Rat CYP2D1 Research Articles

In vitro metabolism of a novel phosphodiesterase-5 inhibitor DA-8159 in rat liver preparations using liquid chromatography/electrospray mass spectrometry.

The in vitro metabolism of a new erectogenic, DA-8159, has been studied by LC with UV detection and on-line LC-electrospray mass spectrometry using rat hepatic microsomal incubation and rat liver perfusion. Both rat liver microsomal incubation of DA-8159 in the presence of NADPH and single-pass liver perfusion of DA-8159 resulted in the formation of three metabolites (M1-3). M1 was tentatively identified as hydroxy-DA-8159. M2 and M3 were identified as N-demethyl-DA-8159 and 5-(2-propyloxy-5-aminosulphonylphenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo(4,3-d)pyrimidin-7-one (DA-8164), respectively, on the basis of LC-MS/MS analysis with authentic standards. Rat CYP2D1 was a major isozyme for the formation of hydroxy-DA-8159 and N-demethyl-DA-8159. CYP2C12 and CYP1A1 catalysed the oxidation of DA-8159 to DA-8164.

Inactivation of rat cytochrome P450 2D enzyme by a further metabolite of 4-hydroxypropranolol, the major and active metabolite of propranolol.

Repetitive administration of propranolol (PL) in rats decreases the activities of cytochrome P450 (CYP) 2D enzyme(s) in hepatic microsomes. We examined the properties of 4-hydroxypropranolol (4-OH-PL) as an inactivator of rat liver microsomal CYP2D enzyme(s) using bunitrolol (BTL) 4-hydroxylation and PL 5- and 7-hydroxylations as indices of CYP2D enzyme activity. Rat microsomal BTL 4-hydroxylase activity was inhibited by the addition of 4-OH-PL to the incubation medium. The inhibition was greater after preincubation of microsomes with 4-OH-PL in the presence of NADPH than in its absence. The type of inhibition kinetics of BTL 4-hydroxylase by 4-OH-PL was changed from a competitive type to a noncompetitive type by the preincubation. The inhibition of rat liver microsomal PL 5- and 7-hydroxylases by 4-OH-PL was blocked efficiently by co-incubation with quinine, a typical inhibitor of rat CYP2D enzyme(s), or to a lesser extent by BTL. However, quinidine, a diastereomer of quinine, did not significantly protect against the enzyme inactivation. The protective capacities of the substrate and inhibitors reflected their affinities for rat CYP2D enzyme(s). BTL hydroxylase was not affected by either 1,4-naphthoquinone or 1,4-dihydroxynaphthalene which are possible metabolites of 4-OH-PL. These results provide further evidence to support the notion that PL is biotransformed by rat CYP2D enzyme(s) to 4-OH-PL, which is further oxidized to a chemically reactive metabolite in the active site. The inactivation of CYP is likely the result of covalent binding of the reactive species to an amino acid residue of the active site.

Interaction of bisphenol A with rat hepatic cytochrome P450 enzymes

The effect of bisphenol A (BPA) on the kinetics of cytochrome P450 (P450)-dependent monooxygenases in rat liver microsomes was studied. Testosterone 16β-hydroxylase (TS16BH) and testosterone 2α-hydroxylase (TS2AH) activities were extensively inhibited by BPA at 100 μM (69% and 74%, respectively). The inhibition type was mixed for both P450-dependent monooxyganases. The K i of TS16BH and TS2AH from Lineweaver–Burk plots were 25.9 and 24.9 μM, respectively. The activities of acetanilide 4-hydroxylase (AA4H), 7-ethoxycoumarin O-deethylase (ECOD), bufuralol 1′-hydroxylase (BF1′H), chlorzoxazone 6-hydroxylase (CZ6H) and testosterone 6β-hydroxylase (TS6BH) were also effectively inhibited by BPA at 100 μM (43–52%). The inhibition type of these P450-dependent monooxygenases was mixed or uncompetitive, and the K is (50.5–88.5 μM) were higher than those of TS16BH and TS2AH. By contrast, the values of IC 50 and K i of testosterone 7α-hydroxylase (TS7AH) and lauric acid ω-hydroxylase (LAOH) for BPA were >1000 μM. These results suggest that BPA interacts with rat hepatic CYP1A2, CYP2A2, CYP2B2, CYP2C11, CYP2D1, CYP2E1 and CYP3A2 in vitro.

Regional and cellular distribution of CYP2D subfamily members in rat brain

1. Human CYP2D6 is present in brain, metabolizes many drugs and has been implicated in Parkinson's and Alzheimer's diseases and some cancers. It is still unclear which of the six known rat CYP2D subfamily members is are homologous to human CYP2D6. 2. In this study, RT-PCR, Southern and Western blotting and immunohistochemical techniques were used to study the distribution of CYP2D subfamily member mRNA and proteins across 10 rat brain regions. CYP2D subfamily mRNA and protein levels were correlated with brain dextromethorphan O-demethylation (DOD), a measure of human CYP2D6 and rat CYP2D1 activities. 3. The data showed a strong relationship between CYP2D1 and CYP2D1-18 with brain DOD activity. In addition, it was shown that CYP2D proteins are present in brain mitochondrial as well as microsomal membranes. CYP2D subfamily member mRNA and proteins varied across brain regions and were highly concentrated in specific cell types. 4. These data strongly suggest that CYP2D1 and not CYP2D5 mediates DOD activity in rat brain, and may be the rat homologue of human CYP2D6. The highly localized nature of CYP2D indicates that in specific neurones enzyme levels may approach hepatic levels and, hence, contribute to local alterations in brain drug metabolism.

Oxidation of Optically Active Substrates by Human and Rat CYP2D Enzymes.

Oxidation of Optically Active Substrates by Human and Rat CYP2D Enzymes.

THE PROPERTY OF EXPRESSED CYP2D6*10 AND ITS POSSIBILITY AS A TOOL FOR PREDICTING HUMAN PHARMACOKINETICS

Interindividual differences in the pharmacokinetics of venlafaxine, a new antidepressant, were shown during early clinical trials in Japan. Venlafaxine is metabolized mainly by CYP2D6 to an active metabolite, O-desmethylvenlafaxine (ODV). We have found that the CYP2D6*10 genotype influenced the pharmacokinetics of venlafaxine in Japanese population. We have recently expressed the CYP2D6*10A, *10C, *1(wild) and other two variants in yeast cell. The CO-reduced difference spectra of the variants conteining amino acid substitution Pro34->Ser resulted in a dominant absorption at 420nm as well as 450nm, suggesting that the substitution caused a unstabl form proteine. In addition, exchange of the proline residue to serine at same position in rat CYP2D1 also increased P420, denatured P450. The apparent Km values of CYP2D6* 10A, CYP2D6* 10C and CYP2D6* 1(wild) for bufuralol 1'-hydroxylation were 6.39, 63.1 and 1.31μM, and for venlafaxine O-demethylation 83.6, 82.2 and 9.91 p.M, respectively. These findings suggest that the decreased disposition of venlafaxine in vivo was caused not only instability but also increased Km value of CYP2D6* 10. We proposed that these enzymes in vitro may be used as a tool for predicting the effect of the CYP2D6*10 genotypes on human in vivo pharmacokinetics.

CHARACTERIZATION OF OXIDATION OF OPTICALLY ACTIVE SUBSTRATES BY RAT AND HUMAN CYP2D ENZYMES EXPRESSED IN SACCHAROMYCES CEREVISIAE

To validate the usefulness of recombinant CYP enzymes in metabolic studies of optically active drugs, enantiomer selectivity was examined for oxidation of optically active R-blokers by rat CYP2D enzymes (CYP2D1, -2D2, -2D3 and -2D4) and human CYP2D6 expressed in yeast cells, and was compared with the profile of liver microsomes (Ms). In bunitrolol (BTL) 4hydroxylation rate, selectivity of [(+)-BTL S(-)-PL] in a low PL concentration range. CYP2D2 having the highest PL oxidation activities among the rat CYP2D enzymes showed selectivity of [R(+)PL S(-)-PL] in a low PL concentration range, whereas recombinant CYP2D6 did not show clear selectivity. PL oxidation by human CYP2D6 showed biphasic kinetics. Kinetics of racemic bufuralol (BUF) were monophasic for rat CYP2D2 and human CYP2D6 but biphasic for rat CYP2D4. Considering these results, different enantioselectivity between liver Ms and recombinant CYP2D enzymes and biphasic kinetics for recombinant CYP enzymes may be caused by different microenvironments surrounding CYP enzymes between liver and yeast cells.

Cytochrome P450 isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of NS-21

1. Cytochrome P450 (P450) isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of (±)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2- hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21) have been identified in rat and man. 2. Anti-CYP2C11 antibody inhibited the N-deethylation of S- and R-NS-21 in rat hepatic microsomes by 84 and 66% respectively, indicating that CYP2C11 is mainly responsible for these activities in male rats. 3. Of several human recombinant P450 isoforms, CYP3A4 had the activities for the Ndeethylation of S- and R-NS-21. In addition, triacetyloleandomycin (TAO), an inhibitor of the CYP3A subfamily, significantly inhibited the N-deethylation of S- and R-NS-21 in human hepatic microsomes by 67 and 69%, respectively. CYP3A4 therefore contributes to it in man. 4. Quinine, an inhibitor of the rat CYP2D subfamily, significantly inhibited the cyclohexane-4-cis-hydroxylation of S-NS-21 by 48% in rat hepatic microsomes. In contrast, this inhibitor had little effect on the cyclohexane-4-trans-hydroxylation of S-NS21, and the cyclohexane-4-cis-and trans-hydroxylation of R-NS-21. 5. Human recombinant CYP3A4 catalysed the cyclohexane-4-trans-hydroxylation of S-NS-21, and CYP2D6 supported the cyclohexane-4-cis- and trans-hydroxylation of S-NS-21. Quinidine, an inhibitor of human CYP2D6, had little effect on these latter activities in human hepatic microsomes. TAO significantly inhibited the cyclohexane-4-trans-hydroxylation of S-NS-21 by 75%, indicating that CYP3A4 catalyses this reaction.

In vitro biotransformation of atrazine by rat liver microsomal cytochrome P450 enzymes

We studied atrazine (ATZ) metabolism in male and female rat liver microsomes in vitro, and the major metabolite was deisopropylatrazine (DeiPr-ATZ) with deethylatrazine (DeEt-ATZ) and 1-hydroxyisopropylatrazine (iPrOH-ATZ) as minor metabolites in both sexes. The enzyme kinetics of ATZ biotransformation were examined by means of Eadie–Hofstee analyses. Although no remarkable sex difference of Michaelis–Menten values for each pathway was observed, Cl ints ( V max/ K m) for DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ were slightly higher in female than in male rats. The formation of DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ from ATZ was substantially inhibited by SKF-525A, metyrapone, diallyl sulfide, 7-ethoxycoumarin, benzphetamine, nicotine, testosterone and lauric acid in both sexes. Cimetidine effectively inhibited the formation of all metabolites in male rats. On the other hand, the inhibition rates of the formation of DeiPr-ATZ and iPrOH-ATZ by cimetidine in female rats were lower than those in male rats, and DeEt-ATZ was hardly affected by the chemicals. In contrast with the results for cimetidine, the inhibition of ATZ biotransformation by bufuralol was more effective in female than in male rats. Anti-rat CYP2B1 and CYP2E1 antibodies effectively inhibited DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ formations in both sexes. Anti-rat CYP2C11 antibody also inhibited the three metabolites in both sexes, with the inhibition rates higher in male than in female rats, similar to cimetidine. In the case of anti-rat CYP2D1 antibody, the inhibitory effect on ATZ biotransformation in male rats was less than that in female rats. On the other hand, anti-rat CYP1A2, CYP3A2 and CYP4A1 antibodies did not affect the ATZ biotransformation in either sex. There was no significant correlation between the formation rate of ATZ metabolites and P450 isoform levels in either sex. These results may mean that CYP2B2, CYP2C11, CYP2D1 (only iPrOH-ATZ formation) and CYP2E1 in male rats, and CYP2B2, CYP2D1 and CYP2E1 in female rats are involved ATZ metabolism in liver, and that the substrate specificity of P450 isoforms for ATZ is broad.

Expression and Characterization of Canine Cytochrome P450 2D15

CYP2D15 is the canine ortholog of human CYP2D6, the human CYP2D isoform involved in the metabolism of drugs such as antiarhythmics, adrenoceptor antagonists, and tricyclic antidepressants. Similar to human, canine CYP2D15 is expressed in the liver, with detectable levels in several other tissues. Three different CYP2D15 cDNA clones were obtained by RT-PCR from dog liver RNA. Two clones corresponded to variant full-length CYP2D15 cDNAs (termed CYP2D15 WT2 and CYP2D15 V1); the third was identified as a splicing variant missing exon 3 (termed CYP2D15 V2). Recombinant baculoviruses were constructed containing full-length cDNAs and used to express CYP2D15 WT2 and CYP2D15 V1 inSpodoptera frugiperda(Sf9) cells with expression levels of up to 0.14 nmol/mg cell protein. As with human CYP2D6, the recombinant CYP2D15 enzymes exhibited bufuralol 1′-hydroxylaseand dextromethorphanO-demethylase activities whencoexpressed with rabbit NADPH:P450 oxidoreductase. For bufuralol 1′-hydroxylase, apparentKmvalues were 4.9, 3.7, and 2.5 μM and theVmaxvalues were 0.14, 0.034, and 0.60 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. For dextromethorphanO-demethylase, apparentKmvalues were 0.6, 0.6, and 2.0 μM and theVmaxvalues were 0.18, 0.034, and 0.057 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. The human CYP2D6-specific inhibitor quinidine and the rat CYP2D1-specific inhibitor quinine were both shown to be inhibitors of bufuralol 1′-hydroxylase activity for dog liver microsomes, CYP2D15 WT2, and the CYP2D15 V1 variant with nearly equal potency. Thus, the dog expresses a CYP2D ortholog possessing enzymatic activities similar to human CYP2D6, but is affected by the inhibitors quinine and quinidine in a manner closer to that of rat CYP2D1.

Tissue distributions of CYP2D1, 2D2, 2D3 and 2D4 mRNA in rats detected by RT-PCR

The tissue distributions of four isoforms (CYP2D1/5, 2D2, 2D3 and 2D4/18) in rat CYP2D subfamily were investigated. Twelve kinds of tissue (liver, kidney, brain, lung, heart, spleen, adrenal gland, small intestine mucosa, bladder, testis, ovary and gonecystis) were removed from Sprague–Dawley male and female rats. The expression of CYP2D mRNA in these tissues was detected by RT-PCR. Specific primers were designed to recognize the four isoforms individually. In liver, kidney and small intestine mucosa, the mRNA expression of all four CYP2D isoforms was detected as high-intensity PCR products. mRNA of CYP2D1/5 was expressed in all tissues used in this study except the brain, although the intensity of PCR products varied among tissues. mRNAs of CYP2D2 and CYP2D3 were mainly expressed in liver, kidney and small intestine mucosa, which were exposed to xenobiotics such as drugs, food components and environmental contaminations. mRNA of CYP2D4/18 was expressed in liver, kidney, small intestine mucosa and brain. In brain, only mRNA of CYP2D4/18 was expressed. CYP2D4/18 mRNA was also expressed in ovary, testis and gonecystis. The tissue distributions help to clarify the differences in physiological and pharmacological functions between CYP2D isoforms.

Cytochrome P450-dependent drug oxidation activities in liver microsomes of various animal species including rats, guinea pigs, dogs, monkeys, and humans.

Levels of cytochrome P450 (P450 or CYP) proteins immunoreactive to antibodies raised against human CYP1A2, 2A6, 2C9, 2E1, and 3A4, monkey CYP2B17, and rat CYP2D1 were determined in liver microsomes of rats, guinea pigs, dogs, monkeys, and humans. We also examined several drug oxidation activities catalyzed by liver microsomes of these animal species using eleven P450 substrates such as phenacetin, coumarin, pentoxyresorufin, phenytoin, S-mephenytoin, bufuralol, aniline, benzphetamine, ethylmorphine, erythromycin, and nifedipine; the activities were compared with the levels of individual P450 enzymes. Monkey liver P450 proteins were found to have relatively similar immunochemical properties by immunoblotting analysis to the human enzymes, which belong to the same P450 gene families. Mean catalytic activities (on basis of mg microsomal protein) of P450-dependent drug oxidations with eleven substrates were higher in liver microsomes of monkeys than of humans, except that humans showed much higher activities for aniline p-hydroxylation than those catalyzed by monkeys. However, when the catalytic activities of liver microsomes of monkeys and humans were compared on the basis of nmol of P450, both species gave relatively similar rates towards the oxidation of phenacetin, coumarin, pentoxyresorufin, phenytoin, mephenytoin, benzphetamine, ethylmorphine, erythromycin, and nifedipine, while the aniline p-hydroxylation was higher and bufuralol 1'-hydroxylation was lower in humans than monkeys. On the other hand, the immunochemical properties of P450 proteins and the activities of P450-dependent drug oxidation reactions in dogs, guinea pigs, and rats were somewhat different from those of monkeys and humans; the differences in these animal species varied with the P450 enzymes examined and the substrates used. The results presented in this study provide useful information towards species-related differences in susceptibilities of various animal species regarding actions and toxicities of drugs and xenobiotic chemicals.

Multiple forms of human P450 expressed in Saccharomyces cerevisiae: Systematic characterization and comparison with those of the rat

We systematically characterized the levels and substrate specificity of P450s from humans and rats to extrapolate drug metabolism data from experimental animals to humans. Human P450s (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4) were expressed in Saccharomyces cerevisiae and purified. Rat P450s were purified from hepatic microsomes of rats. We investigated the catalytic activities of purified P450s in a reconstituted system. Human CYP2B6 and rat CYP2B1 had high lidocaine N-deethylation activity. Human and rat CYP2D forms had high debrisoquine 4-hydroxylation activity. Human CYP3A4 and rat CYP3A2 had high testosterone 2β- and 6β-hydroxylation activities in a modified reconstituted system with a lipid mixture. The hydroxylation site of testosterone by CYP2B6 (16α- and 16β-positions) agreed with that by rat CYP2B1. Human CYP2E1 had the highest lauric acid (ω-1)-hydroxylation activity and also had catalytic properties similar to those of rat CYP2E1. Human CYP2A and 2C forms had catalytic properties in testosterone metabolism different from those of rats. Antibodies raised against purified P450s were used to measure the levels of hepatic P450s. The level of CYP3A4 was the highest in human hepatic microsomes, comprising 30–40% of the total P450. CYP2C9 comprised 10–20% of the total. The levels of CYP1A2, 2A6, 2C8, 2D6, and 2E1 were moderate (5–15% of total P450). CYP2B6 content was very low. The information of this study is useful for drug metabolism and toxicological studies.

PURIFICATION AND CHARACTERIZATION OF YEAST-EXPRESSED HUMAN P450S AND COMPARISON OF THEM WITH PURIFIED RAT P450S

Rats are commonly used as experimental animals in an attempt to predict drug metabolism and toxicity in man and much data for drug metabolism with rats have been accumulated. Extrapolation of the data to humans can be improved by a comparative studies of characteristics and properties of drug metabolizing enzymes in experimental animals such as rats. In this study, we compared catalytic activities of purified human recombinant P450 with those of rats. Furthermore, we assayed the levels of P450 in human hepatic microsomes with specific antibody prepared with recombinant human P450. Like in rat hepatic microsomes, CYP2C and 3A forms were major P450s in human hepatic microsomes. Human and rat CYP3A forms had similar characteristics; they required phospholipid mixtures and cytochrome b5 for having efficient catalytic activity in a reconstituted system. Also, these forms had similar catalytic acitivity. However, rat CYP2C11 had different catalytic properties from human CYP2C8, 2C9, and 2C18. CYP2C11 had high activity toward testosterone and some drugs including lidocaine, but human CYP2C forms did not. Therefore, difference in drug metabolism between human and rat may occur when CYP2C11 contributes to metabolism. In drug metabolism, CYP2D6 is important, because it exhibits genetic polymorphism and metabolism of some drugs such as debrisoquine was delayed in poor metabolizer who has a mutated CYP2D6 gene. Rat CYP2D1 and human 2D6 seem to have similar catalytic activity. CYP2E1 is ethanol-inducible in both rat and human. Human and rat CYP2E1 had similar catalytic activities. Rat CYP2B1 and human CYP2B6 have high activity toward drugs including lidocaine. Rat CYP2B1 is strongly induced by phenobarbital and by many other drugs. In human, CYP2B6 may be induced by barbiturate as well as CYP3A4. In this case, drug metabolism maybe enhanced.

Characterization of two P-450 isozymes placed in the rat CYP2D subfamily

Two P-450s with debrisoquine 4-hydroxylation activity, designated P-450 UT-7 and UT-7b, were purified and partially purified, respectively, from hepatic microsomes of untreated male rats. Both purified P-450s with an apparent molecular weight of 49 000, were associated with another protein with an apparent molecular weight of 29 000 which was designated 29 k-protein. The CO-reduced spectra of both P-450 UT-7 and UT-7b showed a peak at 448 nm. The NH2-terminal amino acid sequences of P-450 UT-7 and UT-7b were the same as the amino acid sequences of CYP2D1 and CYP2D2 deduced from the cDNA, respectively, except for the lack of a terminal methionine for P-450 UT-7b. In a reconstituted systems, P-450 UT-7 and UT-7b catalyzed lidocaine 3-hydroxylation and N-deethylation in the presence of the 29 k-protein. The Km and Vmax values for lidocaine 3-hydroxylation were 3.6 μM and 0.50 nmol/min/nmol of P-450 for P-450 UT-7, and 3.6 μM and 0.93 nmol/min/ nmol of P-450 for P-450 UT-7b, respectively. Antibody against P-450 UT-7, which also cross-reacted with P-450 UT-7b, inhibited lidocaine 3-hydroxylation in liver microsomes from untreated male rats, but had little effect on lidocaine N-deethylation. These findings suggested that lidocaine 3-hydroxylation in hepatic microsomes from untreated male rats was catalyzed by P-450 UT-7 and/or UT-7b. P-450 UT-7 not containing 29 k-protein was obtained as the non-absorbed fraction from hydroxylapatite HPLC. The activities of debrisoquine 4-hydroxylation as well as lidocaine 3-hydroxylation and N-deethylation in a reconstituted system with P-450 UT-7 without 29 k-protein were one-fifth of those of P-450 UT-7 containing 29 k-protein at the same substrate concentration. These findings suggested that the 29 k-protein was essential to express the maximal metabolic activities. However, the lidocaine metabolic activity in a reconstituted system with P-450 UT-7 containing 29 k-protein and in hepatic microsomes were not inhibited by 29 k-protein antibody.