Identification of multiple glutathione conjugates of 8-amino- 2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline maleate (nomifensine) in liver microsomes and hepatocyte preparations: evidence of the bioactivation of nomifensine.

8-Amino-2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline maleate (nomifensine), an antidepressant drug, was withdrawn from the market because of increased incidence of hemolytic anemia, as well as kidney and liver toxicity. Although the nature of the potentially reactive metabolites formed after nomifensine metabolism remains unknown and no glutathione (GSH) adducts of these nomifensine reactive metabolites have been reported, bioactivation has been postulated as a potential mechanism for the toxicity of nomifensine. This study was conducted to probe the potential bioactivation pathways of nomifensine in human and animal hepatocytes and in liver microsomes using GSH as a trapping agent. Two types of GSH conjugates were characterized by liquid chromatography/tandem mass spectrometry: 1) aniline oxidation followed by GSH conjugation leading to the formation of nomifensine-GSH sulfinamides (M1 and M2); and 2) arene oxidation followed by GSH conjugation yielding a range of arene C-linked GSH adducts (M3-M9). Nine GSH adducts (M1-M9) were identified in liver microsomes of humans, dogs, monkeys, and rats and in human and rat hepatocytes. In dog hepatocyte preparations, six GSH adducts (M1-M6) were identified. The GSH adducts in dog and rat liver microsomes were formed primarily through aniline and arene oxidation, respectively. Both pathways contributed significantly to the formation of the GSH adducts in human and monkey liver microsomes. The bioactivation pathways proposed here account for the formation of the observed GSH conjugates. These investigations have confirmed the aniline and the arene groups in nomifensine as potential toxicophores capable of generating reactive intermediates.

Relationships between striatal dopamine denervation and frontal executive tests in Parkinson's disease.

Indirect evidence from human and monkey investigations supports the idea that impaired frontal tasks in Parkinson's disease (PD) may result from striato-frontal disruption caused by dopamine (DA) denervation of the caudate nucleus. To directly investigate this hypothesis, we used PET with 11C-S-Nomifensine (11C-S-NMF), a sensitive marker of striatal DA denervation, in 10 non-demented PD patients in whom two frontal executive tests, the object alternation (OA) and the conditional associative learning (CAL) tasks, thought to reflect mainly set-shifting/inhibition and planning, respectively, were given. In addition, the central executive function of verbal working memory was assessed with the Brown Peterson paradigm (BPP). We found a highly significant correlation between right caudate 11C-S-NMF specific binding and OA performance, less significant and reverse-direction correlations between CAL performance and putamen 11C-S-NMF binding, and no significant correlation with BPP performance. Thus, caudate DA denervation may subtend poor set-shifting/inhibition process in PD. Our results also point to distinct and complex relationships between striatal DA and specific frontal tasks.

Resolution, absolute stereochemistry, and enantioselectivity of 2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinolin-4-ol.

Racemic 2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinolin-4-ol (PI-OH) (1) was found to be an effective potentiator of the contractile response of norepinephrine (NE) on rat anococcygeus muscle. This paper describes the resolution of racemic PI-OH by an HPLC method to give the optically pure enantiomers (+)-1 and (-)-1. The absolute configuration of (+)-1 was R as determined by CD analysis and by single-crystal X-ray diffractometric analysis of the methiodide 6 derived from (+)-1. Examination of the effects of the enantiomers to potentiate the contraction of the rat anococcygeus muscle by NE showed a high degree of enantioselectivity. The NE potentiation was found to reside exclusively in (R)-(+)-1; the activity ratio being 21 at 3 x 10(-6) M, whereas (S)-(-)-1 did not show any potentiating and inhibiting activity.

4-Hydroxy-2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (PI-OH): a new potentiator of sympathomimetic amines on the rat anococcygeus muscle.

1. The potentiating effects of racemic 4-hydroxy-2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (PI-OH), cocaine, desipramine and nomifensine on the concentration-response curves of the rat anococcygeus muscle to noradrenaline (NA) and field stimulation were examined. 2. PI-OH and cocaine concentration-dependently potentiated the responses of anococcygeus muscle to NA and field stimulation, but the activity of PI-OH was stronger than that of cocaine on both responses. 3. At high concentrations the potentiating activities of desipramine and nomifensine were less, a fact that was explained by their postsynaptic inhibitory properties; the actions of nomifensine and desipramine as antagonists against NA were competitive and non-competitive, respectively. 4. It is concluded that PI-OH may be an ideal potentiator of the response to NA in adrenergically-innervated tissues because it has no side effects such as postsynaptic inhibition.

New substituted 1-phenyl-3-benzazepine analogues of SK&F 38393 and N-methyl-thienopyridine analogues of dihydroxynomifensine with selective affinity for the D-1 dopamine receptor in human post-mortem brain.

3H-SCH 23390 and 3H-spiperone were used to label D-1 and D-2 dopamine receptors, respectively, in human post-mortem brain. SK&F 82958 and SK&F 77434, new substituted 1-phenyl-1H-3-benzazepines, showed selective affinity for the D-1 receptor. While S- and R- enantiomers of dihydroxynomifensine showed only weak stereoselective affinity for the D-1 receptor, the thieno [2,3-c]-and thieno [3,2-c]- analogues SK&F 89145 and SK&F 89641 showed selective D-1 affinity comparable to that of the benzazepines. These novel agents provide new information on structure-affinity relationships and D-1 receptor topography, and constitute new tools for functional studies.

Metabolism of nomifensine after oral and intravenous administration.

The metabolism of nomifensine was studied after single oral and intravenous administration and after 2 weeks of oral dosing. The three principal metabolites reached maximum plasma concentrations rapidly (in 1 to 1.5 hours) after nomifensine administration. Less than 10% was detected as a free, unconjugated form. All three metabolites were eliminated rapidly (elimination t1/2 values between 6.8 and 9.0 hours). Only very low concentrations of free metabolites were found in plasma after 24 hours of nomifensine administration. AUC values for free metabolites were between 0.27 to 0.46 hr X mumol/L after all nomifensine schedules. Two weeks of dosing had no significant influence on the elimination t1/2 or AUC values of the metabolites, indicating no change in the hydroxylation and methylation reactions. In addition, there were no changes in the conjugation reactions during prolonged nomifensine dosing. Nomifensine has a very short t1/2 and no tendency for accumulation after repeated doses. We conclude that nomifensine's clinical pharmacokinetic profile is not significantly changed by the kinetic behavior of its three main metabolites after the usual maintenance doses.

Selective and sensitive high-performance liquid chromatographic assay for the metabolites of nomifensine in human plasma.

A selective high-performance liquid chromatographic method for the determination of the three metabolites of nomifensine in human plasma is described. All metabolites and the internal standard, mexiletine, are extracted with diethyl ether and then back-extracted into an acidic aqueous phase. After subsequent extraction into diethyl ether the metabolites are analysed by high-performance liquid chromatography. A reversed-phase C18 column is used with a mobile phase of dioxane-methanol-potassium phosphate buffer (pH 2.25). The sensitivity of the method is 0.007 micromol/l for all metabolites. Extraction efficiencies are 84.6%, 75.8%, and 78.2% for 4'-hydroxynomifensine, 4'-hydroxy-3'-methoxynomifensine and 3'-hydroxy-4'-methoxynomifensine, respectively. The reproducibility of the method is good, the coefficients of variation (%) varying between 2.1% and 9.9% in the concentration range 0.05-1.00 micromol/l. The procedure was applied to human plasma samples from a volunteer who had received a single oral dose of nomifensine. The method is accurate and sensitive for pharmacokinetic studies on the metabolites of nomifensine.

Synthesis and antidepressant activity of 4-aryltetrahydrothieno[2,3-c]pyridine derivatives.

A series of substituted 4-aryltetrahydrothieno[2,3-c]pyridines was prepared by acid-catalyzed cyclization of 1-aryl-2-[(2-thienylmethyl)amino]ethanol derivatives. The compounds were examined for their antidepressant activity, as demonstrated by their ability to inhibit the uptake of norepinephrine (NE) and serotonin (5-HT) and to prevent tetrabenazine-induced ptosis (TBZ) in mice. Significant inhibition of both neurotransmitters is observed for several of the tested compounds, while some of them are selective inhibitors of either NE or 5-HT uptake. Optimal activity is associated with the introduction of lipophilic substituents into the 4-position of the phenyl ring and less lipophilic substituents into the 2-position of the thiophene ring (11, 23). Compound 33 bearing substituents in positions 2 and 6 of the phenyl ring is inactive. This might be a consequence of an out of plane conformation of this compound.

Synthesis, resolution, absolute stereochemistry, and enantioselectivity of 3',4'-dihydroxynomifensine.

3',4'-Dihydroxynomifensine, 8-amino-1,2,3,4-tetrahydro-4-(3,4-dihydroxyphenyl)-2-methylisoquinoli ne (1a), is an agonist of dopamine receptors in central and peripheral systems. Since this dopamine receptor agonist bears an asymmetric center at position 4, its synthesis and resolution were undertaken as part of a study directed toward determining the mode of interaction of these agents with the receptor(s). The enantiomers of 3',4'-dihydroxynomifensine are of particular interest, as they provide additional probes of present conceptual models of the dopamine receptor(s). Initial attempts to prepare 1a were inefficient or unsuccessful; instead, an isomeric compound, 1,2,4,5-tetra-hydro-2-(3,4-dihydroxyphenyl)-4- methyl-3H-1,4-benzodiazepine (9), was obtained. For this reason, a new route to 3',4'-dihydroxynomifensine was employed. The racemic dimethoxy intermediate 1d, thus obtained, was resolved. Methoxyl cleavage of the isomers of 1d afforded the enantiomers of 1a. Enantiomeric excess of these antipodes or appropriate derivatives was examined by NMR, CD, and HPLC methods. CD analysis suggests an enantiomeric excess greater than 99%. Determination of the absolute configuration of the enantiomers of 1a was determined by single-crystal X-ray diffractometric analysis. Examination of the isomers in several pharmacological test systems revealed a high degree of enantioselectivity. D-1 dopaminergic activity resides almost exclusively in the S enantiomer. The findings of the study have been employed to suggest an accessory binding site on the dopamine receptor(s) that differs from that advanced earlier. This accessory binding site may be specific for the D-1 subpopulation of dopamine receptors.

Dopaminergic properties of nomifensine.

Nomifensine and a proposed dihydroxy metabolite produced stimulation of motor behavior in mice with nomifensine being more potent. Weak cage-climbing behavior (stereotypy) was also produced. The stimulatory effects were greater in mice in which dopamine receptor sensitivity was increased by long-term haloperidol. Both of the analogs were potent inhibitors of dopamine and norepinephrine uptake in vitro with nomifensine approximately 3 times more potent than the metabolite. In contrast, the two analogs had weak affinity for the post-synaptic dopamine receptor (as measured by displacement of ligand binding in vitro) with dihydroxynomifensine approximately 6 times more potent than nomifensine. These results suggest that the behavioral effects of nomifensine are largely dependent on presynaptic catecholamine mechanisms but that weak direct dopamine agonist properties do exist, particularly in vivo where the drug may be metabolized to a more active form.

Dopamine agonist properties of N-alkyl-4-(3,4-dihydroxyphenyl)-1,2,3,4-tetrahydroisoquinolines.

A series of homologous N-alkyl-4-(3,4-dihydroxyphenyl)-1,2,3,4-tetrahydroisoquinolines was synthesized and examined for a dopamine-like ability to dilate the renal artery. The N-methyl derivative was equipotent to the 3',4'-dihydroxy derivative of the antidepressant agent nomifensine, indicating that the 8-amino group of the latter is not essential for dopamine-like activity. The N-ethyl homologue was reduced in potency when compared to the N-methyl, and the N-n-propyl, surprisingly, was essentially devoid of activity. This was unexpected in view of the fact that in all series of dopamine-like agents reported to date, N-alkylation, when one of the alkyls was an n-propyl group, either allowed retention or enhancement of potency.

Direct effect of a nomifensine derivative on dopamine receptors.

A study was made of the effects of nomifensine, 4'-hydroxynomifensine and 3',4'-dihydroxynomifensine on dopamine receptors in rat striatum and nucleus accumbens, using the dopamine-sensitive adenylate cyclase assay. Nomifensine and its 4'-hydroxy metabolite were both inactive as dopaminergic agonists. 3',4'-dihydroxynomifensine was, however, a potent agonist, being approximately 2 to 4 times less active than dopamine. The effects of dopamine and of 3',4'-dihydroxynomifensine were blocked by fluphenazine. It is concluded that the dopaminergic activity of 3',4'-dihydroxynomifensine is dependent upon the presence of the two hydroxyl groups.