UPLC/MS/MS identified three glucuronide metabolites. The metabolites were further characterised by β-glucuronidase and mild alkaline hydrolysis. The acyl glucuronide of PAAH was shown to be the major metabolite.

5.?This study demonstrates the in vitro metabolism of PAA and PAAH and represents the first systematic study of the metabolism of an active constituent of an Australian Aboriginal medicinal plant.     

Development of an optimized procedure for the preparation of rat intestinal microsomes: comparison of hepatic and intestinal microsomal cytochrome P450 enzyme activities in two rat strains

1. The objective of this study was to characterize cytochrome P450 (CYP) activities in both intestinal and hepatic microsomes from Wistar and Sprague–Dawley rats.

2. Specific probes for measuring CYP activities were selected using rat recombinant CYP.

3. The intestinal microsome preparation was optimized getting a more relevant and reproducible abundance of CYPs to measure CYP activities.

4. Testosterone, propranolol, diclofenac, and midazolam were determined as specific substrates of rat CYP2C11, CYP2D2, CYP2C6, and CYP3A, respectively. Ethoxyresorufin and pentoxyresorufin were not specific substrates of CYP1A2 and CYP2B1, respectively. Hepatic and intestinal microsomes expressed active CYP1A1, CYP1A2, CYP2B1, and CYP3A2. Only liver expressed active CYP2C6, CYP2C11, and CYP2D2. Wistar liver expressed more active CYP1A and CYP3A2, but less active CYP2B1 than Wistar intestine. Sprague–Dawley liver expressed more active CYP2B1 and CYP3A2, but less active CYP1A than Sprague–Dawley intestine.

5. In conclusion, CYP activities were qualitatively equivalent but not quantitatively in both strains.

     

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。     

The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes

1.?The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes.

2.?100?μM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC₅₀) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00?μM, and the inhibition kinetic parameters (K_i) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11?μM, respectively.

3.?Metabolism of morusin exhibited significant species differences. The quantities of M₁ from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The K_m values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83?μM, and V_max for these species were 0.09, 1.23, 1.43, 0.15, and 0.75?nmol/min/mg, respectively. CL_int (intrinsic clearance) values (V_max/K_m) for morusin obeyed the following order: monkey?>?rat?>?minipig?>?dog?>?human. CL_H (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12?mL/min/kg body weight, respectively.

4.?This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.     

Interspecies variation in phase I metabolism of bufalin in hepatic microsomes from mouse,rat, dog,minipig, monkey,and human

Abstract

1.?Bufalin (BF), one of the major bioactive compounds in traditional Chinese medicine (TCM) Chansu, has been found with various pharmacological and toxicological effects. This study aims to investigate the species differences in phase I metabolism of BF in hepatic microsomes from human and five common experimental animals.

2.?Metabolite profiling demonstrated that two major metabolites were formed in liver microsomes from human and animal species in NADPH-generating system. Two major metabolites were identified as 5β-hydroxyl-bufalin and 3-keto-bufalin, with the help of authentic standards. CYP3A was assigned as the main isoform involved in both 5β-hydroxylation and 3-oxidation in all studied liver microsomes. The apparent kinetic parameters including substrate affinity and catalytic efficiency for 5β-hydroxylation and 3-oxidation of BF were also determined.

3.?In summary, CYP3A mediated 5β-hydroxylation and 3-oxidation were two major metabolic pathways of BF in hepatic microsomes from human and five studied animals, but kinetic analysis demonstrated that the intrinsic clearances of these two metabolic pathways were much different among various species. The qualitative and quantitative interspecies study indicated that minipig exhibited the similar metabolic profile, kinetic behaviors and intrinsic metabolic clearances of BF phase I biotransformation in comparison with that of human.     

2,5-Dihydroxy-4-methoxybenzophenone: a novel major in vitro metabolite of benzophenone-3 formed by rat and human liver microsomes

1.?When benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) was incubated with liver microsomes of untreated rats in the presence of NADPH, the 5-hydroxylated metabolite, 2,5-dihydroxy-4-methoxybenzophenone (5-OH-BP-3), was formed as a major novel metabolite of BP-3. The 4-desmethylated metabolite, 2,4-dihydroxybenzophenone (2,4-diOH-BP), previously reported as the major in vivo metabolite of BP-3, was also detected. However, the amount of 5-OH-BP-3 formed in vitro was about the same as that of 2,4-diOH-BP.

2.?The oxidase activity affording 5-OH-BP-3 was inhibited by SKF 525-A and ketoconazole, and partly by quinidine and sulfaphenazole. The oxidase activity affording 2,4-diOH-BP was inhibited by SKF 525-A, ketoconazole and α-naphthoflavone, and partly by sulfaphenazole.

3.?The oxidase activity affording 5-OH-BP-3 was enhanced in liver microsomes of dexamethasone-, phenobarbital- and 3-methylcholanthrene-treated rats. The activity affording 2,4-diOH-BP was enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats.

4.?When examined recombinant rat cytochrome P450 isoforms catalyzing the metabolism of BP-3, 5-hydroxylation was catalyzed by P450 3A2, 1A1, 2B1, 2C6 and 2D1, while 4-desmethylation was catalyzed by P450 2C6 and 1A1.