Cytochrome P450-mediated metabolism of haloperidol and reduced haloperidol to pyridinium metabolites

Haloperidol (HP) has been reported to undergo cytochrome P450 (P450)-mediated metabolism to potentially neurotoxic pyridinium metabolites; however, the chemical pathways and specific enzymes involved in these reactions remain to be identified. The aims of the current study were to (i) fully identify the cytochrome P450 enzymes capable of metabolizing HP to the pyridinium metabolite, 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-oxobutylpyridinium (HPP(+)), and reduced HP (RHP) to 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-hydroxybutylpyridinium (RHPP(+)); and (ii) determine whether 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-oxobutyl-1,2,3,6-tetrahydropyridine (HPTP) and 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-hydroxybutyl-1,2,3,6-tetrahydropyridine (RHPTP) were metabolic intermediates in these pathways. In vitro studies were conducted using human liver microsomal preparations and recombinant human cytochrome P450 enzymes (P450s 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19 2D6, 2E1, 3A4, 3A5, and 3A7) expressed in bicistronic format with human NADPH cytochrome P450 reductase in Escherichia coli membranes. Pyridinium formation from HP and RHP was highly correlated across liver preparations, suggesting the same enzyme or enzymes were responsible for both reactions. Cytochrome P450s 3A4, 3A5, and 3A7 were the only recombinant enzymes which demonstrated significant catalytic activity under optimized conditions, although trace levels of activity could be catalyzed by NADPH-P450 reductase alone. NADPH-P450 reductase-mediated activity was inhibited by reduced glutathione but not catalase or superoxide dismutase, suggesting O(2)-dependent oxidation. No evidence was obtained to support the contention that HPTP and RHPTP are intermediates in these pathways. K(m) values for HPP(+) (34 +/- 5 microM) and RHPP(+) (64 +/- 4 microM) formation by recombinant P450 3A4 agreed well with those obtained with human liver microsomes, consistent with P450 3A4 being the major catalyst of pyridinium metabolite formation in human liver.     

Characterization of the cytochrome P450 isoenzymes involved in the in vitroN-dealkylation of haloperidol

Aims The present study was carried out to identify the cytochrome P450 isoenzyme(s) involved in the N-dealkylation of haloperidol (HAL). Methods In vitro studies were performed using human liver microsomes and c-DNA-expressed human P450 isoforms. N-dealkylation of HAL was assessed by measuring the formation of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP). Results There was a tenfold variation in the extent of CPHP formation amongst the nine human liver microsomal preparations. The CPHP formation rates as a function of substrate concentration, measured in three livers, followed monophasic enzyme kinetics. K_m and V_max values ranged respectively from 50 to 78 μm and from 180 to 412 pmol mg⁻¹ min⁻¹. CPHP formation rates in the nine liver preparations were significantly correlated with dextromethorphan N-demethylase activity (a marker of CYP3A4 activity), but not with the activity of dextromethorphan O-demethylase (CYP2D6), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Ketoconazole, an inhibitor of CYP3A4, inhibited competitively CPHP formation (K_i=0.1 μm ), whereas sulphaphenazole (CYP2C9), furafylline (CYP1A2) and quinidine (CYP2D6) gave only little inhibition (IC₅₀>100 μm ). CPHP formation was, moreover, enhanced by α-naphtoflavone, an effect common to CYP3A4 mediated reactions. Anti-CYP3A4 antibodies strongly inhibited CPHP formation, whereas no inhibition was observed in the presence of CYP2D6 antibodies. Among the recombinant human CYP isoforms tested, CYP3A4 exhibited the highest activity with respect to CPHP formation rate, with no detectable effect of other CYP isoforms (CYP1A2, CYP2D6 and CYP2C9). HAL inhibited dextromethorphan O-demethylase (CYP2D6) with IC₅₀ values between 2.7 and 8.5 μm, but not (IC₅₀>100 μm ) dextromethorphan N-demethylase (CYP3A4), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Conclusions These results strongly suggest that the N-dealkylation of HAL in human liver microsomal preparations is mediated by CYP3A4.     

Involvement of CYP3A4 in the metabolism of bromperidol in vitro

In this study, human cytochrome P450 isoenzymes (CYP1A2, CYP2C19, CYP2D6, and CYP3A4) expressed in a cell line were used to elucidate their roles in the metabolism of bromperidol. We found that CYP3A4 catalyzes the N-dealkylation of bromperidol and its metabolite, reduced bromperidol. CYP3A4 also catalyzes the dehydration of bromperidol to bromperidol 1,2,3,6-tetrahydropyridine, metabolizes bromperidol to bromperidol pyridinium, and catalyzes the oxidation of reduced bromperidol back to bromperidol. CYP1A2, CYP2C19, and CYP2D6 do not catalyze these reactions.     

Enzyme-catalyzed bioactivation of cyclic tertiary amines to form potential neurotoxins.

The pyridinium metabolites formed in the MAO-B catalyzed oxidation of 1-methyl-4-substituted-1,2,3,6-tetrahydropyridinyl derivatives, such as the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), cause the selective degeneration of nigrostriatal neurons, presumably by inhibition of mitochondrial respiration and depletion of ATP stores. The possibility that other partially oxidized piperidinyl derivatives also may be biotransformed to toxic pyridinium metabolites has led us to examine the metabolic fate of the neuroleptic agent haloperidol (HP) and its tetrahydropyridinyl dehydration product 4-(4-chlorophenyl)-1[4-(4-fluorophenyl)-4-oxobutyl]- 1,2,3,6-tetrahydropyridine (HPTP). In vitro metabolic studies employing tissue preparations isolated from rodents, baboons and humans have documented that cytochrome P4503A enzymes catalyze the biotransformation of both HP and HPTP to yield the corresponding pyridinium metabolite HPP+. An analogous biotransformation profile has been observed with "reduced haloperidol" (RHP), an abundant, circulating metabolite of HP formed by the stereospecific reduction of the benzoyl carbonyl group of HP. In vivo studies also have documented these pathways in humans, baboons and rodents. Although both HPP+ and RHPP+ are found in the urine and plasma of HP treated patients and HP or HPTP treated baboons, attempts to identify an MPTP-type lesion in baboons following long-term treatment with HPTP have failed. On the other hand, evidence for a lesion of the nucleus basalis of Meynert has been obtained. Additionally, the urinary excretion of abnormal organic acids and acylcarnitine conjugates suggests that HP and/or metabolites derived from HP interfere with energy production pathways.     

Assessment of the contributions of CYP3A4 and CYP3A5 in the metabolism of the antipsychotic agent haloperidol to its potentially neurotoxic pyridinium metabolite and effect of antidepressants on the bioactivation pathway.

As a plausible explanation for the large interindividual variability in the pharmacokinetics of the neuroleptic agent haloperidol, the contributions of CYP3A isozymes (CYP3A4 and the polymorphic CYP3A5) predominantly involved in haloperidol bioactivation to the neurotoxic pyridinium species 4-(4-Chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-pyridinium (HPP(+)) were assessed in human liver microsomes and heterologously expressed enzymes. Based on recent reports on drug-drug interactions between haloperidol and antidepressants including selective serotonin reuptake inhibitors, the inhibitory effects of antidepressants on the CYP3A4/5-mediated haloperidol bioactivation were also evaluated. HPP(+) formation followed Michaelis-Menten kinetics in microsomes, recombinant CYP3A4, and CYP3A5 with K(m) values of 24.4 +/- 8.9 microM, 18.3 +/- 4.9 microM, and 200.2 +/- 47.6 microM, respectively, and V(max) values of 157.6 +/- 13.2 pmol/min/mg of protein, 10.4 +/- 0.6 pmol/min/pmol P450, and 5.16 +/- 0.6 pmol/min/pmol P450, respectively. The similarity in K(m) values between human liver microsomal and recombinant CYP3A4 incubations suggests that polymorphic CYP3A5 may not be an important genetic contributor to the interindividual variability in CYP3A-mediated haloperidol clearance pathways. Besides HPP(+), a novel 4-fluorophenyl-ring-hydroxylated metabolite of haloperidol in microsomes/CYP3A enzymes was also detected. Its formation was consistent with previous reports on the detection of O-sulfate and -glucuronide conjugates of a fluorophenyl ring-hydroxylated metabolite of haloperidol in human urine. Finally, all antidepressants except buspirone inhibited the CYP3A4/5-catalyzed oxidation of haloperidol to HPP(+) in a concentration-dependent manner. Based on the estimated IC(50) values for inhibition of HPP(+) formation in microsomes, the antidepressants were ranked in the following order: fluoxetine, nefazodone, norfluoxetine, trazodone, and fluvoxamine. These inhibition results suggest that clinically observed drug-drug interactions between haloperidol and antidepressants may arise via the attenuation of CYP3A4/5-mediated 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-4-piperidinol biotransformation pathways.     

Sensitive determination of 4-(4-bromophenyl)-4-hydroxypiperidine, a metabolite of bromperidol, in rat plasma by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

The purpose of this study was to determine the level of 4-(4-bromophenyl)-4-hydroxypiperidine (BPHP), a bromperidol (BRO) metabolite, in rat plasma by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples with benzene, derivatization with NBD-F was conducted in borate buffer (pH 8.0) at 60 degrees C for 3 min. Mexiletine was utilized through the procedure as an internal standard (IS). Retention times of the BPHP and IS derivatives were 7.7 and 11.5 min, respectively. The regression equation for BPHP showed good linearity in the range of 0.01-1 mg/ml with the detection limit of 0.003 microg/ml. The coefficient of variation was less than 12.0%. The recovery was satisfactory. This method was applied for a pharmacokinetic study of BPHP in comparison with 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), the corresponding haloperidol (HAL) metabolite, in rats. The ratio of the area under the plasma concentration curve (AUC) after p.o. administration of BPHP to the AUC after i.p. administration of BPHP (46%) was lower than that of CPHP (56%), indicating that intestinal absorption of BPHP is lower than that of CPHP. The ratio of BRO metabolism to BPHP (48%) was 1.8-fold higher than that of HAL metabolism to CPHP (27%); the ratio was estimated as (AUCp.o.,A-->B/AUCp.o.,B)x100, where AUCp.o.,A-->B is the AUC value of BPHP or CPHP after p.o. administration of BRO or HAL, and AUCp.o.,B is the AUC of BPHP or CPHP after administration of BPHP or CPHP, respectively. Our method provides a sensitive procedure for determination of BPHP in rat plasma and is suitable for pharmacokinetic studies of BPHP after BRO administration.     

Influence of lipophilicity on the interactions of N-alkyl-4-phenyl-1,2,3,6-tetrahydropyridines and their positively charged N-alkyl-4-phenylpyridinium metabolites with cytochrome P450 2D6.

The relationship between lipophilicity and CYP2D6 affinity of cyclic tertiary (N-alkyl-4-phenyl-1,2,3,6-tetrahydropyridines) and quaternary (N-alkyl-4-phenylpyridinium) amines was examined. The 1,2,3,6-tetrahydropyridine scaffold was chosen due to its common occurrence in the structures of CYP2D6 ligands such as the Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the dehydrated haloperidol metabolite N-[4-(4-fluorophenyl)-4-oxobutyl]-4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine (HPTP). Likewise, the pyridinium framework is found in and 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium and N-methyl-4-phenylpyridinium (MPP(+)), the positively charged metabolites of MPTP and haloperidol. The lack of CYP2D6 inhibition by MPTP and its pyridinium metabolite MPP(+) was due to their hydrophilic nature since higher N-alkyl homologs revealed substantial increases in inhibitory potency against recombinant CYP2D6-mediated bufuralol-1'-hydroxylation. The reasonable correlation between lipophilicity and CYP2D6 inhibition by pyridiniums and 1,2,3,6-tetrahydropyridines was only limited to straight chain N-alkyl analogs, since certain N-alkylaryl analogs of lower lipophilicity were better CYP2D6 inhibitors. CYP2D6 substrate properties of straight chain N-alkyltetrahydropyridines were also governed by lipophilicity, and N-heptyl-4-phenyl-1,2,3,6-tetrahydropyridine was the optimal substrate (K(mapp) = 0.63 microM). Metabolism studies indicated that the N-heptyl analog underwent monohydroxylation on the aromatic ring and on the N-heptyl group suggesting that 1,2,3,6-tetrahydropyridines can bind in more than one conformation in the CYP2D6 active site. Increased lipophilicity of haloperidol metabolites did not correlate with inhibitory potency since the more lipophilic HPTP metabolite was less potent as an inhibitor than reduced-haloperidol and reduced-HPTP. Furthermore, HPTP and reduced-HPTP, of comparable lipophilicity to the N-heptyltetrahydropyridine analog were inactive as CYP2D6 substrates. This observation suggests that steric constraints rather than lipophilicity are responsible for the lack of CYP2D6 substrate properties of cyclic tertiary amines tethered to bulky N-substituents. This phenomenon appears to be a common theme among several cyclic tertiary amine-containing anti-depressants and should be taken into consideration when designing central nervous system agents devoid of CYP2D6 substrate properties.     

Size-dependent effects of nanoparticles on the activity of cytochrome P450 isoenzymes

Nanoparticles are known to be able to interfere with cellular metabolism and to cause cytotoxicity and moreover may interfere with specific cellular functions. Serious effects on the latter include changes in liver cell function. The cytochrome P450 system is expressed in many cells but is especially important in hepatocytes and hormone-producing cells. The interaction of polystyrene nanoparticles with the most important drug-metabolizing cytochrome P450 isoenzymes, CYP3A4, CYP2D6, CYP2C9 and CYP2A1 expressed individually in insect cells (BACULOSOMES^®) was studied by the cleavage of substrates coupled to a fluorescent dye. The data obtained for individual isoenzymes were compared to metabolism in microsomes isolated from normal liver and from the hepatoma cell line H4-II-E-C3. Small (20-60 nm) carboxyl polystyrene particles but not larger (200 nm) ones reached high intracellular concentrations in the vicinity of the endoplasmic reticulum. These small particles inhibited the enzymatic activity of CYP450 isoenzymes in BACULOSOMES^® and substrate cleavage in normal liver microsomes. They moreover increased the effect of known inhibitors of the cytochrome P450 system (cimetidine, phenobarbital and paclitaxel). Substrate cleavage by the hepatoma cell line H4-II-E-C3 in contrast was undetectable, making this cell line unsuitable for this type of study.Our results thus demonstrate that nanoparticles can inhibit the metabolism of xenobiotics by the CYP450 system in model systems in vitro. Such inhibition could also potentially occur in vivo and possibly cause adverse effects in persons receiving medication.     

Selective dehydrogenation/oxygenation of 3-methylindole by cytochrome p450 enzymes.

3-Methylindole (3 MI) is a selective pulmonary toxicant, and cytochrome P450 (P450) bioactivation of 3 MI, through hydroxylation, epoxidation, or dehydrogenation pathways, is a prerequisite for toxicity. CYP2F1 and CYP2F3 exclusively catalyze the dehydrogenation of 3 MI to 3-methyleneindolenine, without detectable formation of the hydroxylation or epoxidation products. It was not known whether 3 MI is simply an excellent dehydrogenation substrate for all P450 enzymes, or whether certain cytochrome P450s responsible for 3 MI bioactivation have unique active sites that only catalyze the dehydrogenation of the molecule, while other P450s would catalyze only the oxygenation of 3 MI. Therefore, the kinetics of product formation by the CYP2F1 and CYP2F3 enzymes were compared with other cytochrome P450 enzymes. The enzymes tested were CYP1A1, CYP1A2, CYP1B1, and CYP2E1. The CYP1A1 and CYP1A2 enzymes produced all three 3 MI metabolites: the dehydrogenation product, 3-methyleneindolenine (V(max)/K(m) = 4 and 22, respectively); the hydroxylation product, indole-3-carbinol (V(max)/K(m) = 42 and 100, respectively); and the epoxidation product, 3-methyloxindole (V(max)/K(m) = 4 and 72, respectively). These CYP1A enzymes catalyzed oxygenation of 3 MI at much faster rates than dehydrogenation. CYP1B1 produced indole-3-carbinol (V(max)/K(m) = 85) and 3-methyloxindole (V(max)/K(m) = 7), and CYP2E1 only produced 3-methyloxindole (V(max)/K(m) = 98), but neither enzyme catalyzed the formation of the dehydrogenated product. Six additional P450 enzymes that were tested formed none of the dehydrogenation product. The ability of the various CYP1 family enzymes to catalyze the formation of all three major 3 MI metabolites, along with the specific oxygenation by CYP2E1, illustrates that dehydrogenation of 3 MI is not a substrate-directed process, but that the members of the CYP2F family possess unique active sites that specifically catalyze only the dehydrogenation mechanism.     

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: formation of the 4-hydroxy, 4'-hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen.

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 microM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 microM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 microM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 microM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.     

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 microM and 4.1 pmol min(-1) pmol(-1) P450, 44 microM and 10 pmol min(-1) pmol(-1) P450, and 34 microM 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.     

Characterization of the P450 System in Göttingen Minipigs

Abstract: It is essential to establish the activity and regulation of the cytochrome P450 system of species selected for toxicological and pharmacological studies. The minipig has become a popular substitute for the traditional non-rodent species although little information is available on its P450 system. The total P450 and the enzyme activity of the most important drug-metabolizing isoenzymes: CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were measured in liver microsomes from 4 minipigs and 8 conventional pigs of both sexes. Immunochemical levels were determined for 4 of the isoenzymes. The total P450 activity was slightly higher in minipigs compared to conventional pigs but no sex difference was detected. CYP1A2 activity (7-ethoxyresorufin) was 4 times higher in female minipigs than in male minipigs. The activity in male minipigs was almost identical to the activity in conventional pigs. The activity of CYP2E1 (chlorzoxazone) was 4 times higher in female than in male minipigs and 2 times higher in female than in male conventional pigs. No activity of CYP2D6 (debrisoquine) and CYP2C19 (mephenytoin) could be detected. The CYP3A4 activity (testosterone) detected in minipigs was higher than the activity in conventional pigs. A weak sex difference was seen in both strains. Western blotting using anti-human CYP2E1 and CYP3A4 confirmed the results obtained in the enzyme activity assays, while only CYP1A2 correlated with the activity in the conventional strain. The total P450 enzyme activity was close to the levels reported for human beings, as were the activities of CYP2E1 and CYP3A4.     

Hypoxia-induced down-regulation of CYP1A1/1A2 and up-regulation of CYP3A6 involves serum mediators

1. Acute moderate hypoxia modifies the catalytic activity and expression of certain isoenzymes of hepatic cytochrome P450 (P450). The aim of this study was to document whether hypoxia affects hepatic P450 directly or through the release of serum mediators. 2. Rabbits were subjected to a FiO(2) of 8% for 48 h, sacrificed, and serum and hepatocytes were isolated; hepatocytes from control and rabbits with hypoxia were incubated with serum from control and hypoxic rabbits for 4 and 24 h, and total P450 content, CYP1A1, 1A2 and 3A6 activities and expressions were assessed. Sera were fractionated by size exclusion chromatography and fractions tested for their ability to modify activity and amount of P450, and serum mediators were identified through neutralization experiments. 3. Total serum and fractions with proteins of 15-23 and 65-94 kDa of M(r) reduced P450 content and expression of CYP1A1, 1A2 and 3A6, as well as CYP1A1, 1A2 and 3A6 mRNA. Total serum and the fraction with 32-44 kDa proteins increased CYP3A6 activity and protein and mRNA. The serum mediators implicated in the decrease in activity and expression of CYP1A1, 1A2 and 3A6 were interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta) and IL-2. Erythropoietin (Epo) was partly responsible for the increase in P450 content and CYP3A6 expression. 4. In conclusion, acute moderate hypoxia diminishes the activity and expression of CYP1A1, 1A2 and CYP1A1, 1A2 mRNA, and increases CYP3A6 protein, activity and CYP3A6 mRNA. Several mechanisms contribute to these changes in P450, among them the release of cytokines acting as serum mediators.     

Metabolism of diallyl disulfide by human liver microsomal cytochromes P-450 and flavin-containing monooxygenases.

The metabolism of diallyl disulfide (DADS), a garlic sulfur compound, was investigated in human liver microsomes. Diallyl thiosulfinate (allicin) was the only metabolite observed and its formation followed Michaelis-Menten kinetics with a Km = 0.61 +/- 0.2 mM and a Vmax = 18.5 +/- 4.2 nmol/min/mg protein, respectively (mean +/- S.E. M., n = 4). Both flavin-containing monooxygenase and the cytochrome P-450 monooxygenases (CYP) were involved in DADS oxidation, but the contribution of CYP was predominant. The cytochrome P-450 isoforms involved in this metabolism were investigated using selective chemical inhibitors, microsomes from cells expressing recombinant CYP isoenzymes, and studying the correlation of the rate of DADS oxidation with specific monooxygenase activities of human liver microsomes. Diethyldithiocarbamate and tranylcypromine inhibited allicin formation, whereas other specific inhibitors had low or no effect. Most of the different human microsomes from cells expressing CYP were able to catalyze this reaction, but CYP2E1 showed the highest affinity with a substantial activity. Furthermore, allicin formation by human liver microsomes was correlated with p-nitrophenol hydroxylase activity, a marker of CYP2E1, and tolbutamide hydroxylase activity, a marker of CYP2C9. Among these approaches only CYP2E1 was identified in each case, which suggested that DADS is preferentially metabolized to allicin by CYP2E1 in human liver. However the minor participation of other CYP forms and flavin-containing monooxygenases is likely.     

Oxidation of tamoxifen by human flavin-containing monooxygenase (FMO) 1 and FMO3 to tamoxifen-N-oxide and its novel reduction back to tamoxifen by human cytochromes P450 and hemoglobin.

Tamoxifen (TAM), used as the endocrine therapy of choice for breast cancer, undergoes metabolism primarily forming N-desmethyltamoxifen, 4-hydroxytamoxifen, alpha-hydroxytamoxifen, and tamoxifen-N-oxide (TNO). Our earlier studies demonstrated that flavin-containing monooxygenases (FMOs) catalyze the formation of TNO. The current study demonstrates that human FMO1 and FMO3 catalyze TAM N-oxidation to TNO and that cytochromes P450 (P450s), but not FMOs, reduce TNO to TAM. CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 all reduced TNO, with CYP2A6, CYP1A1, and CYP3A4 producing the greatest reduction. A portion of TAM formed by CYP3A4-mediated reduction of TNO was further metabolized, but not TAM formed by the other P450s. TNO reduction by P450s is extremely rapid with considerable TAM formation detected at the earliest time point that products could be measured. TAM formation exhibited a lack of linearity with incubation time but increased linearly as a function of TNO and P450 concentration. TNO was converted into TAM by reduced hemoglobin (Hb) and NADPH-P450 oxidoreductase, suggesting involvement of the same heme-Fe(2+) complex in both Hb and P450s. The findings raise the question of whether the reductive activity may be nonenzymatic. Results of this in vitro study demonstrate the potential of TAM and TNO to be interconverted metabolically. FMO seems to be the major enzymatic oxidant, whereas several P450 enzymes and even reduced hemoglobin are capable of reducing TNO back to TAM. The possibility that these processes may comprise a metabolic cycle in vivo is discussed in this article.     

In vitro study on the involvement of CYP1A2, CYP2D6 and CYP3A4 in the metabolism of haloperidol and reduced haloperidol

Objective: To investigate in vitro which CYP isoforms (CYP1A2, CYP2D6 and CYP3A4) are involved in the biotransformation of haloperidol (HAL) and reduced haloperidol (RHAL). Methods: The biotransformation of HAL and RHAL is evaluated by measuring HAL and RHAL remaining after incubation with human liver microsomes and with supersomes from human baculovirus-infected cells expressing human P ₄₅₀ isoforms. The influence of chemical- and immuno-inhibition of specific isoforms on the disappearance of HAL and RHAL was also studied. Results: After 60-min incubation of 2 μM and 20 μM HAL or RHAL with human liver microsomes, for HAL, 58% and 64%, respectively, remained in the incubation mixture, for RHAL, 53% and 66%, respectively. Ketoconazole had the most pronounced inhibitory effect on the biotransformation of both substrates, while for quinidine and furafylline there was only a weak or no influence. Anti-CYP3A4 antibodies inhibited strongly the biotransformation of HAL and RHAL, while the influence of anti-CYP2D6 antibodies was much less pronounced. After incubation with supersomes of recombinant CYP3A4, HAL and RHAL disappeared rapidly; disappearance was slow after incubation with CYP2D6 supersomes, and negligible with CYP1A2 supersomes. Conclusion: The results show that CYP3A4 is the most important CYP isoenzyme involved in the biotransformation of HAL and RHAL, and that the metabolism by CYP2D6 is only a minor pathway; CYP1A2 has no or only a negligible influence. Received: 12 April 1999 / Accepted in revised form: 2 August 1999     

Disposition of haloperidol pyridinium and reduced haloperidol pyridinium in schizophrenic patients: no relationship with clinical variables during short-term treatment

In an open clinical trial, serum concentrations of haloperidol pyridinium (C(HP+)) and reduced haloperidol pyridinium (C(RHP+)), as well as haloperidol (CH) and reduced haloperidol (C(RH)), were measured in 57 schizophrenic and schizoaffective inpatients during 6 weeks of short-term treatment. Psychopathology was monitored with the Brief Psychiatric Rating Scale (BPRS), and extrapyramidal adverse effects were assessed with the Extrapyramidal Symptom Rating Scale (EPS). Significantly linear relationships were found between haloperidol dose (D) and pyridinium metabolite serum concentrations, as well as between C(H) and the pyridinium metabolite serum concentrations. C(HP+) (range, 0.2-4.9 ng/mL) and C(RHP+) (range, 0.03-6.23 ng/mL) were low compared with C(H) and C(RH), being as mean values approximately 7% and 14% of C(H) and C(RH), respectively. Additionally, the values of C(RHP+) and the slope of the correlation of C(H) with the C(RHP+)/C(HP+) ratio were considerably lower than in a previous report of long-term treatment with haloperidol. This is explained by the shorter time of treatment of the present study. Carbamazepine comedication was found to not influence relative pyridinium metabolite serum concentrations C(HP+)/D and C(RHP+)/D. However, the aromatization ratios of haloperidol (C(HP+)/C(H)) and reduced haloperidol (C(RHP+)/C(RH)) were increased by concomitant carbamazepine. As the main result, no relationships between the pyridinium metabolite serum concentrations and clinical variables (BPRS change, EPS, dose of biperiden) were detected. For instance, the aromatization ratios C(HP+)/C(H) and C(RHP+)/C(RH) did not predict clinical improvement or extrapyramidal adverse effects. Therefore, no confirmation of the "pyridinium hypothesis," which suggests haloperidol pyridinium metabolites to be the origin of adverse effects and decreased therapeutic effect, can be derived from this study. However, the authors emphasize that pyridinium metabolites cannot be excluded as the origin of decreased therapeutic effect in long-term treatment and of adverse effects not investigated in the present study, such as tardive dyskinesia. Finally, it is concluded that the serum concentration of the parent drug remains the main variable of interest in the therapeutic drug monitoring of haloperidol during short-term treatment.     

Interaction of cisapride with the human cytochrome P450 system: metabolism and inhibition studies.

Using human liver microsomes (HLMs) and recombinant cytochrome P450s (CYP450s), we characterized the CYP450 isoforms involved in the primary metabolic pathways of cisapride and documented the ability of cisapride to inhibit the CYP450 system. In HLMs, cisapride was N-dealkylated to norcisapride (NORCIS) and hydroxylated to 3-fluoro-4-hydroxycisapride (3-F-4-OHCIS) and to 4-fluoro-2-hydroxycisapride (4-F-2-OHCIS). Formation of NORCIS, 3-F-4-OHCIS, and 4-F-2-OHCIS in HLMs exhibited Michaelis-Menten kinetics (K(m): 23.4 +/- 8.6, 32 +/- 11, and 31 +/- 23 microM; V(max): 155 +/- 91, 52 +/- 23, and 31 +/- 23 pmol/min/mg of protein, respectively). The average in vitro intrinsic clearance (V(max)/K(m)) revealed that the formation of NORCIS was 3.9- to 5. 9-fold higher than that of the two hydroxylated metabolites. Formation rate of NORCIS from 10 microM cisapride in 14 HLMs was highly variable (range, 4.9-133.6 pmol/min/mg of protein) and significantly correlated with the activities of CYP3A (r = 0.86, P =. 0001), CYP2C19, and 1A2. Of isoform-specific inhibitors, 1 microM ketoconazole and 50 microM troleandomycin were potent inhibitors of NORCIS formation from 10 microM cisapride (by 51 +/- 9 and 44 +/- 17%, respectively), whereas the effect of other inhibitors was minimal. Of 10 recombinant human CYP450s tested, CYP3A4 formed NORCIS from 10 microM cisapride at the highest rate (V = 0.56 +/- 0. 13 pmol/min/pmol of P450) followed by CYP2C8 (V = 0.29 +/- 0.08 pmol/min/pmol of P450) and CYP2B6 (0.15 +/- 0.04 pmol/min/pmol of P450). The formation of 3-F-4-OHCIS was mainly catalyzed by CYP2C8 (V = 0.71 +/- 0.24 pmol/min/pmol of P450) and that of 4-F-2-OHCIS by CYP3A4 (0.16 +/- 0.03 pmol/min/pmol of P450). Clearly, recombinant CYP2C8 participates in cisapride metabolism, but when the in vitro intrinsic clearances obtained were corrected for abundance of each CYP450 in the liver, CYP3A4 is the dominant isoform. Cisapride was a relatively potent inhibitor of CYP2D6, with no significant effect on other isoforms tested, but the K(i) value derived (14 +/- 16 microM) was much higher than the clinically expected concentration of cisapride (<1 microM). Our data suggest that CYP3A is the main isoform involved in the overall metabolic clearance of cisapride. Cisapride metabolism is likely to be subject to interindividual variability in CYP3A expression and to drug interactions involving this isoform.     

In vitro metabolism of the calmodulin antagonist DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) by human liver microsomes: involvement of cytochromes p450 in atypical kinetics and potential drug interactions.

Human cytochrome P450 (P450) isozyme(s) responsible for metabolism of the calmodulin antagonist 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e) and kinetic profiles for formation of its six primary metabolites [M3, M5, M6, M7, M8, and DY-9836 (3-[2-[4-(3-chloro-2-methylphenyl)piperazinyl]ethyl]-5,6-dimethoxyindazole)] were identified using human liver microsomes and recombinant P450 enzymes. In vitro experiments, including an immunoinhibition study, correlation analysis, and reactions with recombinant P450 enzymes, revealed that CYP3A4 is the primary P450 isozyme responsible for the formation of the DY-9760e metabolites, except for M5, which is metabolized by CYP2C9. Additionally, at clinically relevant concentrations, CYP2C8 and 2C19 make some contribution to the formation of M3 and M5, respectively. The formation rates of DY-9760e metabolites except for M8 by human liver microsomes are not consistent with a Michaelis-Menten kinetics model, but are better described by a substrate inhibition model. In contrast, the enzyme kinetics for all metabolites formed by recombinant CYP3A4 can be described by an autoactivation model or a mixed model of autoactivation and biphasic kinetics. Inhibition of human P450 enzymes by DY-9760e in human liver microsomes was also investigated. DY-9760e is a very potent competitive inhibitor of CYP2C8, 2C9 and 2D6 (Ki 0.25-1.7 microM), a mixed competitive and noncompetitive inhibitor of CYP2C19 (Ki 2.4 microM) and a moderate inhibitor of CYP1A2 and 3A4 (Ki 11.4-20.1 microM), suggesting a high possibility for human drug-drug interaction.     

In vitro identification of the human cytochrome P-450 enzymes involved in the N-demethylation of azelastine.

Azelastine hydrochloride [4-[(4-chlorophenyl)methyl]-2-(hexahydro-1-methyl-1H-azepin-4yl )-1-(2 H)-phthalazinone monohydrochloride], is a long-acting antiallergic and antiasthmatic drug. The human cytochrome P-450 (CYP) isoform responsible for azelastine N-demethylation, the major metabolic pathway for azelastine, has been examined. Eadie-Hofstee plots of azelastine N-demethylation in human liver microsomes were biphasic. In microsomes from baculovirus-infected insect cells, recombinant CYP3A4, 2D6, 1A2, and 2C19 exhibited high azelastine N-demethylase activity. The K(m) values of the recombinant CYP2D6 (3.75 microM) and CYP3A4 (43.7 microM) were relatively close to that of high-affinity (14.1 microM) and low-affinity (54.7 microM) components in human liver microsomes, respectively. Azelastine N-demethylase activity was inhibited only by the anti-CYP3A antibody, in contrast to antibodies for CYP1A, 2D6, and 2C. In addition, desmethylazelastine formation was significantly inhibited by ketoconazole and troleandomycin but only weakly by omeprazole, sulfaphenazole, and furafylline. These observations suggested that the N-demethylation of azelastine is most extensively catalyzed by the CYP2D6 and 3A4 isoforms in humans.