Rationally designed multi-targeted agents against neurodegenerative diseases.

Neurodegenerative diseases are complex disorders with several pathoetiological pathways leading to cell death. Rationally designed multi-targeted agents, or "multi-targeted designed drugs" (MTDD) show significant promise in preclinical studies as neuroprotective and disease-modifying agents. In this review, we highlight the use of chemical scaffolds that lend themselves exquisitely to the development of MTDDs in neurodegeneration. Notably, synthetic polycyclic cage compounds have served as scaffolds for novel voltage-gated calcium channel blockers, NMDA receptor antagonists, and sigma-receptor ligands - attractive targets in neurodegeneration. In an entirely different approach, compounds containing the thiazolidinedione moiety (referred to as glitazones) alter mitochondrial function through the mitochondrial protein mitoNEET, an attractive new drug target for the treatment of neurodegenerative diseases. The design strategy for yet another agent, ladostigil, employed the amalgamation of active chemical moieties of the AChE inhibitor rivastigmine, and the monoamine oxidase-B (MAO-B) inhibitor rasagiline, leading to a single compound that targets both enzymes simultaneously. Natural products have also served as design templates for several MTDD design studies. In particular, the stilbene scaffold has become popular in particular due to the neuroprotective effects of the non-flavonoid natural product resveratrol. Recently, stilbene scaffold-based compounds were developed to reduce - through chelation with metal ions that interact with beta-amyloid - both metal-induced beta-amyloid protein aggregation, and ROS generated from this aggregate. Other subtle modifications of the stilbene motif led to the creation of reversible, non-competitive MAO inhibitors. Finally, compounds derived from the xanthine scaffold afford neuroprotection in Parkinson's disease through mechanisms that include dual adenosine A2A receptor antagonism and MAO-B inhibition.

NGP1-01, a multi-targeted polycyclic cage amine, attenuates brain endothelial cell death in iron overload conditions.

Development and progression of neurodegenerative disorders have, amongst other potential causes, been attributed to a disruption of iron regulatory mechanisms and iron accumulation. Excess extracellular iron may enter cells via nontraditional routes such as voltage-gated calcium channels and N-methyl-d-aspartate (NMDA) receptors leading to intracellular oxidative damage and ultimately mitochondrial failure. Nimodipine, an L-type calcium channel blocker has been shown to reduce iron-induced toxicity in neuronal and brain endothelial cells. Our current study investigates NGP1-01, a multimodal drug acting as an antagonist at both the NMDA receptor and the L-type calcium channel. Our previous studies support NGP1-01 as a promising neuroprotective agent in diseases involving calcium-related excitotoxicity. We demonstrate here that NGP1-01 (1 and 10µM) pretreatment abrogates the effects of iron overload in brain endothelial cells protecting cellular viability. Both concentrations of NGP1-01 were found to attenuate iron-induced reduction in cellular viability to a similar extent, and were statistically significant. To further verify the mechanism, the L-type calcium channel agonist FPL 64176 was administered to promote iron uptake. Addition of NGP1-01 dose-dependently reduced FPL 64176 stimulated uptake of iron. These data support further evaluation of NGP1-01 as a neuroprotective agent, not only in diseases associated with excitotoxicity, but also in those of iron overload.

Differential effect of nimodipine in attenuating iron-induced toxicity in brain- and blood-brain barrier-associated cell types.

Metal homeostasis is increasingly being evaluated as a therapeutic target in stroke and neurodegenerative diseases. Metal dysregulation has been shown to lead to protein aggregation, plaque formation and neuronal death. In 2007, we first reported that voltage-gated calcium channels act as a facile conduit for the entry of free ferrous (Fe(2+)) ions into neurons. Herein, we evaluate differential iron toxicity to central nervous system cells and assess the ability of the typical L-type voltage-gated calcium channel blocker nimodipine to attenuate iron-induced toxicity. The data demonstrate that iron sulfate induces a dose-dependent decrease in cell viability in rat brain endothelial cells (RBE4; LC(50) = 150 µM), neuronal cells (Neuro-2α neuroblastoma; LC(50) = 400 µM), and in astrocytes (DI TNC1; LC(50) = 1.1 mM). Pre-treatment with nimodipine prior to iron sulfate exposure provided a significant (P < 0.05) increase in viable cell numbers for RBE4 (2.5-fold), Neuro2-α (~2-fold), and nearly abolished toxicity in primary neurons. Astrocytes were highly resistant to iron toxicity compared to the other cell types tested and nimodipine had no (P > 0.05) protective effect in these cells. The data demonstrate variable susceptibility to iron overload conditions in different cell types of the brain and suggest that typical L-type voltage-gated calcium channel blockers (here represented by nimodipine), may serve as protective agents in conditions involving iron overload, particularly in cell types highly susceptible to iron toxicity.

Chronic nicotine exposure alters blood-brain barrier permeability and diminishes brain uptake of methyllycaconitine.

Methyllycaconitine (MLA) is reported to be a selective antagonist for the nicotinic acetylcholine receptor alpha7 subtype and has been found in animal behavioral studies to reduce nicotine self-administration and attenuate nicotine withdrawal symptoms. While MLA crosses the blood-brain barrier (BBB), no studies have assessed brain uptake in animals subjected to chronic nicotine exposure. Given that chronic nicotine administration has been reported to alter BBB parameters that may affect the kinetic BBB passage of MLA, we evaluated MLA brain uptake in naive and S-(-)nicotine-exposed rats (4.5 mg/kg/day for 28 days; osmotic minipumps) using in situ rat brain perfusions. Our results demonstrate that in situ(3)H-MLA brain uptake rates in naive animals approximate to intravenous kinetic data (K(in), 3.24 +/- 0.71 x 10(-4) mL/s/g). However, 28-day nicotine exposure diminished (3)H-MLA brain uptake by approximately 60% (K(in), 1.29 +/- 0.4 x 10(-4) mL/s/g). This reduction was not related to nicotine-induced (3)H-MLA brain efflux or BBB transport alterations. Similar experiments also demonstrated that the passive permeation of (14)C-thiourea was diminished approximately 24% after chronic nicotine exposure. Therefore, it appears that chronic nicotine exposure diminishes the blood-brain passive diffusion of compounds with very low extraction rates (i.e. permeability-limited compounds). These findings imply that the pharmacokinetics of neuropharmaceutical agents that are permeability limited may need to be re-evaluated in individuals exposed to nicotine.

Effects of organophosphorus compounds on ATP production and mitochondrial integrity in cultured cells.

Recent studies in vivo and in vitro suggested that mitochondrial dysfunction follows exposure to organophosphorus (OP) esters. As mitochondrial ATP production is important for cellular integrity, ATP production in the presence of OP neurotoxicants was examined in a human neuronal cell line (SH-SY5Y neuroblastoma cells) and primary dorsal root ganglia (DRG) cells isolated from chick embryos and subsequently cultured to achieve maturation with axons. These cell culture systems were chosen to evaluate toxic effects on the mitochondrial respiratory chain associated with exposure to OP compounds that do and do not cause OP-induced delayed neuropathy (OPIDN), a disorder preceded by inhibition of neurotoxic esterase (NTE). Concentration- and time-response studies were done in neuroblastoma cells exposed to phenyl saligenin phosphate (PSP) and mipafox, both compounds that readily induce delayed neuropathy in hens, or paraoxon, which does not. Phenylmethylsulfonyl fluoride (PMSF) was included as a non-neuropathic inhibitor of NTE. Purified neuronal cultures from 9 day-old chick embryo DRG were treated for 12 h with 1 microM PSP, mipafox, or paraoxon. In situ evaluation of ATP production measured by bioluminescence assay demonstrated decreased ATP concentrations both in neuroblastoma cells and chick DRG neurons treated with PSP. Mipafox decreased ATP production in DRG but not in SH-SY5Y cells. This low energy state was present at several levels of the mitochondrial respiratory chain, including Complexes I, II, III, and IV, although Complex I was the most severely affected. Paraoxon and PMSF were not effective at all complexes, and, when effective, required higher concentrations than needed for PSP. Results suggest that mitochondria are an important early target for OP compounds, with exposure resulting in depletion of ATP production. The targeting of neuronal, rather than Schwann cell mitochondria in DRG following exposure to PSP and mipafox was verified by loss of the mitochondrial-specific dye, tetramethylrhodamine, in these cells. No such loss was seen in paraoxon exposed neurons isolated from DRG or in Schwann cells treated with any of the test compounds.

Brain uptake kinetics of nicotine and cotinine after chronic nicotine exposure.

Blood-brain barrier (BBB) nicotine transfer has been well documented in view of the fact that this alkaloid is a cerebral blood flow marker. However, limited data are available that describe BBB penetration of the major tobacco alkaloids after chronic nicotine exposure. This question needs to be addressed, given long-term nicotine exposure alters both BBB function and morphology. In contrast to nicotine, it has been reported that cotinine (the major nicotine metabolite) does not penetrate the BBB, yet cotinine brain distribution has been well documented after nicotine exposure. Surprisingly, therefore, the literature indirectly suggests that central nervous system cotinine distribution occurs secondarily to nicotine brain metabolism. The aims of the current report are to define BBB transfer of nicotine and cotinine in naive and nicotine-exposed animals. Using an in situ brain perfusion model, we assessed the BBB uptake of [3H]nicotine and [3H]cotinine in naive animals and in animals exposed chronically to S-(-)nicotine (4.5 mg/kg/day) through osmotic minipump infusion. Our data demonstrate that 1) [3H]nicotine BBB uptake is not altered in the in situ perfusion model after chronic nicotine exposure, 2) [3H]cotinine penetrates the BBB, and 3) similar to [3H]nicotine, [3H]cotinine BBB transfer is not altered by chronic nicotine exposure. To our knowledge, this is the first report detailing the uptake of nicotine and cotinine after chronic nicotine exposure and quantifying the rate of BBB penetration by cotinine.

Inhibition of human MAO-A and MAO-B by a compound isolated from flue-cured tobacco leaves and its neuroprotective properties in the MPTP mouse model of neurodegeneration.

Prompted by the findings that smokers have lowered brain and blood platelet monoamine oxidase-A and -B activities compared to non-smokers and that smokers have a lowered incidence of Parkinson's disease, we have examined the neuroprotective properties of an MAO inhibitor, 2,3,6-trimethyl-1,4-naphthoquinone (TMN), which is present in the tobacco plant and smoke in the MPTP C57BL/6 mouse model of neurodegeneration. Dopamine (DA) levels in the striata of mice treated with TMN prior to the administration of MPTP were significantly higher than DA levels in the striata of mice receiving MPTP only, thus indicating a degree of neuroprotection in this model of Parkinson's disease. The potential consequences on MAO activity of long term exposure to this compound need to be evaluated. Furthermore, there is evidence for the presence of other inhibitors in the tobacco leaf and smoke, including compounds with irreversible MAO inhibitory properties. Although there is no evidence to link the lowered activities of MAO to the lowered incidence of Parkinson's disease in smokers, the neuroprotective effects of TMN in the MPTP mouse model suggest that such a relationship is worthy of further evaluation.

Neuroprotection in the MPTP Parkinsonian C57BL/6 mouse model by a compound isolated from tobacco.

Epidemiological evidence suggests a lower incidence of Parkinson's disease in smokers than in nonsmokers. This evidence, together with the lower levels of brain monoamine oxidase (MAO) activity in smokers and the potential neuroprotective properties of MAO inhibitors, prompted studies which led to the isolation and characterization of 2,3,6-trimethyl-1,4-naphthoquinone (TMN), an MAO-A and MAO-B inhibitor which is present in tobacco and tobacco smoke. Results of experiments reported here provide evidence that this compound protects against the MPTP-mediated depletion of neostriatal dopamine levels in the C57BL/6 mouse. These results support the hypothesis that the inhibition of MAO by constituents of tobacco smoke may be related to the decreased incidence of Parkinson's disease in smokers.

Effects of derivatives of NGP 1-01, a putative calcium channel antagonist, on electrically stimulated guinea-pig papillary muscle.

In earlier work we have reported calcium antagonistic properties for the polycyclic compound NGP 1-01. We now have derivatized NGP 1-01 by side-chain substitution to obtain ten novel aromatic and aliphatic imino-keto and amino-ether compounds. Electrophysiological tests were conducted on these compounds using isolated guinea-pig papillary muscle preparations to record calcium-mediated (slow) action potentials (APs). The lipophilicities of the compounds, expressed as chromatographically determined RM values, were measured and the molecular surface areas calculated. Several derivatives showed increased activity compared with NGP 1-01. All compounds with aromatic side-chains (benzyl, phenethyl, phenylpropyl) were active (concentrations required for complete suppression of the AP varied between 1 x 10(-5) M and 5 x 10(-5) M) and compounds with shorter (methyl, butyl) aliphatic side-chains were inactive whilst activity increased dramatically in those compounds with octyl side-chains. Lipophilicity and calculated molecular volumes correlated linearly and bulkier, more lipophilic molecules had increasing activities in the electrophysiological assay. We therefore conclude that bulky substituents on the nitrogen atom increase calcium antagonistic activity in this series of compounds.

Metabolic defects caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and by HPTP (the tetrahydropyridinyl analog of haloperidol), in rats.

The results of previous studies in the baboon have suggested that HPTP, the tetrahydropyridinyl analog of haloperidol causes a urinary biochemical marker profile similar to those seen in humans suffering from inborn errors of mitochondrial respiration. In order to identify a possible relationship between compromised cellular energy production and neuronal damage we now have compared the urinary profiles of rats treated with the pro-neurotoxin, MPTP as well as with HPTP. Significantly increased urinary excretion of lactic acid and 2-ethylhydracrylic acid in MPTP and HPTP treated rats was observed, indicating that both MPTP and HPTP and/or their respective metabolites cause mitochondrial inhibition in the rat.

Studies on the in vivo biotransformation of the tobacco alkaloid beta-nicotyrine.

This paper reports the results of studies on the in vivo metabolic fate of the tobacco alkaloid 1-methyl-2-(3-pyridinyl)pyrrole (beta-nicotyrine) in New Zealand white rabbits. Two previously characterized metabolites, 5-hydroxy-1-methyl-5-(3-pyridinyl)-2-pyrrolidinone (5-hydroxycotinine) and 2-hydroxy-1-methyl-5-(3-pyridinyl)-3-pyrrolin-2-one, were present in low concentrations in the urine of the treated animals. The major urinary metabolite of beta-nicotyrine was identified as cis-3'-hydroxy-1-methyl-5-(3-pyridinyl)-2-pyrrolidinone (cis-3'-hydroxycotinine), the diastereoisomer of the major urinary metabolite of (S)-nicotine. The pathway leading to cis-3'-hydroxycotinine is proposed to proceed via autoxidation of 2-hydroxy-1-methyl-5-(3-pyridinyl)pyrrole, a postulated cytochrome P450-generated metabolite of beta-nicotyrine, followed by reduction of the carbon-carbon double bond present in the resulting 3-hydroxy-3-pyrrolin-2-one species. This proposal is supported by the in vivo biotransformation of 2-acetoxy-1-methyl-5-(3-pyridinyl)pyrrole, a latent form of the putative hydroxypyrrole intermediate, to cis-3'-hydroxycotinine. The in vivo conversion of 5-hydroxy-1-methyl-5-(3-pyridinyl)-3-pyrrolin-2-one to 5-hydroxycotinine is offered as evidence that supports the proposed reduction step.

Structure-activity relationships of polycyclic aromatic amines with calcium channel blocking activity.

8-Benzylamino-8, 11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane (1) inhibits the calcium current in L-type calcium channels. A series of nitrobenzylamines (2, 3, 4), methoxybenzylamines (5, 6, 7), methylpyridines (8, 9, 10), and a phenylhydrazine derivative (11) of 8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane was synthesized. By substituting the 8,11-oxapentacyclo-[5.4.0.0(2,6).0(3,10).0(5,9)]undecane skeleton with 3-hydroxyhexacyclo-[6.5.0.0(3,7).0(4,12).0(5,10).0(9,13)]tridec ane (12), 8,13-dioxapentacyclo[6.5.0.0(2,6).0(5,10).0(3,11)]tridecane- 9-one (13), and pentacyclo-[5.4.0.0(2,6).0(3,10).0(5,9)]undecane (14), the effect of the polycyclic skeleton could also be investigated. Increased inhibition of calcium current was observed with aromatic substitution (especially ortho and meta substitution) in the pentacycloundecane series. The calcium channel activities of the methoxy compounds were slightly higher than those of the corresponding nitro compounds while a definite decrease in activity was observed for the phenylhydrazine and aminomethylpyridine derivatives. Increased inhibition of the calcium current was also observed for structures in which the polycyclic 'cages' were enlarged. Structure-activity relationships in this series of compounds therefore appear to be dominated by geometric or steric constraints.

Melatonin fails to protect against long-term MPTP-induced dopamine depletion in mouse striatum.

Several laboratories recently have reported that melatonin may possess neuroprotective properties. The present paper presents the results of our studies on the long term in vivo neuroprotective effects of melatonin in a well-defined neurotoxicity model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the C57BL/6 mouse. MPTP is bioactivated by brain monoamine oxidase B (MAO-B) to its neurotoxic pyridinium metabolite 1-methyl-4-phenylpyridinium (MPP(+)) which destroys dopaminergic nerve terminals leading to the depletion of neostriatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC). Our initial study compared striatal DA and DOPAC levels in MPTP-only-treated animals and animals treated with melatonin 30 min prior to and 3 times hourly post-MPTP. DA/DOPAC levels measured 7 days after MPTP were similar in both groups. A second study was designed to address the possibility that melatonin cleared from the brain prior to MPP(+). Animals, that had been administered the same regimen of melatonin as in the first study plus a fourth post-MPTP melatonin dose, were maintained on melatonin in drinking water until 5 days post-MPTP. Striatal DA/DOPAC levels of these melatonin-plus-MPTP treated animals also were the same as the MPTP-only-treated animals. In vitro studies confirmed that melatonin is not an inhibitor of MAO-B. These data demonstrate that melatonin does not have any significant protective effects against the long-term striatal DA and DOPAC depletion induced by MPTP in the C57BL/6 mouse.

Mitochondrial ultrastructure and density in a primate model of persistent tardive dyskinesia.

The use of neuroleptic drugs to treat schizophrenia is almost invariably associated with extrapyramidal movement disorders. One of these disorders, tardive dyskinesia (TD), can persist long after neuroleptic withdrawal suggesting that permanent neurological damage is produced. However, there appears to be no convincing pathology of TD and its pathogenesis remains unknown. Findings that neuroleptics interfere with normal mitochondrial function and produce mitochondrial ultrastructural changes in the basal ganglia of patients and animals suggest that mitochondrial dysfunction plays a role in TD. We have established a model for persistent TD in baboons that appears to involve compromised mitochondrial function. In this study, we evaluated two animals treated for 41 weeks with a derivative of haloperidol and two treated with vehicle only. Treatment was then withdrawn and the animals observed for a further 17-18 weeks. Treated animals developed abnormal orofacial signs that were consistent with TD. These symptoms persisted during the drug-free period. The animals were euthanased, the brains perfused-fixed then post-fixed in 4% paraformaldehyde and the caudate and putamen prepared for electron microscopy. Regardless of whether mitochondria were located in neural soma, excitatory terminals, glia or in non-somal neuropil there was no consistent difference either in size or number between treated and control animals. Thus, even if mitochondria in striatal neurons undergo ultrastructural alterations during neuroleptic therapy, these changes do not persist after drug withdrawal.

Species-dependent differences in monoamine oxidase A and B-catalyzed oxidation of various C4 substituted 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridinyl derivatives.

In an attempt to provide a better understanding of the scope and limitations of animal models used in some drug development programs and to further our understanding of potential metabolic bioactivation reactions, we have undertaken studies to profile the monoamine oxidase A and B (MAO-A and -B, respectively) activities in liver and brain mitochondrial preparations obtained from a variety of species using a series of 1-methyl-4-aryl-1,2,3, 6-tetrahydropyridinyl substrates. Mitochondrial preparations were incubated with substrates at 37 degrees C in the presence or absence of clorgyline, (R)-deprenyl, or a mixture of these two propargylamines to inhibit MAO-A, MAO-B, or both enzymes. The rates of formation of the corresponding dihydropyridinium metabolites were estimated spectrophotometrically. MAO-B was found to be the principal enzyme present in all tissues. Human liver displayed more MAO-A activity than the liver of any other species studied; subhuman primates displayed little or no detectable MAO-A activity. The properties of the preparations from rat liver were most similar to those from human liver with respect to the MAO-A/MAO-B ratios and the kinetic parameters of the four substrates used to profile enzymatic activity. The kinetic properties of mitochondrial preparations from bovine liver, a commonly used source of purified MAO-B preparations, were consistently different from all of the other species studied. The mitochondrial preparations from rabbit brain and liver also were unusual in that they displayed relatively low MAO activities. Additionally, these enzyme activities were considerably less susceptible to inhibition by clorgyline and (R)-deprenyl. Finally, an exceptionally low MAO-B liver/brain V(max)/K(m) ratio was observed with the mitochondria obtained from the C57BL/6 mouse, an effect that may contribute to the susceptibility of this strain to the toxic effects of the parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine.

p-Fluorophenylglycine in the urine of baboons treated with HPTP, the tetrahydropyridine analog of haloperidol.

We report the presence of p-fluorophenylglycine (p-FPG) in the urine of six baboons treated with HPTP, the tetrahydropyridine dehydration product of haloperidol (HP). Oxidative N-dealkylation, the major metabolic pathway of HP, gives rise to 3-(4-fluorobenzoyl)propionic acid (p-FBPA). Subsequent beta-oxidation of p-FBPA produces p-fluorophenylacetic acid (p-FPA). The presence of p-FPA argues for the formation also of p-fluorophenylglyoxylic acid (p-FPGA) derived from beta-oxidation of p-FBPA. Plasma aminotransferases should convert p-FPGA to p-FPG. The presence of p-FPG in these animals suggest the presence of phenylglycine aminotransferases in the baboon and possibly also in other primates, including the human. Reports by other authors found that treatment with alpha-phenylglycine (alpha-PG), an "unnatural" amino acid, leads to striatal dopamine (DA) depletion in rabbits--an effect explained on the basis of alpha-PG competing with DA for the neuronal vesicular storage sites. We performed in vitro DA release assays in mouse striatal synaptosomal preparations but found that neither alpha-PG nor p-FPG released any DA. It therefore remains unclear whether p-FPG may be a contributing factor to neurologic side-effects such as tardive dyskinesia (TD) found in patients after long-term HP treatment.

Clinical and neuropathological abnormalities in baboons treated with HPTP, the tetrahydropyridine analog of haloperidol.

Tardive dyskinesia (TD) is relatively common among psychiatric patients on maintenance therapy with typical neuroleptics and persists in more than 20% even after withdrawal of the medication. Such persistence suggests an underlying pathology due to neurotoxicity. We present evidence for such a neurotoxic mechanism in a baboon model of TD. Four baboons were treated chronically with the dehydration product of haloperidol, 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6- tetrahydropyridine (HPTP), which is metabolized, similarly to haloperidol, to two neurotoxic pyridinium species. The animals developed orofacial dyskinesia which persisted after HPTP was ceased. Serial sections of the entire brain from the four treated animals and four vehicle-treated controls revealed volume loss in the basal forebrain and hypothalamus. Histological evaluation demonstrated a reduction in the density of magnocellular neurons in the anterior region of the nucleus basalis of Meynert (NbM). We speculate that the loss of these NbM neurons may be associated with the persistent orofacial dyskinesia observed in the HPTP-treated animals. These findings may contribute to a better understanding of neuroleptic-induced TD.

Enzyme-catalyzed bioactivation of cyclic tertiary amines to form potential neurotoxins.

The pyridinium metabolites formed in the MAO-B catalyzed oxidation of 1-methyl-4-substituted-1,2,3,6-tetrahydropyridinyl derivatives, such as the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), cause the selective degeneration of nigrostriatal neurons, presumably by inhibition of mitochondrial respiration and depletion of ATP stores. The possibility that other partially oxidized piperidinyl derivatives also may be biotransformed to toxic pyridinium metabolites has led us to examine the metabolic fate of the neuroleptic agent haloperidol (HP) and its tetrahydropyridinyl dehydration product 4-(4-chlorophenyl)-1[4-(4-fluorophenyl)-4-oxobutyl]- 1,2,3,6-tetrahydropyridine (HPTP). In vitro metabolic studies employing tissue preparations isolated from rodents, baboons and humans have documented that cytochrome P4503A enzymes catalyze the biotransformation of both HP and HPTP to yield the corresponding pyridinium metabolite HPP+. An analogous biotransformation profile has been observed with "reduced haloperidol" (RHP), an abundant, circulating metabolite of HP formed by the stereospecific reduction of the benzoyl carbonyl group of HP. In vivo studies also have documented these pathways in humans, baboons and rodents. Although both HPP+ and RHPP+ are found in the urine and plasma of HP treated patients and HP or HPTP treated baboons, attempts to identify an MPTP-type lesion in baboons following long-term treatment with HPTP have failed. On the other hand, evidence for a lesion of the nucleus basalis of Meynert has been obtained. Additionally, the urinary excretion of abnormal organic acids and acylcarnitine conjugates suggests that HP and/or metabolites derived from HP interfere with energy production pathways.

Studies on the pyrrolinone metabolites derived from the tobacco alkaloid 1-methyl-2-(3-pyridinyl)pyrrole (beta-nicotyrine).

Previous studies have established that the tobacco alkaloid 1-methyl-2-(3-pyridyl)pyrrole (beta-nicotyrine) is biotransformed by rabbit lung and liver microsomal preparations to an equilibrium mixture of the corresponding 3- and 4-pyrrolin-2-ones. Autoxidation of these pyrrolin-2-ones generates the chemically stable 5-hydroxy-5-(3-pyridinyl)-3-pyrrolin-2-one. This paper summarizes efforts to document more completely the pathway leading to this hydroxypyrrolinone. Chemical and spectroscopic evidence implicates the 2-hydroxy-1-methyl-5-(3-pyridinyl)pyrrole (2-hydroxy-beta-nicotyrine) as the key intermediate in this reaction pathway. Of potential toxicological interest is the detection of radical species derived from the autoxidation of this compound.