Members Of CYP3A Subfamily Research Articles

Stimulatory and Inhibitory Effects of Steroid Hormones and Human Cytochrome P450 (CYP) 3A Inhibitors on Cortisol 6β-Hydroxylation Catalyzed by CYP3A Subfamilies.

The inhibitory and stimulatory effects of several compounds, including steroid hormones and azole antifungal agents, on cortisol 6β-hydroxylation activity by cytochrome P450 (CYP) 3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared with those on testosterone 6β-hydroxylation to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. 6β-Hydroxylation activities of cortisol and testosterone by CYP3A4, CYP3A5, and CYP3A7 in the absence or presence of dehydroepiandrosterone (DHEA), α-naphthoflavone (ANF), ketoconazole, itraconazole, and voriconazole were measured using high-performance liquid chromatography. Lower concentrations of DHEA and ANF increased cortisol 6β-hydroxylation activities catalyzed by CYP3A4 but not those catalyzed by CYP3A5 and CYP3A7. The inhibition strength of azole antifungal agents against cortisol 6β-hydroxylation catalyzed by all CYP3A subfamilies was similar to that of testosterone 6β-hydroxylation. Although the Michaelis constant (Km) increased 2-fold in the presence of 20 μM DHEA compared to that of the control, the maximal velocity (Vmax) values gradually increased with increasing DHEA. For ANF, both Km and Vmax values increased, although the Km value decreased at 2.5 μM concentrations. Ketoconazole and itraconazole competitively inhibited cortisol 6β-hydroxylation mediated by CYP3A4 with similar inhibition constants. The inhibitory/stimulatory pattern among CYP3A subfamily members differed between cortisol and testosterone, and CYP3A4 was found to be the most sensitive in terms of inhibition by azole antifungals among the CYP3A subfamily members investigated.

Comparison of the Stimulatory and Inhibitory Effects of Steroid Hormones and α-Naphthoflavone on Steroid Hormone Hydroxylation Catalyzed by Human Cytochrome P450 3A Subfamilies.

The inhibitory and stimulatory effects of steroid hormones and related compounds on the hydroxylation activity at the 6β-position of two steroid hormones, progesterone and testosterone, by CYP3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. Hydroxylation activities of progesterone and testosterone by CYP3A4, CYP3A5, and CYP3A7 were estimated using HPLC. The Michaelis constants (Km) for progesterone 6β-hydroxylation by CYP3A5 were markedly decreased in the presence of dehydroepiandrosterone (DHEA) and α-naphthoflavone (ANF), whereas progesterone and DHEA competitively inhibited testosterone 6β-hydroxylation mediated by CYP3A4, and progesterone competitively inhibited CYP3A5-mediated activity, which was weaker than that for CYP3A4. ANF noncompetitively inhibited testosterone 6β-hydroxylation mediated by both CYP3A4 and CYP3A5. Progesterone and testosterone 6β-hydroxylation mediated by CYP3A7 was inhibited or unaffected by DHEA, pregnenolone, and ANF. These results suggested that DHEA and ANF stimulated progesterone 6β-hydroxylation by CYP3A5 but not by CYP3A4 and CYP3A7; however, progesterone, DHEA, and ANF inhibited testosterone 6β-hydroxylation mediated by all CYP3A subfamily members. The inhibitory/stimulatory pattern of steroid-steroid interactions is different among CYP3A subfamily members and CYP3A5 is the most sensitive in terms of activation among the CYP3A subfamily members investigated.

Comparison of steroid hormone hydroxylation mediated by cytochrome P450 3A subfamilies

Hydroxylation activity at the 6β-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (CYP) 3A4, polymorphic CYP3A5, and fetal CYP3A7 were compared to understand the catalytic properties of the major forms of human CYP3A subfamily. Testosterone, progesterone, and cortisol 6β-hydroxylation activities of recombinant CYP3A4, CYP3A5, and CYP3A7 were determined by liquid chromatography. Michaelis constants (Km) for CYP3A7-mediated 6β-hydroxylation of testosterone, progesterone, and cortisol were similar to those of CYP3A4 and CYP3A5. The maximal velocity (kcat) and kcat/Km values for CYP3A4 were the highest, followed by CYP3A5 and those for CYP3A7 were the lowest among three CYP3A subfamily members. A decrease in Km values for progesterone 6β-hydroxylation by CYP3A4, CYP3A5, and CYP3A7 in the presence of testosterone was observed, and the kcat values for CYP3A5 gradually increased with increasing testosterone. This indicated that testosterone stimulated progesterone 6β-hydroxylation by all three CYP3A subfamily members. However, progesterone inhibited testosterone 6β-hydroxylation mediated by CYP3A4, CYP3A5, and CYP3A7. In conclusion, the kcat values, rather than Km values, for 6β-hydroxylation of three steroid hormones mediated by CYP3A7 were different from those for CYP3A4 and CYP3A5. In addition, the inhibitory/stimulatory pattern of steroid-steroid interactions would be different among CYP3A subfamily members.

Piperine Is a Mechanism-Based Inactivator of CYP3A.

Piperine (PPR) is the representative alkaloid component of the piper species (family: Piperaceae). Our rapid screening study found PPR caused time-dependent inhibition of cytochrome P450s (CYP) 3A and 2D6, and CYP3A was inactivated the most. Further study demonstrated that PPR is a time-, concentration-, and NADPH-dependent inhibitor of CYP3A, and significant loss (49.5% ± 3.9%) of CYP3A activity was observed after 20minute incubations with 80 μM PPR at 37°C. The values of K I and k inact were 30.7 μM and 0.041 minutes-1, respectively. CYP3A competitive inhibitor ketoconazole showed protective effect against the enzyme inactivation. Superoxide dismutase/catalase and GSH displayed minor protection against the PPR-caused enzyme inactivation. Ferricyanide partially reduced the enzyme inhibition by PPR. Additionally, NADPH-dependent formation of reactive metabolites from PPR were found in human liver microsomal incubation mixtures. An ortho-quinone intermediate was trapped by NAC in microsomal incubations with PPR. DM-PPR, demethylene metabolite of PPR, showed weak enzyme inactivation relative to that caused by PPR. It appears that both carbene and ortho-quinone intermediates were involved in the inactivation of CYP3A caused by PPR. SIGNIFICANCE STATEMENT: CYP3A subfamily members (mainly CYP3A4 and CYP3A5) play a critical role in drug metabolism. Piperine (PPR), a methylenedioxyphenyl derivative combined with an unsaturated ketone, is the major active ingredient of pepper. PPR revealed time-, concentration-, and NADPH-dependent inhibitory effect on CYP3A. Carbene and quinone metabolites were both involved in the observed CYP3A inactivation by PPR. Apparently, the unsaturated ketone moiety did not participate in the enzyme inactivation. The present study sounds an alert of potential risk for food-drug interactions.

Molecular cloning and 3D model of first cytochrome P450 from CYP3A subfamily in saltwater crocodile (Crocodylus porosus)

Cytochrome P450s (CYPs) play critical role in oxidative metabolism of numerous xenobiotics and endogenous compounds. The first CYP3A subfamily member in saltwater crocodile has been cloned and modelled for three-dimensional (3D) structure. The full-length cDNA was obtained employing reverse transcription polymerase chain reaction (RT-PCR) strategy and rapid amplification of cDNA ends (RACE). The cDNA sequence of 1659 nucleotides includes 132 nucleotides from 5′ untranslated region (UTR), an open reading frame of 1527 nucleotides encoding 509 amino acids designated as CYP3A163. The alignment of CYP3A163 sequence with CYP3A subfamily across the lineages exhibit the loss of 1 residue in birds and 7 residues in mammals in comparison to reptiles suggesting the adaptation processes during evolution. The amino acid identity of CYP3A163 with Alligator mississippiensis CYP3A77 and Homo sapiens CYP3A4 is 91% and 62% respectively. The 3D structure of CYP3A163 modelled using human CYP3A4 structure as a template with Phyre2 software, represents high similarity with its functionally important motifs and catalytic domain. Both sequence and structure of CYP3A163 display the common and conserved features of CYP3A subfamily. Overall, this study provides primary molecular and structural data of CYP3A163 required to investigate the xenobiotic metabolism in saltwater crocodiles.

Xyloketal B, a marine compound, acts on a network of molecular proteins and regulates the activity and expression of rat cytochrome P450 3a: a bioinformatic and animal study.

Natural compounds are becoming popular for the treatment of illnesses and health promotion, but the mechanisms of action and safety profiles are often unknown. Xyloketal B (XKB) is a novel marine compound isolated from the mangrove fungus Xylaria sp., with potent antioxidative, neuroprotective, and cardioprotective effects. However, its molecular targets and effects on drug-metabolizing enzymes are unknown. This study aimed to investigate the potential molecular targets of XKB using bioinformatic approaches and to examine the effect of XKB on the expression and activity of rat cytochrome P450 3a (Cyp3a) subfamily members using midazolam as a model probe. DDI-CPI, a server that can predict drug–drug interactions via the chemical–protein interactome, was employed to predict the targets of XKB, and the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to analyze the pathways of the predicted targets of XKB. Homology modeling was performed using the Discovery Studio program 3.1. The activity and expression of rat hepatic Cyp3a were examined after the rats were treated with XKB at 7 and 14 mg/kg for 8 consecutive days. Rat plasma concentrations of midazolam and its metabolite 1′-OH-midazolam were determined using a validated high-performance liquid chromatographic method. Bioinformatic analysis showed that there were over 324 functional proteins and 61 related signaling pathways that were potentially regulated by XKB. A molecular docking study showed that XKB bound to the active site of human cytochrome P450 3A4 and rat Cyp3a2 homology model via the formation of hydrogen bonds. The in vivo study showed that oral administration of XKB at 14 mg/kg to rats for 8 days significantly increased the area under the plasma concentration-time curve (AUC) of midazolam, with a concomitant decrease in the plasma clearance and AUC ratio of 1′-OH-midazolam over midazolam. Further, oral administration of 14 mg/kg XKB for 8 days markedly reduced the activity and expression of hepatic Cyp3a in rats. Taken together, the results show that XKB could regulate networks of molecular proteins and related signaling pathways and that XKB downregulated hepatic Cyp3a in rats. XKB might cause drug interactions through modulation of the activity and expression of Cyp3a members. More studies are warranted to confirm the mechanisms of action of XKB and to investigate the underlying mechanism for the regulating effect of XKB on Cyp3a subfamily members.

Cytochrome P450 CYP3A in marsupials: Cloning and identification of the first CYP3A subfamily member, isoform 3A70 from Eastern gray kangaroo (Macropus giganteus)

Australian marsupials are unique fauna that have evolved and adapted to unique environments and thus it is likely that their detoxification systems differ considerably from those of well-studied eutherian mammals. Knowledge of these processes in marsupials is therefore vital to understanding the consequences of exposure to xenobiotics. Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of both xenobiotics and endogenous substrates. In this study we have cloned and characterized CYP3A70, the first identified member of the CYP3A gene subfamily from Eastern gray kangaroo (Macropus giganteus). A 1665 base pair kangaroo hepatic CYP3A complete cDNA, designated CYP3A70, was cloned by reverse transcription-polymerase chain reaction approaches, which encodes a protein of 506 amino acids. The CYP3A70 cDNA shares approximately 71% nucleotide and 65% amino acid sequence homology to human CYP3A4 and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Transfection of the CYP3A70 cDNAs into 293T cells resulted in stable cell lines expressing a CYP3A immuno-reactive protein that was recognized by a goat anti-human CYP3A4 polyclonal antibody. The anti-human CYP3A4 antibody also detected immunoreactive proteins in liver microsomes from all test marsupials, including the kangaroo, koala, wallaby, and wombat, with multiple CYP3A immunoreactive bands observed in kangaroo and wallaby tissues. Relatively, very low CYP catalytic activity was detected for the kangaroo CYP3A70 cDNA-expressed proteins (19.6 relative luminescent units/μg protein), which may be due to low protein expression levels. Collectively, this study provides primary molecular data regarding the Eastern kangaroo hepatic CYP3A70 gene and enables further functional analyses of CYP3A enzymes in marsupials.

Comparative study on altered hepatic metabolism of CYP3A substrates in rats with glycerol-induced acute renal failure

To examine the mechanism accounting for the diverse alteration of hepatic metabolism of CYP3A substrates observed with renal function being severely impaired, the hepatic drug metabolizing activity was evaluated using liver microsomes prepared from rats with glycerol-induced acute renal failure (ARF). Midazolam, nifedipine and rifabutin were employed as representative CYP3A substrates. When the Michaelis-Menten parameters, K(m) and V(max) , were examined in the incubation study, the K(m) values of midazolam and nifedipine in ARF rats were shown to decrease by 50.9% and 29.9% compared with the normal value, respectively. The V(max) values of midazolam and nifedipine in ARF rats also decreased by 49.3% and 28.0%, respectively, showing that their decreased K(m) values accompanied the decreased V(max) values. The parameters of nifedipine seemed to alter to a lesser extent than those of midazolam. As for rifabutin metabolism, the decrease in the K(m) value was observed in ARF rats, but it did not accompany the decrease in the V(max) value. Then, the hepatic expressions of the CYP3A subfamily were examined with western blotting using anti-CYP3A1 and anti-CYP3A2 antibodies. It was revealed that the hepatic expression of CYP3A2 decreased, while that of CYP3A1 was unaffected. Additionally, a band signal deduced to originate from CYP3A9 was clearly detected in ARF, but not in normal rats. Considering each substrate having different specificities for CYP3A subfamily member proteins, individual alterations of hepatic CYP3A subfamily expression seem to underlie the diverse alterations of hepatic metabolism of CYP3A substrates in ARF rats.

WITHDRAWN: Cytochrome P450 CYP3A in marsupials: Characterisation of the first identified CYP3A subfamily member, isoform 3A70 from Eastern grey kangaroo (Macropus giganteus)

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Cytochrome P450 CYP3A in marsupials: Cloning and characterisation of the second identified CYP3A subfamily member, isoform 3A78 from koala (Phascolarctos cinereus)

Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. Previously, we cloned and characterised the CYP2C, CYP4A, and CYP4B gene subfamilies from marsupials and demonstrated important species-differences in both activity and tissue expression of these CYP enzymes. Recently, we isolated the Eastern grey kangaroo CYP3A70. Here we have cloned and characterised the second identified member of marsupial CYP3A gene subfamily, CYP3A78 from the koala (Phascolarctos cinereus). In addition, we have examined the gender-differences in microsomal erythromycin N-demethylation activity (a CYP3A marker) and CYP3A protein expression across test marsupial species. Significant differences in hepatic erythromycin N-demethylation activity were observed between male and female koalas, with the activity detected in female koalas being 2.5-fold higher compared to that in male koalas (p<0.01). No gender-differences were observed in tammar wallaby or Eastern grey kangaroo. Immunoblot analysis utilising anti-human CYP3A4 antibody detected immunoreactive proteins in liver microsomes from all test male and female marsupials including the koala, tammar wallaby, and Eastern grey kangaroo, with no gender-differences detected across test marsupials. A 1610bp koala hepatic CYP3A complete cDNA, designated CYP3A78, was cloned by reverse transcription-polymerase chain reaction approaches. It displays 64% nucleotide and 57% amino acid sequence identity to the Eastern grey kangaroo CYP3A70. The CYP3A78 cDNA encodes a protein of 515 amino acids, shares approximately 68% nucleotide and 56% amino acid sequence identity to human CYP3A4, and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Collectively, this study provides primary molecular data regarding koala hepatic CYP3A78 gene and enables further functional analyses of CYP3A enzymes in marsupials. Given the significant role that CYP3A enzymes play in the metabolism of both endogenous and exogenous compounds, the clone provides an important step in elucidating the metabolic capacity of marsupials.

Effects of the CYP3A5 genetic polymorphism on the pharmacokinetics of diltiazem

Background The cytochrome P450 (CYP) 3A subfamily plays an important role in the metabolism of various endogenous and exogenous compounds. Among CYP3A subfamily members, CYP3A5 is polymorphically expressed and the CYP3A5*3 and CYP3A5*6 alleles are known not to express functional CYP3A5. Thus, these mutant alleles are thought to be responsible for interindividual variability of CYP3A activity. Methods Subjects possessing CYP3A5*1/*1, *1/*3 or *3/*3 received oral administration of diltiazem hydrochloride (60 mg), and plasma and urinary concentrations of diltiazem and its metabolite N-desmethyldiltiazem were measured. Before drug intake, cortisol metabolic clearance in each subject was measured to estimate in vivo CYP3A4 activity. Results The mean values of total oral diltiazem clearance of subjects with *1/*1, *1/*3 and *3/*3 were 183.4 ± 39.4, 197.9 ± 49.6 and 293.6 ± 141.1 (l/h), respectively, and were not significantly different among the 3 genotype groups. The cortisol metabolic clearance was not significantly different among the three genotype groups, indicating that the CYP3A4 activity is not significantly different. Conclusion The results suggest that CYP3A5*3 has only a minor effect on the pharmacokinetics and metabolism of diltiazem. Although our results did not indicate significance of CYP3A5, the effects of CYP3A5*3 on the metabolism of other CYP3A substrates remain to be investigated.

Cloning, tissue distribution, and functional studies of a new cytochrome P450 3A subfamily member, CYP3A45, from rainbow trout ( Oncorhynchus mykiss) intestinal ceca

In trout and mammals, the major extrahepatic expression site for CYP3A forms is in the intestine. A cDNA encoding a new CYP3A subfamily member was isolated from rainbow trout intestinal ceca by reverse transcriptase-polymerase chain reaction (RT-PCR), followed by rapid amplification of cDNA ends (RACE)-PCR. In a set of two primers for PCR, a consensus sequence in the highly conserved regions in 17 CYP3A sequences was used for one primer, and the second primer was designed based on adapter sequence ligated on the 5 ′ and 3 ′ cDNA ends. The 3 ′ and 5 ′ end nucleotide sequences of RACE-PCR products were used for the priming sites for the full-length cDNA in RT-PCR. The resulting 2615-bp cDNA contained an open reading frame of 1554 bp encoding a 518-amino acid residue protein ( M r=59057.13, p I=6.15) with 26 amino acid differences from that of the previously cloned rainbow trout CYP3A27. The cDNA was assigned as CYP3A45 by the P450 Nomenclature Committee. The deduced amino acid sequence of rainbow trout CYP3A45 was 94% identical with trout CYP3A27, 72% with killifish CYP3A56, and 71% with both medaka CYP3A40 and killifish CYP3A30 in positional alignment comparisons. Northern blotting by a CYP3A45-specific nucleotide probe showed that the major expression site was the intestinal ceca rather than the liver in both male and female trout. Recombinant baculovirus containing a CYP3A45 cDNA (Bv-3A45) was constructed under polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and used to express CYP3A45 protein in Spodoptera frugiperda cells. The Western blot showed that the expressed CYP3A45 protein comigrated with purified LMC5 P450 and was recognized by anti-LMC5 polyclonal antibodies. The expressed CYP3A45 showed catalytic activities for the 6β-, 2β-, and 16β-hydroxytestosterones of 1.76, 0.193, and 0.078 nmol/min/nmol CYP3A45, respectively. In summary, a second form of CYP3A with steroid hydroxylase activity, CYP3A45, has been cloned from rainbow trout and the major site of expression was in the intestinal tissues.

Biotransformation of alprazolam by members of the human cytochrome P4503A subfamily

1. To aid in the prediction of drug interactions with alprazolam, the human CYP involved in the 1'- and 4-hydroxylation of alprazolam were characterized using human liver microsomes, expressed enzymes and selective chemical inhibitors. 2. The formation of 4-hydroxyalprazolam and 1-hydroxyalprazolam at an alprazolam concentration of 62.5 μM were reduced by the prototypic CYP3A inhibitor, troleandomycin (50 μM), by 97 and 99% respectively. Only microsomes from B-lymphoblastoid cells expressing CYP3A4 were capable of catalysing the 1'- and 4-hydroxylation of alprazolam. 3. The formation rates of 1'-hydroxyalprazolam and 4-hydroxyalprazolam at an alprazolam concentration of 1 mM were significantly correlated (n=19, r=0.95, p<0.01) indicating that the same enzyme(s) mediated these biotransformations. A significant (p<0.01) correlation was observed between alprazolam 4- and 1'-hydroxylase activity and CYP3A-mediated midazolam 4-hydroxylase, midazolam 1'-hydroxylase, dextromethorphan N-demethylase and erythromycin N-demethylase activities. 4. In conclusion, in adult human liver the CYP3A subfamily members are the principal enzymes involved in the 1'- and 4-hydroxylation of alprazolam. Thus, clinically significant drug-drug interactions between alprazolam and other CYP3A substrates are to be expected.

Cloning, Sequencing, and Tissue Expression of CYP3A27, a New Member of the CYP3A Subfamily from Embryonic and Adult Rainbow Trout Livers

Screening of λgt11 and λgt22A cDNA libraries of livers from adult females and embryos of rainbow trout (Oncorhynchus mykiss), respectively, using rabbit anti-rainbow trout cytochrome P450 LMC5 polyclonal antibodies showed that there were identical cDNAs of 1802-bp nucleotides with open reading frames coding for proteins containing 518 amino acids (59,206 Da, pI= 6.39). The cDNA was assigned CYP3A27 by the P450 Nomenclature Committee to represent the first CYP3A subfamily member reported for aquatic species. The deduced N-terminal sequence of CYP3A27 was in agreement with 8 of the first 12 confirmed amino acid residues from Edman degradation of LMC5, a P450 previously isolated from juvenile trout liver. In similarity comparisons between species by positional alignment, the deduced amino acid sequence of rainbow trout CYP3A27 was 56.4% identical with dog CYP3A12, 56.0% with monkey CYP3A8, 54.9% with human CYP3A4, 54.7% with rat CYP3A9, and 54.2% with sheep CYP3A24. Marked differences in sex, age, and tissue expression of CYP3A27 in rainbow trout were observed at the mRNA level as shown by Northern blots. The major extrahepatic expression site for CYP3A27 was upper small intestine. Females expressed considerably more CYP3A27 mRNA than male in the fish examined. Southern blot analysis of restriction enzyme-digested rainbow trout genomic DNA demonstrated that multiple CYP3A27-related genes exist in rainbow trout.

The Roles of CYP3A and CYP2B Isoforms in Hepatic Bioactivation and Detoxification of the Pyrrolizidine Alkaloid Senecionine in Sheep and Hamsters

The roles of cytochrome CYP3A and CYP2B isozymes in the bioactivation and detoxification of the pyrrolizidine alkaloid (PA) senecionine (SN) have been investigatedin vitrowith sheep and hamster hepatic microsomes. Our results show that the rate of SN activation measured by (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) formation greatly exceeded the rate of SNN-oxide formation (detoxification) in hamsters. In contrast, SNN-oxide, a detoxification product, was the major metabolite in sheep with much lower DHP production. Immunoinhibition studies with anti-sheep CYP3A and CYP2B antibodies show that members of CYP3A subfamily play the major role in the conversion of PA to pyrrolic metabolites in both species (over 90% in sheep; 68% in hamster). These enzymes also contribute 38.8 and 41.3% of SN N-oxidation in sheep and hamsters, respectively. In contrast, CYP2B isoforms have a limited capacity toward DHP formation in both species (47% in sheep; 32% in hamster), while these enzymes catalyzed only 24.6 and 35.4% SN N-oxidation in sheep and hamster, respectively. Using triacetyloleandomycin (TAO) and gestodene, two highly selective chemical inhibitors of CYP3A isoforms, our data show that 90% of DHP formation was inhibited by either inhibitor in sheep. Gestodene appeared to be more efficient than TAO in the inhibition of DHP production in hamsters. Testosterone 6β-hydroxylase activity, a functional marker of CYP3A, was significantly inhibited by TAO and gestodene in sheep liver microsomes and by gestodene (100 μM) in hamster liver microsomes. These results suggest that CYP3A isozymes have important roles in bioactivation and detoxification of PA in both species, whereas CYP2B subfamily members are less efficient in biotransformation of PA.

Contribution of human CYP3A subfamily members to the 6-hydroxylation of chlorzoxazone

1. The capability of human CYPs other than 2E1 to catalyse the formation of 6hydroxychlorzoxazone (6OHCHZ) was examined in vitro using human liver microsomes. 2. 4-Methylpyrazole, diethyldithiocarbamate (DDC), and rabbit anti-human CYP2E1 antibodies reduced chlorzoxazone 6-hydroxylase activity by 60, 60 and 50 % respectively. The rate of formation of 6OHCHZ by DDC- treated microsomes was reduced further by the 3A inhibitors midazolam, troleandomycin and gestodene and increased by α-naphthoflavone, a 3A4 stimulator. 3. Following preincubation with DDC there were significant correlations (p < 0.05) between the residual CHZ 6-hydroxylase activity and immunoquantified CYP3A levels, and corresponding activities (e.g. midazolam 1′-hydroxylation). Rabbit anti-human CYP3A antibodies alone and in combination with DDC reduced the formation of 6OHCHZ by 47 and 62 % respectively. 4. cDNA expressed CYP3A4, 2E1 and 2D6 exhibited comparable CHZ 6-hydroxylase activity. CHZ modulated 3A4 activity as reflected by midazolam 1′-hydroxylase and 4-hydroxylase activities. 5. CYP3A may make a significant contribution to CHZ 6-hydroxylation and therefore caution should be exercized when chlorzoxazone is employed as a specific 2E1 probe in vitro and in vivo.

CYP2D6- and CYP3A-dependent metabolism of dextromethorphan in humans

The metabolism of dextromethorphan, a drug used to probe genetically determined CYP2D6 activity has been investigated in vivo and in vitro. In vitro, kinetic parameters were determined in adult microsomes: Km for the O-demethylation was much lower than for N-demethylation (7 versus 650 microM) but Vmax was comparable. Fetal liver microsomes actively catalysed the N-demethylation of dextromethorphan, with kinetic parameters (Vmax and Km) quite similar in fetal and adult microsomal preparations while the O-demethylation did not exceed 5% of adult activity. In microsomes, the N-demethylation was inhibited by antibodies raised against CYP3A subfamily members although fetal microsomes were much less sensitive to immunoinhibition than adult microsomes. In vivo, urinary excretion of dextromethorphan and its three demethylated metabolites was examined in 155 adult volunteers and compared between extensive (n = 144, 92.9%) and poor (n = 11, 7.1%) metabolizers. The O-demethylation to dextrorphan is the rate-limiting step of metabolism. In 2D6 poor metabolizers, the N-demethylation to 3-methoxymorphinan is slightly higher than in 2D6 extensive metabolizers but does not compensate defective O-demethylation. The frequency distribution histograms of dextromethorphan/dextrorphan and 3-methoxymorphinan/3-hydroxymorphinan metabolic ratios appeared bimodally distributed, reflecting the participation of CYP2D6 in the O-demethylation reaction. They clearly differed from the random distribution of dextromethorphan/3-methoxymorphinan, and dextrorphan/3-hydroxymorphinan ratios among the population. These data clearly suggest that the N-demethylation of dextromethorphan is dependent on CYP3A and that both CYP2D6 and CYP3A are involved in the overall metabolism of dextromethorphan.