Metabolism Of Bufuralol Research Articles

Tryptophan-75 Is a Low-Energy Channel-Gating Residue that Facilitates Substrate Egress/Access in Cytochrome P450 2D6.

CYP2D6 is a major drug metabolizing enzyme with a buried active site. Channels leading to the active site from various enzyme surfaces are believed to facilitate ligand egress and access to the active site. The present study used molecular dynamics (MD) and in vitro studies with CYP2D6*1 and a Trp75-to-Ala mutant to examine channel gating in CYP2D6 by Trp75. MD simulations measured energy landscapes of Trp75 conformations and simulated substrate passage within channel 2b using bufuralol as a model substrate. Trp75 alternated between multiple stable states that supported substrate transport along channel 2b with low-energy barriers between states (∼ -1 kcal/mol). Trp75 conformations were stabilized primarily by hydrogen bonding between Trp75 and Glu222, Asn226, Ala225, or Gln72. Energy barriers were low between Trp75 conformations, allowing Trp75 to easily move between various conformations over time and to function in both binding to and moving substrates in the 2b channel of CYP2D6. Michaelis-Menten kinetic studies completed with purified enzyme in a reconstituted system showed overall reduced enzyme efficiency for metabolism of bufuralol and dextromethorphan by the Trp75Ala mutant compared with CYP2D6*1. In stopped-flow measurements, k off for dextromethorphan was decreased in the absence of Trp75. Our results support a role for Trp75 in substrate shuttling to the active site of CYP2D6. SIGNIFICANCE STATEMENT: Using combined molecular dynamics and in vitro assays, this study shows for the first time a role for Trp75 as a channel entrance gating residue in the mechanism of substrate binding/unbinding in CYP2D6. Energy landscapes derived from molecular dynamics were used to quantitate the strength of gating, and kinetics assays showed the impact on enzyme efficiency and k off of a Trp75Ala mutation.

CYP2D6 Allelic Variants *34, *17-2, *17-3, and *53 and a Thr309Ala Mutant Display Altered Kinetics and NADPH Coupling in Metabolism of Bufuralol and Dextromethorphan and Altered Susceptibility to Inactivation by SCH 66712.

Metabolic phenotype can be affected by multiple factors, including allelic variation and interactions with inhibitors. Human CYP2D6 is responsible for approximately 20% of cytochrome P450-mediated drug metabolism but consists of more than 100 known variants; several variants are commonly found in the population, whereas others are quite rare. Four CYP2D6 allelic variants-three with a series of mutations distal to the active site (*34, *17-2, *17-3) and one ultra-metabolizer with mutations near the active site (*53), along with reference *1 and an active site mutant of *1 (Thr309Ala)-were expressed, purified, and studied for interactions with the typical substrates dextromethorphan and bufuralol and the inactivator SCH 66712. We found that *34, *17-2, and *17-3 displayed reduced enzyme activity and NADPH coupling while producing the same metabolites as *1, suggesting a possible role for Arg296 in NADPH coupling. A higher-activity variant, *53, displayed similar NADPH coupling to *1 but was less susceptible to inactivation by SCH 66712. The Thr309Ala mutant showed similar activity to that of *1 but with greatly reduced NADPH coupling. Overall, these results suggest that kinetic and metabolic analysis of individual CYP2D6 variants is required to understand their possible contributions to variable drug response and the complexity of personalized medicine.

Impact of age on hepatic cytochrome P450 of domestic male Camborough-29 pigs.

Swine is not only an important species in veterinary medicine research but also a popular animal model for human drug discovery. It is valuable to understand the impact of pig age on abundance and activity of porcine hepatic cytochrome P450 (CYP450). Liver microsomes were prepared from Camborough-29 intact male pigs at the age of 1day and 2weeks and the castrated male pigs at the age of 5, 10, and 20weeks. Hepatic CYP450 content in the liver microsomes was measured using a UV/visible spectroscopic method. The activities of CYP450s were evaluated by metabolism of phenacetin, coumarin, tolbutamide, bufuralol, chlorzoxazone, and midazolam. The porcine hepatic CYP450 content increased with age with a plateau between age 2 and 5weeks. Activities of all CYP450 enzymes increased with age of pigs too. The bufuralol 1'-hydroxylase showed the highest hepatic activities compared with other CYP enzymes at all ages of pigs. The average activities at the age of 20weeks were about five times higher than those at the age of 5weeks for most of the CYP enzymes. With compensation of the ratio of liver to body weights, the overall CYP450 metabolism capability of the pigs may be peaked around ages of 10 to 20weeks. Those findings suggest that metabolism can be significantly different in growing phase of pigs and that the age may be an important factor in porcine medicine evaluation and pig model development.

Defining the in Vivo Role for Cytochrome b5 in Cytochrome P450 Function through the Conditional Hepatic Deletion of Microsomal Cytochrome b5

In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50–90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.

Identification of Human P450 Isoforms Involved in the Metabolism of the Antiallergic Drug, Oxatomide, and Its Kinetic Parameters and Inhibition Constants

Oxatomide is an antiallergic drug used for the treatment of diseases mediated by type I allergy. Recently, terfenadine and astemizole, which have antiallergic actions similar to those of oxatomide, showed side effects on the cardiovascular system. This might be because concomitant drugs such as itraconazole inhibit cytochrome P450 3A4 (CYP3A4), the enzyme responsible for the degradation of terfenadine and astemizole, and thus the blood concentrations of the drugs are abnormally increased. In another article of this issue, we have reported that oxatomide is metabolized by CYP2D6-Val and CYP3A4, and simultaneously inhibits the metabolism of the model substrates for these enzymes. In this study, we performed the kinetic analysis of oxatomide metabolism using microsomes prepared from human liver, and found that the Km and Vmax values were 26.1 microM and 1254.4 pmol/mg protein/min, respectively. Ketoconazole, one of the representative inhibitors for CYP3A4, potently inhibited the metabolism of oxatomide, but other well-known CYP inhibitors did not show significant inhibition. These results suggest that the metabolism of oxatomide is principally catalyzed by CYP3A4. Furthermore, oxatomide inhibited the metabolism of (+/-) bufuralol and testosterone, model substrates for CYP2D6 and CYP3A4, respectively, in a dose-dependent manner with the Ki values of 57.4 and 24.3 microM, respectively. These observations, together with the finding that the putative highest concentration of oxatomide in blood was congruent with 40 ng/ml ( congruent with 93 nM) at 4 h after each dosage during consecutive 6-d administration, encouraged us to conclude that oxatomide won't inhibit CYP2D6 or CYP3A4 at clinical doses.

Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism

The polymorphic human debrisoquine hydroxylase, cytochrome P450 2D6 (CYP2D6), is one of the most important phase I drug metabolising enzymes. It is responsible for metabolising a large number of compounds that mostly share similarity in having a basic N-atom and an aromatic moiety. In homology modelling studies, it has been suggested that in fixation of this aromatic moiety, there may be an important role for phenylalanine 120 (Phe 120). In this study, the role of Phe 120 in ligand binding and catalysis was experimentally examined by mutating it into an alanine. Strikingly, this substitution led to a completely abolished 7-methoxy-4-(aminomethyl)-coumarin (MAMC) O-demethylating activity of CYP2D6. On the other hand, bufuralol metabolism was hardly affected ( K m of 1-hydroxylation mutant: 1.2 μM, wild-type: 2.9 μM, 4-hydroxylation mutant: 1.5 μM, and wild-type: 3.2 μM) and neither was affected dextromethorphan O-demethylation ( K m mutant: 1.2 μM, wild-type: 2 μM, k cat mutant: 4.5 min −1, and wild-type: 3.3 min −1). However, the Phe 120Ala mutant also formed 3-hydroxymorphinan, the double demethylated form of dextromethorphan, which was not detected using wild-type CYP2D6. 3,4-Methylenedioxymethamphetamine (MDMA) was demethylenated by both mutant and wild-type CYP2D6 to 3,4-dihydroxymethamphetamine (3,4-OH-MA K m of mutant: 55 μM and wild-type: 2 μM). In addition, the mutant formed two additional metabolites; 3,4-methylenedioxyamphetamine (MDA) and N-hydroxy-3,4-methylenedioxymethamphetamine ( N-OH-MDMA). Inhibition experiments of dextromethorphan O-demethylation showed a decreased affinity of the Phe 120Ala mutant for quinidine (IC 50 mutant: 240 nM and wild-type, 40 nM), while IC 50s for quinine were equal (1 μM). These data indicate the importance of Phe 120 in the selectivity and regiospecificity in substrate binding and catalysis by CYP2D6.

Metabolism of human cytochrome P450 marker substrates in mouse: a strain and gender comparison

The aim was to characterize mouse gender and strain differences in the metabolism of commonly used human cytochrome (CYP) P450 probe substrates.Thirteen human CYP probe substrates (phenacetin, coumarin, 7-ethoxy-4-trifluoromethyl coumarin, amiodarone, paclitaxel, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, chlorzoxazone, p-nitrophenol, testosterone and lauric acid) were used in activity measurements. The metabolism of the probe substrates was compared in liver microsomes from male and female NMRI, CBA, C57bl/6, 129/SvJ and CD1 strains. The expression of proteins identified on Western blots with commonly available antibodies selective for specific human and rat CYP enzymes were compared in the different mouse strains.Males had higher metabolism than corresponding females for phenacetin O-deethylation (human marker for CYP1A2 activity), and a high correlation was found between phenacetin activity and immunoreactivity in Western blots produced with rat CYP1A2 antibodies.Protein detected by antibodies cross-reacting with human CYP2B6 and rat CYP2B1/2 antibodies was female specific except for the 129/SvJ strain, where it was absent in both genders.Females generally had a higher metabolism of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation (human markers for CYP2D activity).Bufuralol 1′-hydroxylation correlated with a female-dominant mouse CYP, which was detected with antibodies against rat CYP2D4.p-Nitrophenol 2-hydroxylation correlated better than chlorzoxazone 6-hydroxylation with the protein detected with antibodies against rat CYP2E1, indicating that p-nitrophenol is a more specific substrate for mouse CYP2E1.

Downregulation of CYP2D16 by ACTH in the Guinea Pig Adrenal Cortex: Time Course, Reversibility, and Mechanism of Action

Studies were done to investigate the actions of ACTH on the expression of CYP2D16 in the guinea pig adrenal cortex. Guinea pigs were treated with ACTH for 1, 3, or 7 days. In addition, some animals received ACTH for 7 days and were then untreated for an additional 3 or 7 days to test for reversibility of ACTH actions. ACTH treatment caused a time-dependent decrease in the rates of adrenal microsomal bufuralol metabolism, a CYP2D-catalyzed reaction; hepatic bufuralol metabolism was unaffected by ACTH. Adrenal enzyme activity was significantly reduced by ACTH within 1 day and decreased by 80% after 7 days. Western blotting and in situ hybridization analyses revealed corresponding declines in adrenal CYP2D16 protein and mRNA concentrations. Nuclear runoff assays indicated that ACTH treatment inhibited CYP2D16 expression at the transcriptional level. Adrenal 17α-hydroxylase activities were increased by ACTH treatment, but CYP17 protein concentrations were not affected. Following cessation of ACTH administration, the rates of adrenal bufuralol metabolism and CYP2D16 protein and mRNA concentrations returned to control levels within 7 days. The results demonstrate that ACTH has a relatively rapid and reversible effect to inhibit adrenal CYP2D16 transcription, thereby decreasing adrenal xenobiotic metabolism. Thus, the actions of ACTH on CYP2D16 expression are opposite to those on other adrenal P-450 isozymes, indicating unique regulatory mechanisms.

Is cytochrome P450 CYP2D activity present in pig liver?

The presence of CYP2D in pig livers has been studied using different strains of pig, different CYP2D test substrates and monoclonal and polyclonal antibodies. The results of the studies lacked consistency, therefore the aim of this study was to identify the reasons for these inconsistencies. Liver microsomes isolated from conventional pigs and minipigs were tested in Western blotting using both monoclonal and polyclonal antibodies against human CYP2D6. The microsomes were also incubated with three different CYP2D tes t substrates.'The immunoblotting only gave a positive response when hybridised with polyclonal antibody. The pig microsomes did not metabolise debrisoquine, but metabolised two other test substrates, dextromethorphan and bufuralol. No correlation was found between the two enzyme assays and CYP2D apoprotein level. On the other hand positive correlations were found between dextromethorphan and bufuralol metabolism and the CYP2B immunochemical protein level, indicating that the CYP2B isoenzyme may be involved in the metabolism of these substrates. Further, assays using immunoinhibition and chemical inhibition of these reactions were performed. No response was obtained in the immunoinhibition assay. When using chemical inhibition, however, an average inhibition percentage of 83 were obtained with orphenadrine, a human CYP2B inhibitor. Average Ki values of 26.9 microM and 43.6 microM for orphenadrine indicate that it was a potent inhibitor. A rat and a mouse CYP2B inhibitor, resveratrol and pilocarpine, inhibited the reaction with an average of 40 and 70 percentage respectively. Orphenadrine did not inhibit CYPIA, CYP2A, CYP2E and CYP3A activities up to more than maximum 12 percentage, showing that it was almost selective for dextromethorphan metabolism. These results indicate that dextromethorphan and bufuralol metabolism may be catalysed by CYP2B and not CYP2D.

Inhibition kinetics of monoclonal antibodies against cytochromes P450.

Monoclonal antibodies (MAbs) inhibitory to individual cytochromes P450 (P450s) are of tremendous utility in identification of P450s responsible for the metabolism of a given drug or drug candidate in pharmaceuticals. In the present study, two inhibitory MAbs against CYP2D6 (MAb(2D6-50,) IgG(2b) and MAb(2D6-184), IgG(2a)) were developed by hybridoma technology to exhibit their high specificity and potency. The MAbs were further employed to assess the quantitative role (47-93%) of CYP2D6 to the metabolism of bufuralol in human liver microsomes from seven donors. Together with the MAb inhibitory to CYP3A4 as previously reported (Mei et al., 1999), the MAbs were used to study the inhibition kinetics of dextromethorphan O-demethylation (CYP2D6), testosterone 6beta-hydroxylation (CYP3A4) and aflatoxin B1 3-hydroxylation (CYP3A4), respectively, with an adequate size of sample measurement. A kinetic model was proposed to fit the experimental observations with three-dimensional nonlinear regression, thereby resulting in a solution of kinetic parameters, i.e., K(I), K(S), V(max), alpha, and beta (changes in K(I) or K(S) and V(max) in the presence of the MAb). As a result, dissociation constants (K(I)) of the MAbs for the enzymes and the maximal inhibition (beta) values for the P450-catalyzed reactions were predicted to have 0.04 to 0.25 microM and > or =94%, respectively. The results have demonstrated that the model can accurately predict the kinetic parameters and provide some insights into the understanding of the mechanism of MAb interaction with P450 enzyme in nature and the applications of the MAbs in qualitative and quantitative identification of P450s involved in drug metabolism.

Expression, Purification, and Biochemical Characterization of A Human Cytochrome P450 CYP2D6-NADPH Cytochrome P450 Reductase Fusion Protein

Cytochrome P450 CYP2D6 metabolizes a wide range of pharmaceutical compounds. A CYP2D6 fusion enzyme (CYP2D6F), containing an amino-terminal human CYP2D6 sequence and a carboxyterminal human NADPH-cytochrome P450 oxidoreductase (CPR) moiety, was constructed. High levels of expression were achieved in Escherichia coli (60–100 nmol/liter) and the enzyme was catalytically active with optimal activities achieved in the presence of the antioxidant, GSH. Turnover values for bufuralol 1′-hydroxylation, metoprolol α-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation, using membranes expressing the fusion enzyme, were 5.6, 0.4, 0.72, and 6.19 min−1, respectively. These values were similar to E. coli membranes which coexpressed human CYP2D6 and CPR (CYP2D6/R). The Km and kcat values for bufuralol metabolism were estimated to be 10.2 μM and 4.1 min−1, respectively. The enzyme was purified using ion-exchange chromatography, affinity chromatography (2′-5′ ADP-Sepharose), and gel filtration. Estimated turnover rates for bufuralol 1′-hydroxylation, metoprolol α-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation were 1.2, 0.52, 0.79, and 0.76 min−1, respectively. Bufuralol 1′-hydroxylase activity by purified CYP2D6F was enhanced by phospholipids and added CPR. The CYP2D6F enzyme was able to stimulate CYP3A4 testosterone 6β-hydroxylase activity in a reconstitution system indicating that electron transfer may be largely intermolecular. The catalytically self-sufficient CYP2D6F enzyme will facilitate investigations of P450-CPR interactions and the development of new biocatalysts.

Interaction of drugs and Chinese herbs: pharmacokinetic changes of tolbutamide and diazepam caused by extract of Angelica dahurica.

The inhibitory effects of Angelica dahurica root extract on rat liver microsomal cytochrome P450 and drug-drug interactions were studied. The 2alpha- and 16alpha-hydroxylase activity of testosterone were most strongly inhibited, with 17.2% and 28-5% of their activity remaining, respectively, after oral administration of A. dahurica extract at a 1 g kg(-1) dose. 6beta-Hydroxylase activity was also inhibited, with 70% of its activity remaining, under the same conditions. In addition, treatment with the extract inhibited the metabolism of tolbutamide, nifedipine and bufuralol. These results showed that the extract inhibited the various isoforms of cytochrome P450 such as CYP2C, CYP3A and CYP2D1. The A. dahurica extract delayed elimination of tolbutamide after intravenous administration at a 10 mg kg(-1) dose to rats. Thus, the extract altered the liver intrinsic clearance. It had little effect, however, on the pharmacokinetic parameters of diazepam after intravenous administration at 10 mg kg(-1). Since diazepam showed high clearance, it underwent hepatic blood flow rate-limited metabolism. Therefore, the change of intrinsic clearance had little effect on hepatic clearance. However, the Cmax value after oral administration of diazepam with extract treatment was four times that with non-treatment. It was suggested that the first-pass effect was changed markedly by the extract. High-dose (1 g kg(-1)), but not low dose (0.3 g kg(-1)), administration of A. dahurica extract increased significantly the duration of rotarod disruption following intravenous administration of diazepam at 5 mg kg(-1). It was concluded that administration of A. dahurica extract has the potential to interfere with the metabolism, by liver cytochrome P450, of other drugs.

Metabolism of artelinic acid to dihydroqinghaosu by human liver cytochrome P4503A

1. Artelinic acid (AL), a water-soluble artemisinin analogue for treatment of multidrug resistant malaria, is metabolized to the active metabolite dihydroqinghaosu (DQHS) solely by CYP3A4/5. Although AL is not metabolized by CYP2C9, it does inhibit diclofenac 4-hydroxylase activity with an IC50 = 115 muM. Interestingly, AL activates CYP2D6-mediated bufuralol metabolism in human liver microsomes but not recombinant CYP2D6-Val by ~30% at AL concentrations up to 100 muM. 2. In human liver microsomes, AL is metabolized to DQHS with a Km = 157 +/- 44 muM and Vmax = 0.77 +/- 0.56 nmol DQHS/min/mg protein. Human recombinant CYP3A4 catalysed the conversion of AL to DQHS with a Km = 102 +/- 23 muM and a Vmax = 1.96 +/- 0.38 nmol DQHS/min/nmol P450. The kinetic parameters (Km and Vmax) for DQHS formation from CYP3A5 were 189 +/- 19 muM and 3.60 +/= 0.42 nmol DQHS/min/nmol P450 respectively. 3. Inhibition studies suggest that azole antifungals and calcium channel blockers may present clinically significant drug-drug interactions. In human liver microsomes, ketoconazole and miconazole were potent competitive inhibitors of DQHS formation with a Ki = 0.028 and 0.124 muM respectively. Verapamil is a non-competitive inhibitor of DQHS formation in human liver microsomes with a Ki = 15muM.

A monoclonal antibody inhibitory to human P450 2D6: a paradigm for use in combinatorial determination of individual P450 role in specific drug tissue metabolism.

Human cytochrome P450 2D6 metabolizes more than 50 common drugs and is polymorphically expressed, with 5-10% of the population lacking expression caused by mutant genes. This may result in a defective and toxic response in deficient individuals treated with 2D6 drug substrates. Baculovirus-expressed 2D6 was used to immunize mice for hybridoma production and two clones yielded monoclonal antibodies, that were positive against 2D6 by ELISA and inhibited 2D6 catalysed metabolism of bufuralol, dextromethorphan and phenanthrene by more than 90%. The inhibitory activity was highly specific to 2D6 and the monoclonal antibodies did not bind to 11 other P450s, nor inhibit seven human P450s tested. Analysis of eight human liver microsome samples showed that their basal bufuralol 1'-hydroxylase activity varied from 6.7-83.5 pmol min-1 nmol-1 P450. The monoclonal antibody 512-1-8 inhibited 2D6-dependent bufuralol 1'-hydroxylase in these samples by 10-70% indicating a widely variable role for 2D6 in human liver bufuralol 1'-hydroxylase activity and a role for other P450s in bufuralol metabolism. Independent analysis of several recombinant human P450s showed that 2D6, 2C8, 2C9, 2C19 and 1A2 exhibited bufuralol 1'-hydroxylase activity with 2D6 and 2C19 being the most active. Further analysis of three liver samples was made with individual inhibitory monoclonal antibodies. Inhibitory antibodies to 2D6, 2B6, 2E1, 2C8/9/19, 3A4 and 1A2 were added to the microsomes either singly or additively. Inhibitory activity of bufuralol 1'-hydroxylase was observed with antibodies to 2D6 (14-76%), 2C8/9/19 (24-69%) and 1A2 (2-25%) indicating a variable and different role for each of these P450s in the bufuralol 1'-hydroxylase of human liver. The monoclonal antibodies to 2B6, 2E1 and 3A4 were not inhibitory, indicating that these enzymes play no role in bufuralol 1'-hydroxylase metabolism. When the three antibodies to 2D6, 2C8/9/19 and 1A2, respectively, were all added, the total bufuralol 1'-hydroxylase of the liver samples was inhibited by more than 90%, indicating that the latter P450s catalyse all of liver bufuralol 1'-hydroxylase metabolism. These studies demonstrate that inhibitory monoclonal antibodies offer a simple and precise method for assessing the quantitative role of each P450 in the metabolism of a P450 substrate in a tissue, which include drugs, carcinogens, mutagens, toxic chemicals and endobiotics.

Functional High Level Expression of Cytochrome P450 CYP2D6 Using Baculoviral Expression Systems

Cytochrome P-450 CYP2D6 plays a central role in the metabolism of many widely used therapeutic drugs including β-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. Recombinant baculoviruses have been constructed containing the full-length human CYP2D6 cDNA and used to express CYP2D6 inSpodoptera frugiperda(Sf9) cells. High levels of recombinant protein have been produced using either polyhedrin or basic protein promoters (0.05–0.20 nmol/mg cell protein; 0.05–0.15 nmol/liter). The enzyme is catalytically active toward CYP2D6 substrates such as bufuralol and metoprolol. In order to optimize catalytic activity human reductase was coexpressed with CYP2D6 in Sf9 cells; reductase activity was in the region of 1000–1500 units per mg cell protein, while spectrally active CY2D6 was in the range 10–20 pmol/mg cell protein. TheKmandKcatvalues for bufuralol metabolism were estimated as 4.7 μMand 12.23 min−1, respectively. The use of the conventional very late promoters such as the polyhedrin promoter generate a large proportion of inactive CYP2D6. The problem was to a degree circumvented using the “late” basic promoter which is active earlier in the baculovirus infection cycle. The yield of functional CYP2D6 was at least as high as with very late promoters, but the proportion of inactive protein was reduced. Bufuralol hydroxylase activity could be measured directly by HPLC analysis of cell culture media supplemented with bufuralol, and we have developed a plate assay system which provides a simple method for the analysis of drug metabolism reactions using Sf9 cells. Expression using baculovirus provides a valuable source of functional CYP2D6 for work aimed at elucidating the structure and function of the enzyme.

(+)-Bufuralol 1′-hydroxylation activity in human and rhesus monkey intestine and liver

(+)-Bufuralol 1′-hydroxylation, a commonly used marker of hepatic CYP2D6 activity, was investigated in human and rhesus monkey intestinal microsomes and compared with that in hepatic microsomes. The cumene hydroperoxide (CuOOH)-mediated metabolism of (+)-bufuralol suggested that at least two enzymes were responsible for bufuralol 1′-hydroxylation in both human and monkey intestinal microsomes. In contrast, the kinetics of the CuOOH-mediated metabolism in human and monkey livers were monophasic. The K m values for the higher affinity component of the intestinal enzyme(s) of both species were similar to, while the corresponding V max, values were much lower than, those obtained with the livers. Bufuralol metabolism mediated by NADPH exhibited biphasic kinetics and was less efficient than that observed in the presence of CuOOH in both human and monkey intestines, in agreement with the observations in the livers. Inhibition of bufuralol hydroxylase activity in the intestine and liver preparations from the same species by known CYP2D6 inhibitors/ substrates was qualitatively similar. Quinidine was the most potent inhibitor of (+)-bufuralol 1−'-hydroxylation in all tissues studied. Western immunoblots using anti-CYP2D6 peptide antibody revealed a protein band in human and monkey intestinal microsomes of the same molecular weight as that observed in the liver preparations. The intestinal CYP2D protein content appeared to be much less than that of liver, and correlated with the (+)-bufuralol hydroxylase activity. Immunoinhibition studies indicated significant (up to 50%) inhibition of the CuOOH-mediated (+)-bufuralol metabolism in human and monkey intestines only by anti-CYP2D6, and not by anti-CYP2A6, or anti-CYP2E1. Inhibition of the bufuralol 1′-hydroxylase activity by anti-rat CYP3 A1 was only slight (20%) in human, but marked (60–65%) in monkey intestinal microsomes. The hepatic metabolism of (+)-bufuralol in humans and monkeys was only inhibited (75%) by anti-CYP2D6, but not by anti-CYP3A1. Overall, the results suggest that (1) tissue and species differences exist in the catalysis of (+)-bufuralol 1′-hydroxylation, and (2) CYP2D6-related enzymes are partially or primarily responsible for the bufuralol hydroxylase activity in human and monkey intestines or monkey liver.

Inhibitory effects of antiparasitic drugs on cytochrome P450 2D6.

The interaction of antiparasitic drugs with the polymorphic cytochrome P450 2D6 was studied in human liver microsomes. Of ten different drugs tested, three quinolines, oxamniquine, primaquine and chloroquine inhibited microsomal CYP2D6-catalysed formation of 1'hydroxybufuralol at concentrations that might have clinical consequences in drug use. These drugs inhibited competitively bufuralol metabolism with Ki values of 22, 23 and 15 microM, respectively, indicative of high affinity for the CYP2D6-active site. The results imply that oxamniquine, primaquine and chloroquine could be substrates of cytochrome P4502 D6 or that they are potent non-substrate inhibitors of the enzyme similar to quinidine. In either case, the inhibition of CYP2D6 by these agents could lead to interference with in vivo population-phenotyping procedures in the tropical regions where treatment with the drugs is common.

PREDICTION OF DRUG-DRUG INTERACTION CAUSED BY CYP3A INHIBITION: FROM IN VITRO TO IN VIVO, FROM ANIMAL TO HUMAN

Inhibitory effect of E4080 on the cytochrome P450 was studied using laboratory animals in vitro and in vivo.In vitro study E4080 has a high affinity for P450 heme in rat, dog, monkey and human liver microsomes. In rat microsomes, E4080 inhibited testosterone 6β and 16β-hydroxylase which are involved in CYP2B and CYP3A. Using human microsomes, E4080 inhibited metabolism of 7-benzyloxyresolfin, tolbutamide, bufuralol and testosterone. These results suggest that E4080 inhibits CYP 2B6, 2C9, 2D6 and 3A4 in human liver microsomes.In vivo study E4080 prolonged antipyrine half-life and decreased antipyrine clearance in rats, dogs and monkeys at the doses of 10mg/kg which exert pharmacological effect postulated from the minimal effective plasma concentrations(1-2 μg/ml ). The results may be suggested that E4080 interacts with antipyrine metabolism which is thought to be carried out by CYP 1A, 2C and 3A. In the clinical trial E4080 (100mg/man) decreased the excretion of urinary 6β-hydroxycortisol which is a marker of human hepatic cytochrome P4503A induction and inhibition.In conclusion, it is suspected that E4080 inhibits CYP3A not only in animals, but also in human and should be noticed drug-drug interaction in clinical use.

Identification of a new variant CYP2D6 allele lacking the codon encoding Lys-281: possible association with the poor metabolizer phenotype

A variant CYP2D6(C) P450 protein was found in a liver characterized by deficient microsomal metabolism of bufuralol and sparteine, prototypical substrates for the debrisoquine-sparteine drug oxidation polymorphism. This protein was present at decreased levels in liver and had a slightly different relative mobility on SDS-polyacrylamide gels. The cDNA cloning and sequencing of the variant, designated CYP2D6(C), revealed that its mRNA lacked a single codon resulting in deletion of Lys281. This was the result of a three base pair deletion at the 3' end of CYP2D6 exon 5. The CYP2D6(C) P450, produced in HepG2 cells using vaccinia virus mediated cDNA expression displayed Km values toward bufuralol, debrisoquine and sparteine that were not significantly different from wild type CYP2D6. These data suggest that the poor metabolizer phenotype in livers expressing CYP2D6(C) is not due to a catalytically defective enzyme but perhaps due to decreased levels of the P450 protein in microsomal membranes. Low microsomal CYP2D6(C) contents could result from deficient membrane insertion or decreased stability of the P450 protein. A polymerase chain reaction-based procedure, developed to detect CYP2D6(C) alleles, indicates that this variant probably represents less than 1.5% of all CYP2D6 alleles.

Cytochrome P450IID subfamily in non-human primates: Catalytical and immunological characterization

Interindividual variations of debrisoquine metabolism was recently identified in non-human primates tested in vivo. The catalytical and immunological characterization of cytochrome P450IID subfamily was undertaken in hepatic microsomes from extensive metabolizer primates. The NADPH/ O 2 mediated metabolism of debrisoquine, dextromethorphan and bufuralol was similar to the kinetics reported in humans. The CuOOH mediated metabolism of bufuralol suggested that at least two enzymes are responsible for bufuralol 1'-hydroxylation. Eleven compounds were tested for their capacity to modify P450IID function vitro. Eight competitive inhibitors of P450IID6 in man were all and exclusively competitive inhibitors of P450IID subfamily in non-human primates. Quinidine, which is the strongest competitive inhibitor in man, exhibited the higher inhibitory potency in monkey ( K i = 0.75 μM). Anti-LKM antibody against P450IID subfamily cross-reacted with two proteins of 49 and 47 kDa, and sera containing anti-LKM antibody against these two proteins inhibited dextrorphan formation in vitro. These data provide evidence for catalytical and immunological similarities between human and monkey microsomes and indicate that the primate system could be a model for enzymatic studies of P450IID.