rifampin and alpha-naphthoflavone

1. Amiodarone (AMI) is a potent anti-arrhythmic drug and mono-N-desethylamiodarone (MDEA) is its only known metabolite. It was found recently that in rabbit liver microsomes MDEA was biotransformed to n-3-hydroxybutyl-MDEA (3OH-MDEA). 2. In liver microsomes isolated from the untreated rabbit, the formation of 3OH-MDEA obeyed Michaelis-Menten enzyme kinetics with Km = 6.39 +/- 1.07 microM and Vmax = 0.56 +/- 0.21 nmolmin(-1) mg(-1) protein. 3. Furthermore, (1) among chemicals usually used as inhibitors of cytochrome P450, only midazolam (MDZ), cyclosporin A and ketoconazole inhibited the MDEA hydroxylase activity significantly (>60% inhibition), (2) MDZ, a substrate of CYP3A, inhibited the 30OH-MDEA formation competitively (Ki = 10 +/- 5 microM), (3) the formation rates of 3OH-MDEA correlated positively with those of 1'OH-MDZ (r = 0.81; n = 6), and (4) MDEA hydroxylase activity of microsomes isolated from rabbit rifampicin-induced cultured hepatocytes was 4-fold more active than the control. 4. Since CYP3A6 is mainly induced by rifampicin in rabbit-cultured hepatocytes, the data suggest that this isoform is involved in the biotransformation of MDEA to 3OH-MDEA. 5. Since alpha-naphthoflavone, cimetidine and quinidine also partially inhibited the MDEA hydroxylase activity, it is possible that other CYPs, such as 1A, 2C and 2D, may also be active in the metabolism of amiodarone.

Topics: Amiodarone; Animals; Anti-Anxiety Agents; Anti-Arrhythmia Agents; Aryl Hydrocarbon Hydroxylases; Benzoflavones; Chromatography, High Pressure Liquid; Cimetidine; Cyclosporine; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ketoconazole; Kinetics; Microsomes, Liver; Midazolam; Models, Chemical; Oxidoreductases, N-Demethylating; Protein Isoforms; Quinidine; Rabbits; Rifampin; Time Factors

The binding of the amino steroid, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), to rabbit liver cytochrome P-450 3c was studied using purified P-450 3c and liver microsomes prepared from rifampicin-treated B/J rabbits. 22-ABC binds to purified cytochrome P-450 3c producing a type II spectral change reflecting the coordination of the amine with the heme iron of the protein. In the absence of allosteric effectors, the binding is characterized by a Ks of 5 microM. In the presence of alpha-naphthoflavone or progesterone, the Ks decreases to 0.8 microM, indicating that these two compounds serve as positive effectors of the binding of 22-ABC to cytochrome P-450 3c. The antibiotic rifampicin induces cytochrome P-450 3c in rabbit liver microsomes, and the benzo(a)pyrene hydroxylase, estradiol 2-hydroxylase, and progesterone 6 beta-hydroxylase activities of these microsomes are stimulated by alpha-naphthoflavone. Moreover, the progesterone 6 beta-hydroxylase activity catalyzed by these microsomes exhibits a dependence on substrate concentration that is consistent with activation of the enzyme by the substrate, progesterone. The magnitude of the type II spectral change elicited by 22-ABC for microsomes prepared from rifampicin-treated B/J rabbits is greater than that observed for microsomes from untreated rabbits. For microsomes from rifampicin-treated rabbits, the apparent binding constant for 22-ABC was decreased 5-fold in the presence of alpha-naphthoflavone. We propose that the effects of alpha-naphthoflavone and progesterone on the binding of 22-ABC to cytochrome P-450 3c mimic the effects of the two positive effectors on the metabolism of substrates by increasing the affinity of the enzyme for substrate.

Topics: Animals; Benzoflavones; Binding Sites; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Microsomes, Liver; Pregnenes; Pregnenolone; Progesterone; Protein Binding; Rabbits; Rifampin; Spectrophotometry

Rifampicin induces cytochrome P-450 3c, progesterone 16 alpha- and 6 beta-hydroxylation, 17 beta-estradiol 2-hydroxylation, benzo[a] pyrene hydroxylation, and erythromycin N-demethylation in rabbit liver microsomes. Kinetic analysis of the 6 beta-hydroxylation of progesterone as catalyzed by liver microsomes prepared from rifampicin-treated B/J rabbits exhibits a curvilinear double-reciprocal plot, suggestive of substrate activation. Further experimentation demonstrated that alpha-naphthoflavone could augment the catalytic efficiency [Vmax/Km] observed for the 16 alpha- and 6 beta-hydroxylation of progesterone and the 2-hydroxylation of 17 beta-estradiol, whereas erythromycin N-demethylase activity was partially inhibited. Allosteric activation of these steroid hydroxylases by alpha-naphthoflavone is also found for human liver microsomes, indicating that the activation of these enzymes is conserved in man and rabbit.

Topics: Allosteric Regulation; Animals; Benzoflavones; Cytochrome P-450 Enzyme System; Enzyme Activation; Estradiol; Flavonoids; Humans; In Vitro Techniques; Kinetics; Microsomes, Liver; Progesterone; Rabbits; Rifampin

The metabolism of benzo(a)pyrene by rabbit liver microsomes can be stimulated or inhibited by 7,8-benzo(a)flavone (ANF) depending on the distribution of specific P-450 enzymes present within the microsomes. Treatment of rabbits with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or rifampicin leads to an increase of hepatic microsomal metabolism of benzo(a)pyrene. ANF stimulates the rate of benzo(a)pyrene metabolism catalyzed by microsomes isolated from rabbits treated with rifampicin by 3-fold. In contrast, ANF moderately inhibits the activity of microsomes from TCDD-treated rabbits. Variations in the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits apparently reflect differences in the expression of P-450 1, a constitutive form of P-450. Thus, the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits, which varies from 0.40 to 1.5 nmol/min/mg of protein, is directly correlated with the microsomal concentration of P-450 1. The metabolism of benzo(a)pyrene by microsomes containing high concentrations of P-450 1 is inhibited by a monoclonal antibody specific for this cytochrome to approximately the rate exhibited by microsomes with a low concentration of P-450 1. The benzo(a)pyrene activity stimulated by ANF in microsomes with a low concentration of P-450 1 is not inhibited by the monoclonal antibody. The activity of P-450 1 is inhibited by ANF at concentrations that stimulate other constitutive forms of P-450. Thus, ANF produces offsetting effects on benzo(a)pyrene metabolism in microsomes from untreated animals by stimulating the activity of at least one cytochrome and inhibiting P-450 1-mediated activity.

Topics: Animals; Antibodies, Monoclonal; Benzo(a)pyrene; Benzoflavones; Benzopyrene Hydroxylase; Cytochrome P-450 Enzyme System; Flavonoids; In Vitro Techniques; Microsomes, Liver; Polychlorinated Dibenzodioxins; Rabbits; Rifampin