Evaluation of animal models for intestinal first-pass metabolism of drug candidates to be metabolized by CYP3A enzymes via in vivo and in vitro oxidation of midazolam and triazolam.

1. To search an appropriate evaluation methodology for the intestinal first-pass metabolism of new drug candidates, grapefruit juice (GFJ)- and vehicle (tap water)-pretreated mice or rats were orally administered midazolam (MDZ) or triazolam (TRZ), and blood levels of the parent compounds and their metabolites were measured by liquid chromatography/MS/MS. A significant effect of GFJ to elevate the blood levels was observed only for TRZ in mice. 2. In vitro experiments using mouse, rat and human intestinal and hepatic microsomal fractions demonstrated that GFJ suppressed the intestinal microsomal oxidation of MDZ and especially TRZ. Substrate inhibition by MDZ caused reduction in 1'-hydroxylation but not 4-hydroxylation in both intestinal and hepatic microsomal fractions. The kinetic profiles of MDZ oxidation and the substrate inhibition in mouse intestinal and hepatic microsomal fractions were very similar to those in human microsomes but were different from those in rat microsomes. Furthermore, MDZ caused mechanism-based inactivation of cytochrome P450 3A-dependent TRZ 1'-hydroxylation in mouse, rat and human intestinal microsomes with similar potencies. 3. These results are useful information in the analysis of data obtained in mouse and rat for the evaluation of first-pass effects of drug candidates to be metabolized by CYP3A enzymes.

Separate evaluation of intestinal and hepatic metabolism of three benzodiazepines in rats with cannulated portal and jugular veins: comparison with the profile in non-cannulated mice.

Pharmacokinetic analyses of three kinds of benzodiazepines--midazolam (MDZ), triazolam (TRZ) and alprezolam (APZ)--were performed in rats with cannulated portal and jugular veins. Each drug was administered to the double-cannulated rats, and pharmacokinetic data for the parent drugs and their 1'- and 4-hydroxylated metabolites were compared with those obtained in non-cannulated mice. In bioavailability, the drugs ranked APZ >> TRZ = MDZ in rats, and APZ > TRZ >> MDZ in mice, with the values for MDZ remarkably different between rats and mice (19% in rats versus 2.3% in mice). In contrast, hepatic availability (Fh) was similar (APZ > TRZ > MDZ) in both species. Highly significant relationships were found between the ratio of the area under the plasma concentration-time curve (AUC) for the parent drugs in portal blood (AUC(por)) to that in systemic blood (AUC(sys)) and Fh in rats and mice. The double-cannulated rat is useful for estimating the hepatic availability of drug candidates by determining the AUC values for the parent drugs in portal and systemic blood samples.

Functional characterization of CYP3A4.16: catalytic activities toward midazolam and carbamazepine.

1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus-insect cell system. 2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them. 3. When midazolam was used as a substrate, K(m) and V(max) for 1'-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (V(max)/K(m)) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in K(m) without a difference in V(max). 4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, K(m2) and V(max2) of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance. 5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.

Solubilization and partial characterization of fatty acyl-CoA:sphingosine acyltransferase (ceramide synthetase) from rat liver and brain.

Lignoceroyl-CoA:sphingosine lignoceroyltransferase, which catalyzes synthesis of lignoceroylsphingosine, the ceramide that is a major component of sphingolipids in mammalian tissues, has been solubilized from microsomes of rat brain and liver and partially purified. The microsomes were treated with 1 M sodium thiocyanate in N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer containing 20% glycerol. The supernatant fraction obtained after centrifugation was fractionated by Sepharose CL-4B gel filtration. The ceramide synthetase activity was recovered in a small fraction containing high molecular weight proteins. Analysis of proteins and lipids indicated that the fraction was not simply a fragment of microsomes. The activity for synthesis of lignoceroylsphingosine, which is abundant in nervous system, was compared with that for the synthesis of stearoylsphingosine, which is more enriched in extraneural sphingolipids, in brain and liver microsomes. Despite the difference in relative abundance of molecular species of ceramides in these tissues, the activity for lignoceroylsphingosine synthesis was not more enriched in brain than in liver.

Recent advances in the metabolism of cannabinoids.

This review describes recent advances in the metabolism of cannabinoids. Cannabidiol was metabolized to cannabielsoin, 6 beta-hydroxymethyl-delta 9-tetrahydrocannabinol and an oxepine derivative through epoxide intermediates by hepatic microsomal enzymes containing cytochrome P450 of animals. Cannabidiol inactivated cytochrome P450 UT-2 (CYP2C11) not equal to in male rats and a member of 3A subfamily in mouse liver. These inactivations may be very important because serious drug-drug interactions will occur in the case that cannabidiol is co-administered with drugs which are metabolized mainly by the enzyme system containing these P450 isozymes. A member of cytochrome P450 belonging to 2C subfamily was the major isozymes responsible for the cannabinoid metabolism in many experimental animals and that of 3A subfamily made some contribution to the metabolism of cannabinoids by human hepatic microsomes. Microsomal aldehyde oxygenase, a particular isozyme of cytochrome P450 catalyzing the oxidation of 11-oxo-tetrahydrocannabinol to tetrahydrocannabinol-11-oic acid, was found for the first time by the authors. Cytochrome P450 MUT-2 (CYP2C29) is the major isozyme responsible for the microsomal aldehyde oxygenase activity in mouse hepatic microsomes.

Functional evaluation of cytochrome P450 2D6 with Gly42Arg substitution expressed in Saccharomyces cerevisiae.

A single amino acid-substituted mutant protein, CYP2D6 (G42R) was expressed in Saccharomyces cerevisiae and its enzymatic properties were compared with those of other single (P34S, R296C and S486T) and double amino acid-substituted mutant proteins (P34S/S486T and R296C/S486T) expressed in yeast cells, all of which were known to occur in the CYP2D6 gene as single nucleotide polymorphisms. The protein levels of G42R, P34S and P34S/S486T in microsomal fractions and their oxidation capacities towards debrisoquine as a prototypic substrate and bunitrolol as a chiral substrate were different from those of wild-type CYP2D6, while the R296C, S486T and R296C/S486T behaved similarly to the wild-type in these indices. The CYP contents both in yeast microsomal and in whole cell fractions indicated that some part of G42R protein was localized in the endoplasmic reticulum membrane fraction, whereas most of G42R protein was in some subcellular fractions other than endoplasmic reticulum. In kinetic analysis, the G42R substitution increased apparent Km and decreased Vmax for debrisoquine 4-hydroxylation, while it increased both Km and Vmax for bunitrolol 4-hydroxylation. The P34S substitution did not drastically change Km but decreased Vmax for debrisoquine 4-hydroxylation, whereas Km was increased and Vmax unchanged or decreased for bunitrolol 4-hydroxylation by P34S substitution. These results suggest that the G42R substitution causes a change in the CYP2D6 conformation, which may be different from the change produced by the P34S substitution.

Oxidative metabolism of bunitrolol by complementary DNA-expressed human cytochrome P450 isozymes in a human hepatoma cell line (Hep G2) using recombinant vaccinia virus.

We examined the oxidative metabolism of a beta-blocker bunitrolol (BTL) by 10 human cytochromes P450 (CYP) (1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, 3A3, 3A4 or 3A5), which were individually expressed in Hep G2 cells with a vaccinia virus complementary DNA expression system. Among the 10 isozymes, only CYP2D6 and 1A2 at a substrate concentration of 5 microns, and CYP2C8 and 2C9 in addition to the two isozymes at a BTL concentration of 1 mM, exhibited detectable BTL 4-hydroxylase activities. The activities at 1 mM BTL were on the order of CYP2D6 (100% as relative activity) > CYP1A2 (86%) >> CYP2C8 and 2C9 (7-8%). Enzyme kinetic parameters of CYP2D6 were calculated to be 4.41 microns as a Km value and 0.442 nmol min-1 per nmol CYP as a Vmax value. Kinetic parameters of CYP1A2 were calculated as 295 microns and 0.411 nmol min-1 per nmol CYP for Km and Vm values, respectively. These results suggest that both CYP2D6 and 1A2 primarily catalyse BTL 4-hydroxylation, but that the former is a predominant isozyme responsible for the reaction at a low substrate concentration range of BTL in human liver.

Activation of propranolol and irreversible binding to rat liver microsomes: strain differences and effects of inhibitors.

In summary, strain difference and inhibition studies showed that an enzyme(s) converting propranolol to a reactive metabolite capable of irreversible binding to microsomal macromolecules appeared to be a P450 isozyme(s) which catalyses debrisoquine 4-hydroxylation in rats. It seems likely that cytochrome P450 isozymes responsible for debrisoquine 4-hydroxylation activate propranolol and may be impaired after chronic use of propranolol also in human subjects. The findings obtained in the present study provide a clue for the elucidation of the mechanism of propranolol-induced impairment of the drug metabolizing enzyme system. Further studies using purified debrisoquine 4-hydroxylase are required to identify a P450 isozyme(s) responsible for the metabolic activation of propranolol. We are now performing experiments along this line.

Oxidative metabolism of cinnarizine in rat liver microsomes.

The oxidative metabolism of cinnarizine (CZ) [1-(diphenylmethyl)-4-(3-phenyl-2-propenyl)-piperazine] to 1-(diphenylmethyl)piperazine (M-1), 1-(diphenylmethyl)-4-[3-(4'-hydroxyphenyl)-2-propenyl]piperazine (M-2), benzophenone (M-3) and 1-[4'-hydroxyphenyl)-phenylmethyl]-4-(3- phenyl-2-propenyl)piperazine (M-4) has been studied in rat liver microsomes. In Wistar rats, kinetic analysis revealed sex differences (male > female) in the Km values for formation of all the metabolites and the Vmax values for the formation of M-1, M-3 and M-4. The reactions required NADPH, and were inhibited by carbon monoxide and SKF 525-A. Only M-2 formation was suppressed by sparteine or metoprolol, and was significantly lower in female Dark Agouti rats than in Wistar rats of both sexes. The results suggest that CZ is oxidized by cytochrome P450, and M-2 formation is related to debrisoquine/sparteine-type polymorphic drug oxidation.

Metabolic activation of lidocaine and covalent binding to rat liver microsomal protein.

Incubation of [14C]lidocaine with rat liver microsomes in the presence of an NADPH-generating system resulted in covalent bindings of a 14C-labelled material to microsomal protein. The covalent binding of radioactivity needed NADPH and atmospheric oxygen, and was diminished by purging of carbon monoxide and the addition of SKF-525A. Hence the covalent binding of a 14C-labelled material resulting from a reactive metabolite of lidocaine formed by cytochrome P450-dependent monooxygenation. The covalent binding measured at various concentrations of lidocaine (2.5-30 microM) followed Michaelis-Menten kinetics, and the Km value (4.52 microM) of the activation reaction was close to the Km value (1.78 microM) of lidocaine 3-hydroxylation. The metabolism-dependent covalent binding of lidocaine to microsomal protein as well as lidocaine 3-hydroxylase activity was much lower in the Dark Agouti strain rat, which is known as a poor-metabolizer animal model of debrisoquine 4-hydroxylation, than in the Wistar rat for the corresponding sexes. The covalent binding in male rats was greater than that in females of both strains, but the extent of the sex difference in the binding was smaller than that of the lidocaine N-deethylase activity in Wistar rats. Propranolol and quinidine, specific inhibitors of debrisoquine 4-hydroxylase, markedly inhibited lidocaine 3-hydroxylase activity of Wistar male rats, but not N-deethylase activity. These compounds also inhibited the metabolism-dependent covalent binding of lidocaine to microsomal protein. These strain difference and inhibition studies showed that the reaction converting lidocaine to a reactive metabolite capable of binding covalently to microsomal protein was related to lidocaine 3-hydroxylation, and may be catalysed by cytochrome P450 isozyme(s) belonging to the CYP2D subfamily. The covalent binding of radioactivity to rat liver microsomal protein was diminished by nucleophiles, reduced glutathione and cysteine, indicating that the reactive metabolic intermediate of lidocaine is an electrophilic metabolite such as an arene oxide.

Sex difference in the oxidative metabolism of delta 9-tetrahydrocannabinol in the rat.

Oxidative metabolism of delta 9-tetrahydrocannabinol (THC), one of the major components of marihuana, was studied using liver microsomes of adult male and female rats. There was no significant difference in the rates of the cannabinoid oxidation in terms of nmol per min per nmol of liver microsomal cytochrome P450 or of nmol per min per mg of microsomal protein between male and female rats. delta 9-THC was biotransformed to various metabolites including 11-hydroxy-delta 9-THC (11-OH-delta 9-THC), 8 alpha-OH-delta 9-THC, 8 alpha,11-diOH-delta 9-THC, 3'-OH-delta 9-THC by liver microsomes of male rats, while it was oxidized selectively to 11-OH-delta 9-THC by liver microsomes of female rats. After intraperitoneal administration of delta 9-THC, various metabolites were again found in the liver of the male rat, while in the female rat oxidation of the methyl group at the 9-position was a major metabolic pathway. These results demonstrate that an apparent sex-related difference exists in the oxidative metabolism of delta 9-THC in the rat.

Role of the CYP2D subfamily in metabolism-dependent covalent binding of propranolol to liver microsomal protein in rats.

In vitro covalent binding of a chemically reactive metabolite of propranolol to microsomal macromolecules, which is presumed to cause inhibition of its own metabolism in rats, was diminished in liver microsomes from rats pretreated with propranolol. Covalent binding was suppressed by the addition of an antibody against P450BTL, which is a cytochrome P450 (P450) isozyme belonging to the CYP2D subfamily. SDS-PAGE of microsomal proteins after incubation with [3H]propranolol and NADPH indicated that the binding was non-selective but prominent at the molecular mass of approx. 50 kDa, corresponding to those of the P450 protein. The radioactivity peak was markedly but not completely diminished by the addition of reduced glutathione. In a reconstituted system containing P450BTL, NADPH-cytochrome P450 reductase (fp2) and dilauroylphosphatidylcholine, propranolol 4-, 5- and 7-hydroxylase activities decreased time dependently following preincubation with propranolol in the presence of NADPH, indicating time-dependent inactivation of P450BTL. The covalent binding of a reactive metabolite of [3H]propranolol to the proteins was also observed in this system. SDS-PAGE showed that among the three proteins in the reconstituted system, fp2 and P450BTL consisting of two polypeptides with molecular masses of 49 and 32 kDa, the binding was specific for a polypeptide corresponding to the P450 isozyme with a molecular mass of 49 kDa. In addition, the ratio of the amount of covalently bound radiolabelled materials to that of P450BTL which was estimated from each impaired propranolol hydroxylase activity under the same reconstitutional conditions was calculated to be approx. 1.0. These findings indicate that propranolol is a mechanism-based inactivator of a cytochrome P450 isozyme(s) belonging to the CYP2D subfamily.

Effect of repeated administration of 11-hydroxy-delta 8-tetrahydrocannabinol, an active metabolite of delta 8-tetrahydrocannabinol, on the hepatic microsomal drug-metabolizing enzyme system of mice.

The effects of delta 8-tetrahydrocannabinol (delta 8-THC) and its major and active metabolite, 11-hydroxy-delta 8-tetrahydrocannabinol (11-OH-delta 8-THC), on the hepatic microsomal drug-metabolizing enzyme system were studied in mice. The repeated administration of 11-OH-delta 8-THC (5 mg/kg/day, i.v.) for 3 or 7 days increased significantly the activities of aniline hydroxylase and p-nitroanisole O-demethylase. By the same treatment, cytochrome P-450 content (3 days) or NADPH-cytochrome c reductase activity (7 days) was also increased significantly. The treatment with delta 8-THC for 7 days (5 mg/kg/day, i.v.) significantly increased aniline hydroxylase only. 11-OH-delta 8-THC increased the Vmax, but not the Km, values for both drug-metabolizing enzymes, whereas delta 8-THC decreases significantly the Km value (270 microM) for p-nitroanisole O-demethylase as compared with the control (398 microM). Repeated administration of these cannabinoids for 7 days also increased the metabolism of delta 8-THC by hepatic microsomes; this was attributed to an enhanced formation of 11-OH-delta 8-THC. In contrast, microsomal formation of 7 alpha-OH-delta 8-THC was decreased significantly by treatment with delta 8-THC. 11-OH-delta 8-THC, but not delta 8-THC, treatment increased the metabolism of 11-OH-delta 8-THC by hepatic microsomes. These findings indicate that delta 8-THC and 11-OH-delta 8-THC treatment can induce hepatic microsomal drug-metabolizing enzymes and affect differently the catalytic properties of the enzymes.

Self-catalyzed inactivation of cytochrome P-450 during microsomal metabolism of cannabidiol.

When cannabidiol (CBD) was incubated with hepatic microsomes of mice in the presence of an NADPH-generating system, a significant decrease of cytochrome P-450 content was observed by measuring its carbon monoxide difference spectra. The decrease of cytochrome P-450 by CBD required NADPH and molecular oxygen. The effect was partially inhibited by SKF 525-A but not by various scavengers of active oxygen species, superoxide anion, hydroxyl radical and singlet oxygen. The incubation of CBD with hepatic microsomes did not affect total heme but decreased significantly free sulfhydryl contents in the microsomes. The derivatives of CBD modified in the resorcinol moiety, CBD-monomethyl- and dimethylethers, almost lost the effect on cytochrome P-450, whereas those modified in the terpene moiety, 8,9-dihydro- and 1,2,8,9-tetrahydro-CBDs exhibited some potency to inactivate cytochrome P-450. The inactivation of cytochrome P-450 by CBD and related compounds led to the inhibition of hepatic microsomal p-nitroanisole O-demethylase and aniline hydroxylase activities. These results suggest that the resorcinol moiety of CBD plays some role in the inactivation of cytochrome P-450 by the cannabinoid.

Formation of similar species to carbon monoxide during hepatic microsomal metabolism of cannabidiol on the basis of spectral interaction with cytochrome P-450.

Cannabidiol induced a carbon monoxide-like complex with mouse hepatic microsomal cytochrome P-450 during NADPH-dependent metabolism in vitro on a spectral basis. The reduction by dithionite was required for the maximal development of a spectrum. The complex showed a peak at 450 nm which shifted to 419 or 423 nm, respectively, by further addition of hemoglobin or myoglobin. Cannabidiol-induced complex formation required molecular oxygen, and was decreased by the addition of inhibitors of cytochrome P-450-dependent monoxygenase. Pretreatment of mice with phenobarbital (80 or 100 mg/kg, i.p. for 3 days) but not 3-methylcholanthrene (80 mg/kg, i.p.) increased the complex formation. In contrast, pretreatment with cobaltous chloride (40 mg/kg, i.p. for 3 days) decreased the complex formation. 8,9-Dihydro- and 1,2,8,9-tetrahydrocannabidiols also induced the same spectrum as that of above complex, whereas cannabidiol monomethyl- and dimethylethers reduced this ability. In addition, both cannabidivarin and cannabigerol induced the complex formation, although delta 9-tetrahydrocannabinol, cannabinol and cannabielsoin did not. Olivetol but not d-limonene induced the spectrum of the complex to some extent. These results indicate that cannabidiol induces a carbon monoxide-like complex with cytochrome P-450 during hepatic microsomal metabolism, and suggest that phenobarbital-inducible cytochrome P-450s mediate at least one of the metabolic steps of CBD to form the complex, as well as the importance of the resorcinol moiety of CBD for the complex formation.

A cytochrome P450 isozyme having aldehyde oxygenase activity plays a major role in metabolizing cannabinoids by mouse hepatic microsomes.

A cytochrome P450 (designated P450 MUT-2) which catalyses the oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) to delta 8-THC-11-oic acid has been purified from hepatic microsomes of untreated male mice. Analysis of NH2-terminal sequence suggests that the isozyme is a member of the P450 2C gene subfamily. P450 MUT-2 exhibited aldehyde oxygenase activity for 11-oxo-delta 8-TH, 11-oxo-delta 9-THC, 11-oxo-cannabinol (11-oxo-CBN) and 9-anthraldehyde together with high activity for the hydroxylation of cannabinoids at the 11-position. Antibody against P450 MUT-2 significantly inhibited the microsomal formation of delta 8-THC-11-oic acid from 11-oxo-delta 8-THC, but not that of 9-anthracene carboxylic acid from 9-anthraldehyde. Major metabolic reactions of delta 8-THC, delta 9-THC and CBN with mouse hepatic microsomes were the 11-hydroxylation (all cannabinoids), 7 alpha-(delta 8-THC) or 8 alpha-hydroxylation (delta 9-THC) and epoxide formation (delta 8- and delta 9-THC). All these reactions except for 7 alpha-hydroxylation of delta 8-THC and alpha-epoxide formation from delta 9-THC were also markedly inhibited by the antibody. These results indicate that P450 MUT-2 is a major enzyme for metabolizing cannabinoids by mouse hepatic microsomes.

Possible pharmacokinetic and pharmacodynamic factors affecting parkinsonism inducement by cinnarizine and flunarizine.

Potentialities of cinnarizine [1-(diphenylmethyl)-4-(3-phenyl-2-propenyl)piperazine, CZ] and its fluorine derivative flunarizine [1-[bis(4-fluorophenyl)-methyl]-4-(3-phenyl-2-propenyl)piperazine, FZ] to induce parkinsonism as an adverse effect were evaluated pharmacokinetically and pharmacodynamically in rats. In multiple-dose experiments, CZ or FZ was given to rats at a daily dose of 20 mumol/kg for 1, 5, 10, 15, and 30 days, and CZ, FZ, and the ring-hydroxylated metabolites of their cinnamyl moiety [1-(diphenylmethyl)-4-[3-(4'-hydroxyphenyl)-2-propenyl]piperazine, C-2 and 1-[bis(4-fluorophenyl)methyl]-4-[3-(4'- hydroxyphenyl)propenyl]piperazine, F-2] in the plasma and striatum were determined 24 hr after the final dose. Plasma and striatum concentrations of the above compounds except for FZ reached steady state after 10 doses, but their concentrations of FZ continued to increase throughout the experiments. The concentrations obtained after the 30 doses were in the order of FZ > F-2 > CZ > C-2 for the plasma and of F-2 > FZ > CZ > C-2 for the striatum. The ratios of striatum to plasma concentrations of C-2 and F-2 were 2.4 and 3 times higher than those of the parent drugs. Binding affinities of CZ, FZ, and their 10 metabolites for rat striatal dopamine D-2 receptors (D2-R) were assessed by competitive radioligand-binding studies using [3H]-N-[(2RS,3RS)-1-benzyl-2-methyl-3-pyrrolidinyl]-5-chloro-2-met hoxy- 4-methylamino-benzamide ([3H]-YM-09151-2). The IC50s calculated from their Ki values were in the order of F-2 < C-2 < FZ < CZ < C-4 << F-1, indicating that C-2 and F-2 exhibit higher affinities for D2-R than the parent drugs, whereas affinities of other metabolites were 1 to 2 orders of magnitude less than those of C-2 and F-2. These results suggest some important roles of C-2 and F-2 in the development of parkinsonism as active metabolites during chronic medication with CZ and FZ, respectively.

Immunochemical characterization of a cytochrome P450 isozyme and a protein purified from liver microsomes of male guinea pigs and their roles in the oxidative metabolism of delta 9-tetrahydrocannabinol by guinea pig liver microsomes.

A protein (designated as protein-B) was purified from liver microsomes of adult male guinea pigs by an affinity chromatography with omega-aminooctyl Sepharose 4B, followed by HPLC using DEAE-5PW and hydroxyapatite columns which had been used to purify a cytochrome P450 (P450) isozyme (P450-A) from the same subcellular fraction (Narimatsu et al., Biochem Biophys Res Commun 172: 607-613, 1990). Protein-B had a molecular mass of 49 kDa in SDS-PAGE, but did not show absorbance at 417 nm for heme. Further, it did not show any oxidative activities towards aniline (AN), d-benzphetamine (d-BP), p-nitroanisole (p-NA) or delta 9-tetrahydrocannabinol (delta 9-THC) in a reconstituted system including dilauroylphosphatidylcholine, NADPH-P450 reductase, and cytochrome b5. However, antiserum against protein-B raised in rabbits suppressed liver microsomal oxidative activities towards d-BP and p-NA dose-dependently. The antibody decreased delta 9-THC oxidative activity most effectively, but did not decrease AN hydroxylation activity. Antiserum against P450-A suppressed all the activities towards these four substrates, especially towards delta 9-THC, in liver microsomes of male guinea pigs. Moreover, reconstitution with hemin made it possible for protein-B to produce some oxidative activity toward delta 9-THC. These results suggest that protein-B is also a cytochrome P450 isozyme which has lost a heme moiety during purification steps. Both P450-A and protein-B could have a role as cytochrome P450 isozymes in the oxidative metabolism of drugs, especially that of delta 9-THC by the liver microsomes of adult male guinea pigs.

Induction of propranolol metabolism by the azo dye sudan III in rats.

Effects of the azo dye sudan III, an inducer of cytochrome P450 isozymes belonging to the CYP1A subfamily, on propranolol (PL) in vitro and in vivo metabolism were investigated in rats. The kinetic parameters of the activity for each metabolic pathway were determined in liver microsomes from control and sudan III-treated rats. Sudan III pretreatment increased extensively PL 4-hydroxylase, 5-hydroxylase and N-desisopropylase activities at high but not at low PL concentrations. On the other hand, kinetic parameters of 7-hydroxylase activity were not affected by sudan III pretreatment. Sudan III pretreatment decreased blood concentrations of PL after intraportal infusion of PL at high doses (12.5 and 20 mg/kg), but not at a low dose (5 mg/kg). These observations were consistent with data obtained from the in intro studies showing that sudan III pretreatment induced low-affinity but not high-affinity cytochrome P450 isozymes involved in PL metabolism in rat liver microsomes.

Enzymatic basis for the non-linearity of hepatic elimination of propranolol in the isolated perfused rat liver.

Propranolol (PL) metabolism was studied in the isolated perfused rat liver under single-pass and steady-state conditions. An attempt was made to predict the data observed in the isolated rat liver perfusion at PL infusion rates of 89-1317 nmol/min using the microsomal kinetic parameters obtained in our previous paper (Ishida et al., Biochem Pharmacol 43: 2489-2492, 1992) and the unbound PL fractions in rat liver microsomes and the perfusion medium. The values of kinetic parameters obtained in rat liver microsomes were corrected for the whole liver. Two groups of cytochrome P450 isozymes having high (Km < 0.5 microM)- and low (Km > 20 microM)-affinities participate in the metabolism of PL and sudan III pretreatment induces the low-affinity enzymes rather than the high-affinity enzymes in control rats. Of high-affinity isozyme(s) PL 4-hydroxylase and 7-hydroxylase made a major contribution to the overall activity, while for low-affinity isozymes PL 4-hydroxylase and N-desisopropylase did. A nonlinear relationship between the PL concentrations entering and leaving the liver was predicted from these corrected kinetic parameters using the venous equilibrium model. The outflow concentrations and the metabolic rates of PL for the predicted curves were over-estimated at higher inflow PL concentrations and under-estimated at higher substrate concentrations, respectively. On the other hand, the prediction for them was successfully carried out for the livers whose intrinsic clearance was altered due to the induction of low-affinity enzymes in PL metabolism by sudan III pretreatment. The outflow rates of 4-hydroxypropranolol showed a downward curvature at lower substrate concentrations, followed a linear rise in the livers from control rats, while the outflow rates of 5- and 7-hydroxypropranolol exhibited their respective limiting values. The outflow rates of 4-hydroxypropranolol and N-desisopropylpropranolol were enhanced markedly with increasing the outflow unbound concentration of PL by sudan III pretreatment. These results indicate that non-linear PL first-pass metabolism is due to the saturation of the reactions for the high-affinity enzymes among enzymes engaging in PL ring hydroxylations.