clozapine and norfluoxetine

Allopregnanolone is a neurosteroid which exhibits anxiolytic and anticonvulsant activities through potentiation of the GABA(A) receptor. The reduction of 5alpha-dihydroprogesterone (5alpha-DHP), the last step in allopregnanolone biosynthesis, is catalyzed by 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs). While the mechanism of action of allopregnanolone and the physiological and pharmacological modulation of allopregnanolone concentrations in vivo have been extensively studied, there has been little characterization of the kinetics of human 3alpha-HSD catalyzed allopregnanolone formation. We report here determination of the kinetic mechanism for 5alpha-DHP reduction catalyzed by human 3alpha-HSD type III by using steady-state kinetics studies and assessment of the ability of fluoxetine and various other small molecules to activate 3alpha-HSD type III catalyzed allopregnanolone formation. Enzyme-catalyzed 5alpha-DHP reduction yielded two products, allopregnanolone and 5alpha,20alpha-tetrahydroprogesterone, as measured by using a radiometric thin-layer chromatography assay, while 5beta-DHP reduction yielded the neurosteroid pregnanolone as the only product. 5Beta-DHP reduction proceeded with a catalytic efficiency 10 times higher than that of 5alpha-DHP reduction. Two-substrate kinetic analysis and dead-end inhibition studies for 5alpha-DHP reduction and allopregnanolone oxidation indicated that 3alpha-HSD type III utilized a ternary complex (sequential) kinetic mechanism, with nicotinamide adenine dinucleotide cofactor binding before steroid substrate and leaving after steroid product. Since previous reports suggested that fluoxetine and certain other small molecules increased allopregnanolone concentrations in vivo by activating 3alpha-HSD type III, we investigated whether these small molecules were able to activate human 3alpha-HSD type III. Our results showed that, at concentrations up to 50 microM, fluoxetine, paroxetine, sertraline, norfluoxetine, carbamazepine, clozapine, flurbiprofen, and sulfobromophthalein did not activate the enzyme. These results characterize the role of 3alpha-HSD type III in allopregnanolone formation and suggest that activation of this enzyme by fluoxetine is likely not the mechanism by which fluoxetine increases allopregnanolone concentrations.

Topics: 3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); 3-Hydroxysteroid Dehydrogenases; 5-alpha-Dihydroprogesterone; Carbamazepine; Catalysis; Clozapine; Enzyme Activation; Enzyme Inhibitors; Fluoxetine; Flurbiprofen; Humans; Hydroxysteroid Dehydrogenases; Kinetics; Oxidation-Reduction; Pregnanediones; Pregnanolone; Recombinant Fusion Proteins; Selective Serotonin Reuptake Inhibitors; Substrate Specificity; Ursodeoxycholic Acid

We describe an analytical procedure for the simultaneous quantification of citalopram (seropram), clozapine (leponex), fluoxetine (fluctine), norfluoxetine, maprotiline (ludiomil), desmethylmaprotiline and trazodone (trittico) in human serum within a period of 11.5 minutes using reversed phase HPLC. After 2 liquid/liquid extractions in the sample preparation phase, the drugs and metabolites were separated on a C18 column using a mobile phase consisting of acetonitrile/buffer (30/70, v:v) at 70 degrees C, a flow rate of 1.5 m/min and haloperidol as internal standard. Absorption and native fluorescence signals of the eluted compounds were detected simultaneously at 260 nm and 227/300 nm (excitation/emission), respectively. The calibration ranges for citalopram, clozapine, fluoxetine, norfluoxetine, maprotiline, and desmethylmaprotiline ranged from 50-400 microg/l and for trazodone from 50-3,200 microg/l. The CVs varied between 0.6% and 5.5% (within-run) and between 3.2% and 7.1% (between-run). Recoveries were > 90% for all pharmaceuticals. We noticed no interferences from several commonly used drugs.

Topics: Antidepressive Agents, Second-Generation; Antipsychotic Agents; Chromatography, High Pressure Liquid; Citalopram; Clozapine; Fluoxetine; Humans; Maprotiline; Trazodone