dextromethorphan and alpha-naphthoflavone

Recently, we described a triple mutant of the bacterial cytochrome P450 BM3 as the first mutant with affinity for drug-like compounds. In this paper, we show that this mutant, but not wild-type BM3, is able to metabolise testosterone and several drug-like molecules such as amodiaquine, dextromethorphan, acetaminophen, and 3,4-methylenedioxymethylamphetamine that are known substrates of human P450s. Interestingly, the metabolism of 3,4-methylenedioxymethylamphetamine and acetaminophen could be stimulated up to 70-fold by the addition of caffeine, a known activator of rat P450 3A2. With testosterone metabolism, homotropic cooperativity was observed. This shows that heterotropic and homotropic cooperativity, known to occur in the P450 3A family, can also take place in BM3. BM3 therefore can be used as a model system to study atypical kinetics in mammalian P450s. Second, this study shows that BM3 can be engineered to a drug-metabolising enzyme, making it a promising candidate to use as biocatalyst in drug discovery and synthesis.

Topics: Acetaminophen; Amodiaquine; Arginine; Benzoflavones; Caffeine; Cytochrome P-450 Enzyme System; Dextromethorphan; Enzyme Activation; Humans; Kinetics; Leucine; Molecular Structure; Mutation; N-Methyl-3,4-methylenedioxyamphetamine; Pharmaceutical Preparations; Phenylalanine; Substrate Specificity

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.

Topics: Animals; Benzoflavones; Carbon Radioisotopes; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dextromethorphan; Enzyme Inhibitors; Hepatocytes; Inhibitory Concentration 50; Kinetics; Male; Methylation; Microsomes, Liver; Oxidoreductases, O-Demethylating; Protein Binding; Quinidine; Quinine; Rats; Rats, Sprague-Dawley; Sulfaphenazole; Troleandomycin

Dextromethorphan (DMO), a cough suppressing synthetic analog of codeine, undergoes parallel O-demethylation to dextrorphan (DOP), and N-demethylation to 3-methoxymorphinan (MEM), in humans. 3-hydroxymorphinan, a didemethylated metabolite, is formed secondarily. O-demethylation activity is well established as an index reaction for CYP2D6. However, this pathway appears to be mediated by at least two different enzymes in vitro. N-demethylation activity has recently been proposed to reflect CYP3A3/4 activity. We investigated both pathways in vitro with microsomal preparations from three human livers to assess the value of DMO as a probe drug for CYP2D6 and CYP3A3/4, DMO O-demethylation displayed a biphasic pattern with a high-affinity site reflecting CYP2D6 activity (mean Ki for quinidine, 0.1 +/- 0.13 microM). Kinetic parameters for the two O-demethylation mediating enzymes predict an average relative intrinsic clearance (Vmax/K(m) ratio) of 96% of total O-demethylation mediated via the high-affinity enzyme. Thus, in vitro data confirms the usefulness of DMO O-demethylation as an index reaction to monitor CYP2D6 activity. The Eadie-Hofstee plot of DMO N-demethylation was consistent with single-enzyme Michaelis-Menten kinetics (Vmax varying from 3.3 to 6.8 nmol mg-1 min-1, K(m) from 231 to 322 microM). However, ketoconazole, a CYP3A3/4 inhibitor, reduced N-demethylation only by 60% and had a mean Ki an order of magnitude higher (0.37 microM) compared to other pure CYP3A3/4 mediated reactions. Troleandomycin, a mechanism based CYP3A3/4 inhibitor, inhibited MEM formation by an average maximum of 46%, with an IC50 varying from 1 to 2.6 microM. A polyclonal rat liver CYP3A1 antibody inhibited MEM formation only by approximately 50%. Diethyldithiocarbamate (DDC), a mechanism based CYP2E1 inhibitor, reduced MEM formation at concentrations up to 150 microM between 33 and 43%. Chemical inhibitors of CYP2d6 (quinidine), CYP1A1/2 (alpha-naphthoflavone), and CYP2C9 (sulfaphenazole), as well as a goat rat liver CYP2C11 polyclonal antibody (inhibitory against human CYP2C9 and CYP2C19), had minimal effect on MEM formation rate, thus excluding an involvement of any of these enzymes. DMO N-demethylation is only partly mediated by CYP3A3/4, and therefore is not a reliable index reaction for CYP3A3/4 activity either in vitro or probably in vivo.

Topics: Animals; Antitussive Agents; Benzoflavones; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dextromethorphan; Ditiocarb; Enzyme Inhibitors; Humans; In Vitro Techniques; Methylation; Microsomes, Liver; Mixed Function Oxygenases; Quinidine; Rats; Sulfaphenazole; Tissue Donors