Serotonergic activity of HP 184: does spontaneous release have a role?

Examination of HP 184, [N-n-propyl)-N-(3-fluoro-4-pyridinyl) -1H-3-methylindodel-1-amine hydrochloride], in a variety of tests for serotonergic activity revealed some unique properties of this compound. We report here that 100 microM HP 184 enhanced spontaneous release of [3H]serotonin (5-HT) from rat hippocampal slices. This release was independent of the uptake carrier. In vivo assays confirmed that HP 184 (20 mg/kg, i.p.) lacked significant interactions at the norepinephrine (NE) or 5-HT uptake carrier itself. Notably, HP 184 (15 mg/kg, i.p.) reduced drinking behavior in schedule-induced polydipsic (SIP) rats. We previously reported that some selective 5-HT reuptake inhibitors decrease SIP 30-40% after a 14-21 day treatment. In the current study, HP 184 decreased SIP beginning with the first treatment, and this reduction (30%) was maintained for 28 days. We further investigated HP 184 and serotonin metabolite levels. One hour after i.p. administration of 30 mg/kg HP 184, the ratio of whole brain 5-hydroxyindolacetic acid (5-HIAA) to 5-HT was increased, suggesting serotonergic activation. Under these conditions, the brain:plasma ratio of HP 184 was approximately 2:1, with brain concentrations of 1.6 micrograms/gram. We speculate that the spontaneous release effects of HP 184 may be responsible for the behavioral effects observed.

Application of hyphenated LC/NMR and LC/MS techniques in rapid identification of in vitro and in vivo metabolites of iloperidone.

Iloperidone, 1(-)[4(-)[3(-)[4-(6-fluro-1,2-benzisoxazol-3-yl)-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone, is currently undergoing clinical trials as a potential antipsychotic agent. Iloperidone was found to be extensively metabolized to a number of metabolites by rats, dogs, and humans. LC/MS/MS was used to characterize and identify metabolites of iloperidone present in complex biological mixtures obtained from all three species. Identification of some of the unknown metabolites in rat bile was achieved successfully by combination of LC/NMR and LC/MS with a minimum amount of sample cleanup. The utility of coupling a semipreparative HPLC to LC/MS instrument for further characterization of collected metabolites was demonstrated. It was shown that iloperidone was metabolized by O-dealkylation processes to yield 6-fluoro-3(-)[1-(3-hydroxypropyl)-4-piperidinyl]-1,2-benzisoxazole and 1(-)[4(-)[3(-)[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1- piperidinyl]propoxy]-2-hydroxyphenyl]ethanone. Oxidative N-dealkylation led to the formation of 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole and a secondary metabolite, 3(-)[(4-acetyl-2-methoxy)phenoxy]propionic acid. Iloperidone was reduced to produce 4(-)[3(-)[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]- propoxy]-3-methoxy-alpha-methylbenzenemethanol as the major metabolite in humans and rats. Hydroxylation of iloperidone produced 1(-)[4(-)[3(-)[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1- piperidinyl]propoxy]-2-hydroxy-5-methoxyphenyl]ethanone and 1(-)[4(-)[3(-)[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]-3 -methoxyphenyl]propoxy]-2-hydroxyethanone, the later of which was found to be the principal metabolite in dogs. The identities of all these metabolites were established by comparing the LC/MS retention times and mass spectral data with synthetic standards.

Determination of HP 749, a potential therapeutic agent for Alzheimer's disease, in plasma by high-performance liquid chromatography.

N-(n-Propyl)-N-(4-pyridinyl)-1H-indol-1-amine hydrochloride (HP 749, I), a non-receptor-dependent cholinomimetic agent with noradrenergic activity, is a potential agent for the treatment of Alzheimer's disease. Pharmacokinetic studies in animals and humans showed that I was well absorbed and metabolized primarily to the N-despropyl metabolite (P7480, II) after oral administration. To facilitate the kinetic studies, a sensitive and selective high-performance chromatographic assay was developed. I and II are extracted from plasma by a mixture of cyclohexane-ethyl acetate and chromatographed on an isocratic reversed-phase high-performance liquid chromatographic system employing an analytical phenyl column with acetonitrile-ammonium formate as mobile phase. The concentrations of these two compounds, quantitated by internal standardization, are monitored by ultraviolet detection. The method is linear in the plasma assay over a concentration range of 0.5-500 ng/ml for both compounds with a quantitation limit of 0.5 ng/ml. The precision and accuracy of the calibration curves and/or method are less than 10%. The recovery of I and II from plasma is 63-74 and 63-68%, respectively, over a concentration range of 0.5-500 ng/ml.

Identification of the urinary metabolites of tacrine in the rat.

Tacrine (THA) is a potent cholinesterase inhibitor being studied for the treatment of Alzheimer's disease. The metabolism and excretion of THA were studied in rats following a single oral dose of 20 mg/kg of THA. The results show THA was extensively metabolized in rats after oral administration. Three major urinary metabolites were isolated by HPLC on a semi-prep analytical phenyl column, and subsequent purification of the individual fractions on a semi-prep analytical cyano column. The major metabolic pathways involve the hydroxylation of the saturated ring at positions 1, 2, and 4. The structures of the metabolites 9-amino-1,2,3,4-tetrahydroacridin-1-ol (1-OH-THA), 9-amino-1,2,3,4-tetrahydroacridin-2-ol (2-OH-THA), and 9-amino-1,2,3,4-tetrahydroacridin-4-ol (4-OH-THA) were determined by electron impact mass spectrometry and/or 1H-NMR, and compared with synthetic references. The urinary excretion of THA and metabolites was quantitated by HPLC with UV detection. About 60% of the oral dose was eliminated as total THA, 1-OH-THA, 2-OH-THA, and 4-OH-THA over a 48-hr collection interval; and the non-conjugated THA and hydroxylated metabolites accounted for 45% of the dose.