Pharmacokinetics study of mazindol in plasma, oral fluid, and urine of volunteers.

PURPOSE: There are no pharmacokinetics studies in oral fluid reported in the literature, as well as there are no data on correlation of drug levels in plasma, urine, and oral fluid in order to propose alternative matrices to monitor the use of mazindol by drivers. The present work aimed to study, preliminarily, mazindol's pharmacokinetics in plasma and oral fluid, as well as investigate the correlation of drug levels in urine, plasma, and oral fluid. METHOD: Blood, urine, and oral fluid samples from seven healthy male volunteers were collected at 0, 1, 2, 4, 5, 6, 8, 10, and 24 h after administration of tablets of 2 mg mazindol and analyzed by a previously validated method by LC-MS with liquid-liquid extraction. Levels of the drug found were higher in plasma when compared with oral fluid and higher in urine in relation to plasma. The study of the mazindol's pharmacokinetics showed that the most suitable model to describe the variation of the concentration over time is the compartment open model with absorption and elimination following the first-order kinetics, and confirming literature data, drug is metabolized, being the major metabolite detected, but not quantified. CONCLUSION: It was not found a good correlation between the concentrations of mazindol in urine and plasma, but between plasma and oral fluid, there was a good correlation, suggesting this as an alternative matrix to plasma. However, studies involving more subjects are needed.

Pilot Phase II study of mazindol in children with attention deficit/hyperactivity disorder.

OBJECTIVE: Mazindol has been proposed as a potential treatment of children with attention deficit/hyperactivity disorder (ADHD). The purpose of this pilot study was to assess its pharmacokinetics, short-term efficacy, and safety. SUBJECTS AND METHODS: A total of 24 children (aged 9-12 years) with ADHD (according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, text-revision criteria) received a daily dose of 1 mg for 7 days and were followed for 3 additional weeks. Pharmacokinetic samples were collected after the first administration. ADHD symptoms were assessed using the ADHD Rating Scale (RS)-IV, Conners' Parent Rating Scale - Revised: Long (CPRS-R:L) at screening, baseline, and the end of the study. The Clinical Global Impression - Severity (CGI-S) scale was assessed at baseline, and the CGI - Improvement (CGI-I) scale was assessed at subsequent visits. RESULTS: Twenty-one subjects (aged 10±1 years) were analyzed. Pharmacokinetic data were described by a one-compartment model with first-order absorption, elimination, and lag time. The typical apparent clearance and apparent volume of distribution were 27.9 L/h and 234 L, and increased with fat-free mass and age, respectively. The mean change in score in ADHD RS-IV after 1 week of mazindol was -24.1 (P<0.0001), greater than a 90% improvement from baseline. Reduction of CPRS-R:L and CGI-S scores were -52.1 (P<0.0001) and -2.5 (P<0.01), respectively. Adverse events were mild to moderate, decreased appetite and upper abdominal pain being the most common. CONCLUSION: This preliminary study shows that mazindol might be an effective, well-tolerated, and long-acting (more than 8 hours) agent for the treatment of ADHD in children.

Validated method for determination of mazindol in human plasma by liquid chromatography/tandem mass spectrometry.

A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with electrospray ionization in positive ion multiple reaction monitoring mode was developed for the quantification of mazindol (an anorectic agent) in human plasma. Fluoxetine was adopted as an internal standard (IS), and sample preparation involved one-step liquid/liquid extraction using ethyl acetate. The transition monitored were m/z 285>44 for mazindol and m/z 310>44 for IS. Chromatographic separation was achieved on a Capcell Pak MGII C(18) column using an isocratic mobile phase, consisting of acetonitrile-20mM ammonium formate in water (50:50, v/v, adjusted to pH 3.5 with formic acid) at a flow-rate of 0.2mL/min. The retention times of mazindol and fluoxetine were 1.03min and 1.45min, respectively. The lower limit of quantitation (LLOQ) was 0.1ng/mL using 200microL of plasma, and no interferences were detected in chromatograms. The bench top stability of mazindol was evaluated in buffered and non-buffered plasma. The selectivity, linearity, precision, accuracy, recovery, and stability of the devised method were fully validated and absolute and relative matrix effects were evaluated. The described method provides a fast and sensitive analytical tool for determining mazindol levels in plasma, and was successfully applied to a pharmacokinetic study in 24 healthy human subjects after oral administration of 2mg tablet formulation of mazindol under fasting conditions.

L-DOPA-induced dyskinesia in adult rats with a unilateral 6-OHDA lesion of dopamine neurons is paralleled by increased c-fos gene expression in the subthalamic nucleus.

Levodopa (L-DOPA), the metabolic precursor of dopamine, is widely used as a pharmacological agent for the symptomatic treatment of Parkinson's disease. However, long-term L-DOPA use results in abnormal involuntary movements such as dyskinesias. There is evidence that abnormal cell signaling in the basal ganglia is involved in L-DOPA-induced dyskinesia. The subthalamic nucleus (STN) plays a key role in the circuitry of the basal ganglia and in the pathophysiology of Parkinson's disease. However, the contribution of the STN to L-DOPA-induced dyskinesias remains unclear. The objective of this work was to study the effects of acute or chronic systemic administration of L-DOPA to adult rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of dopamine neurons on c-fos expression in the STN and test the hypothesis that these effects correlate with L-DOPA-induced dyskinesias. c-fos mRNA expression was measured in the STN by in situ hybridization histochemistry at the single cell level. Our results confirm earlier evidence that the chronic administration of L-DOPA to rats with a unilateral 6-OHDA lesion increases c-fos expression in the STN. We also report that c-fos expression can be increased following an acute injection of L-DOPA to 6-OHDA-lesioned rats but not following a chronic injection of L-DOPA to sham-operated, unlesioned rats. Finally, we provide evidence that the occurrence and severity of dyskinesia is correlated with c-fos mRNA levels in the ipsilateral STN. These results suggest that altered cell signaling in the STN is involved in some of the behavioral effects induced by systemic L-DOPA administration.

Genetic interdependence of adenosine and dopamine receptors: evidence from receptor knockout mice.

Dopamine and adenosine receptors are known to share a considerable overlap in their regional distribution, being especially rich in the basal ganglia. Dopamine and adenosine receptors have been demonstrated to exhibit a parallel distribution on certain neuronal populations, and even when not directly co-localized, relationships (both antagonistic and synergistic) have been described. This study was designed to investigate dopaminergic and purinergic systems in mice with ablations of individual dopamine or adenosine receptors. In situ hybridization histochemistry and autoradiography was used to examine the level of mRNA and protein expression of specific receptors and transporters in dopaminergic pathways. Expression of the mRNA encoding the dopamine D2 receptor was elevated in the caudate putamen of D1, D3 and A2A receptor knockout mice; this was mirrored by an increase in D2 receptor protein in D1 and D3 receptor knockout mice, but not in A2A knockout mice. Dopamine D1 receptor binding was decreased in the caudate putamen, nucleus accumbens, olfactory tubercle and ventral pallidum of D2 receptor knockout mice. In substantia nigra pars compacta, dopamine transporter mRNA expression was dramatically decreased in D3 receptor knockout mice, but elevated in A2A receptor knockout mice. All dopamine receptor knockout mice examined exhibited increased A2A receptor binding in the caudate putamen, nucleus accumbens and olfactory tubercle. These data are consistent with the existence of functional interactions between dopaminergic and purinergic systems in these reward and motor-related brain regions.

Enhanced dopamine uptake in the striatum following repeated restraint stress.

In mice administered chronic stress--repeated overnight restraint stress for 7 days--there was a prolonged enhancement of dopamine (DA) uptake into synaptosomes. The mRNA for the DA transporter (DAT) was found to be concomitantly increased in the midbrain, as was the binding of the transporter ligand mazindol to DAT in the nucleus accumbens and caudate-putamen. Kinetic analysis showed an increase in Vmax for DA, with little change in Km. No changes in tyrosine hydroxylase activity and tissue DA or 3,4-dihydroxyphenylacetic acid (DOPAC) content were observed. However, homovanillic acid (HVA) was found to be increased in the striatum of the stressed animals. Enhanced DAT activity attributable to chronic stress was still observed in animals treated with the DA D2 receptor antagonist haloperidol or the glucocorticoid receptor antagonist mifepristone. Modulation of DAT activity may be a physiological mechanism for regulating the concentration of DA that reaches receptors, following periods of stress.

Dopaminergic innervation of the human striatum in Parkinson's disease.

In Parkinson's disease (PD), dopaminergic input to the caudate nucleus and a band of putaminal tissue abutting the external globus pallidus seems well preserved on immunohistochemical staining for the dopamine transporter. Counting of dopaminergic terminals showed that terminal density in these regions in PD was the same as that in controls, which indicates that input is truly preserved and not a consequence of a compensatory upregulation of metabolism in a reduced pool of surviving terminals. When the branching pattern of dopaminergic axons coursing through the globus pallidus was examined, we found no evidence for increased axonal sprouting in PD that might have contributed to preservation of dopaminergic input to the putamen or caudate nucleus. Although terminal counting indicated that anatomic input was preserved to parts of the striatum, dopamine uptake site density in these regions was reduced significantly. This suggests that the impact of disease in these areas is more profound than was thought previously.

The inhibitory role of methylation on the binding characteristics of dopamine receptors and transporter.

Excess methylation has been suggested to play a role in the pathogenesis of Parkinson's disease (PD), since the administration of S-adenosylmethionine (SAM), a biological methyl donor, induces PD-like changes in rodents. It was proposed that SAM-induced PD-like changes might be associated with its ability to react with the dopaminergic system. In the present study the effects of SAM on dopamine receptors and transporters were investigated using rats and cloned dopamine receptor proteins. Autoradiographic examination of SAM indicated its tendency to be localized and accumulated in rat striatal region after the intracerebroventricular injection into rat brain. Moreover, results showed that SAM significantly decreased dopamine D1 and D2 receptor binding activities by decreasing the Bmax and increasing the Kd values. At concentrations of 0.1, 0.25 and 0.5 mM, SAM was able to reduce the Bmax from the control value of 848.1 for dopamine D1-specific ligand [3H] SCH 23390 to 760.1, 702.6 and 443.0 fmol/mg protein, respectively. At the same concentrations, SAM was able to increase the Kd values from 0.91 for the control to 1.06, 3.84 and 7.01 nM of [3H] SCH 23390, respectively. The effects of SAM on dopamine D2 binding were similar to those of dopamine D1 binding. SAM also decreased dopamine transporter activity. The interaction of SAM with dopamine receptor proteins produced methanol from methyl-ester formation and hydrolysis. We propose that the SAM effect might be related to its ability to react with dopamine receptor proteins through methyl-ester formation and methanol production following the hydrolysis of the carboxyl-methylated receptor proteins.

Apparent opposite effects of tetrabenazine and reserpine on the toxic effects of 1-methyl-4-phenylpyridinium or 6-hydroxydopamine on nigro-striatal dopaminergic neurons.

It is well documented that VMAT2 protects nigrostriatal DA neurons against MPP(+) by sequestering it inside vesicles away from its mitochondrial site of neurotoxic action. However, the implication of the VMAT2 in the mechanism of action exerted by 6-OHDA has received little attention. Therefore, the aim of the present study was to determine whether the vesicular sequestration of 6-OHDA would protect dopaminergic neurons from its toxicity similarly to what is observed with MPP(+). We injected mice with 6-OHDA 90 min after TBZ treatment. Since, unexpectedly, TBZ pretreatment prevented 6-OHDA neurotoxicity, we performed a similar experience replacing 6-OHDA with MPP(+) in order to check our experimental protocol. TBZ pretreatment similarly prevented MPP(+) neurotoxicity. This discrepancy with what is commonly describe in the literature, led us to use reserpine. Indeed, the long lasting VMAT2 inhibition induced by reserpine allowed us to inject neurotoxins while mice no longer presented hypothermia. Contrary to TBZ pretreatment, reserpine pretreatment potentiated both 6-OHDA and MPP(+) toxicity on dopaminergic neurons. Hypothermia elicited by TBZ appeared to be responsible, at least in part, for the neuroprotective effect observed. To verify this hypothesis, we investigated the influence of hypothermia on the toxic activity of both neurotoxins. A hypothermia similar to that induced by TBZ was obtained by a forced swimming test of putting mice into cool water (23 degrees C). The hypothermia prevented both 6-OHDA and MPP(+)-induced neurotoxicity. We finally reported that VMAT2 inhibition potentiates both MPP(+) and 6-OHDA neurotoxicity.

Changes in function and ultrastructure of striatal dopaminergic terminals that regenerate following partial lesions of the SNpc.

Following partial substantia nigra lesions, remaining dopaminergic neurones sprout, returning terminal density in the dorsal striatum to normal by 16 weeks. This suggests regeneration and maintenance of terminal density is regulated to release appropriate levels of dopamine. This study examined the structure and function of these reinnervated terminals, defining characteristics of dopamine uptake and release, density and affinity of the dopamine transporter (DAT) and ultrastructural morphology of dopamine terminals in the reinnervated dorsal striatum. Finally, rotational behaviour of animals in response to amphetamine was examined 4 and 16 weeks after substantia nigra pars compacta (SNpc) lesions. Dopamine transport was markedly reduced 16 weeks after lesioning along with reduced density and affinity of DAT. Rate of dopamine release and peak concentration, measured electrochemically, was similar in lesioned and control animals, while clearance was prolonged after lesioning. Ultrastructurally, terminals after lesioning were morphologically distinct, having increased bouton size, vesicle number and mitochondria, and more proximal contacts on post-synaptic cells. After 4 weeks, tendency to rotate in response to amphetamine was proportional to lesion size. By 16 weeks, rotational behaviour returned to near normal in animals where lesions were less than 70%, although some animals demonstrated unusual rotational patterns at the beginning and end of the amphetamine effect. Together, these changes indicate that sprouted terminals are well compensated for dopamine release but that transport mechanisms are functionally impaired. We discuss these results in terms of implications for dyskinesia and other behavioural states.

Expression of striatal preprotachykinin mRNA in symptomatic and asymptomatic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-exposed monkeys is related to parkinsonian motor signs.

Striatal preprotachykinin (PPT) gene expression and [(3)H]mazindol binding were examined in monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Some animals (n = 5) became moderately to severely parkinsonian after receiving large doses of MPTP over 9-30 d and remained symptomatic for a relatively short time (3 weeks to 3 months; acutely symptomatic group). A second group of animals (n = 5) received low doses of MPTP (1.5-12 months), developed cognitive impairments but displayed no gross motor deficits (asymptomatic group), and were killed 3-12 months after their final dose of MPTP. Other animals became moderately to severely parkinsonian after receiving escalating doses of MPTP (>6 months; n = 4) or high doses of MPTP (<1 month; n = 1) and remained symptomatic for 2.5-5.75 years (chronically symptomatic group). All MPTP-treated animals had extensive losses of [(3)H]mazindol binding in dorsal striatal sensorimotor regions with asymptomatic animals generally having a lesser degree of damage. However, PPT mRNA levels differed sharply among treatment groups. Symptomatic animals (acutely and chronically parkinsonian) had significantly decreased PPT mRNA levels in most striatal regions. In asymptomatic animals, PPT mRNA expression was not significantly different from that measured in control animals, despite decreases in [(3)H]mazindol binding in some striatal regions of similar magnitude to those observed in symptomatic animals. These observations suggest that PPT gene expression may be directly related to expression of parkinsonian motor symptomatology regardless of duration of MPTP exposure, duration of the parkinsonism, or extent of dopamine denervation. These results imply that the direct striatal output circuit may have a greater contribution to expression of parkinsonian symptomatology than proposed previously.

The role of conserved tryptophan and acidic residues in the human dopamine transporter as characterized by site-directed mutagenesis.

The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and acidic residues that are completely or highly conserved among Na(+)/Cl(-)-dependent transporters. We have explored the roles of these residues using non-conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184L) lacked plasma membrane immunostaining and were not functional. Both DA uptake and cocaine analog (i.e. 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative selectivity of the hDAT for cocaine analogs and DA by 10-24-fold. In particular, mutation of D345 in the third intracellular loop still allowed considerable [(3)H]DA uptake, but caused undetectable [(3)H]CFT binding. Upon anti-C-terminal-hDAT immunoblotting, D345N appeared as broad bands of 66-97 kDa, but this band could not be photoaffinity labeled with cocaine analog [(125)I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid ([(125)I]RTI-82). Unexpectedly, in this mutant, cocaine-like drugs remained potent inhibitors of [(3)H]DA uptake. CFT solely raised the K(m) of [(3)H]DA uptake in wild-type hDAT, but increased K(m) and decreased V(max) in D345N, suggesting different mechanisms of inhibition. The data taken together indicate that mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding of cocaine to the transporter may not be the only way by which cocaine analogs inhibit DA uptake.

Comparison between the role of the neuronal and inducible nitric oxide synthase in methamphetamine-induced neurotoxicity and sensitization.

The involvement of the neuronal and inducible nitric oxide synthase (nNOS and iNOS, respectively) in methamphetamine (METH)-induced dopaminergic neurotoxicity and behavioral sensitization was investigated. To determine METH-induced neurotoxicity, mice deficient in the nNOS and iNOS genes, nNOS(-/-) and iNOS(-/-) mice, and wild-type controls received either saline or METH (5 mg/kg x 3). After 72 h the level of striatal dopaminergic markers were measured. Administration of METH to nNOS(-/-) mice had no significant effect on the level of striatal dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), or dopamine transporter (DAT) binding sites. However, METH caused 25-40% depletion of dopaminergic markers in iNOS(-/-) mice and 63-69% depletion in the wild-type mice. METH-induced locomotor activity was measured following the administration of a low dose (1 mg/kg) on day 1. Subsequently animals received the high dose of METH (5 mg/kg x 3). On day 4, after a 68-72 h drug free period, animals were challenged with 1 mg/kg METH, and locomotor activity was recorded. The intensity of METH-induced locomotion in nNOS(-/-) mice on day 1 and 4 was similar, suggesting that locomotor sensitization did not develop. However, the intensity of METH-induced locomotion in the iNOS(-/-) and wild-type mice on day 4 was doubled compared to day 1, suggesting the development of sensitization. The present findings indicate that nNOS(-/-) mice are more resistant to METH-induced neurotoxicity and behavioral sensitization than iNOS(-/-) mice. These results suggest a major role for nNOS rather than iNOS in the effects of METH.

Striatal dopaminergic markers in dementia with Lewy bodies, Alzheimer's and Parkinson's diseases: rostrocaudal distribution.

Dementia with Lewy bodies (DLB) is a neuropsychiatric disease associated with extrapyramidal features which differ from those of Parkinson's disease, including reduced effectiveness of L-dopa and severe sensitivity reactions to neuroleptic drugs. Distinguishing Alzheimer's disease from DLB is clinically relevant in terms of prognosis and appropriate treatment. Dopaminergic activities have been investigated at coronal levels along the rostrocaudal striatal axis from a post-mortem series of 25 DLB, 14 Parkinson's disease and 17 Alzheimer's disease patients and 20 elderly controls. [(3)H]Mazindol binding to the dopamine uptake site was significantly reduced in the caudal putamen in DLB compared with controls (57%), but not as extensively as in Parkinson's disease (75%), and was unchanged in Alzheimer's disease. Among three dopamine receptors measured (D1, D2 and D3), the most striking changes were apparent in relation to D2. In DLB, [(3)H]raclopride binding to D2 receptors was significantly reduced in the caudal putamen (17%) compared with controls, and was significantly lower than in Parkinson's disease at all levels. D2 binding was significantly elevated at all coronal levels in Parkinson's disease compared with controls, most extensively in the rostral putamen (71%). There was no change from the normal pattern of D2 binding in Alzheimer's disease. The only significant alteration in D1 binding ([(3)H]SCH23390) in the groups examined was an elevation (30%) in the caudal striatum in Parkinson's disease. There were no differences in D3 binding, measured using [(3)H]7-OH-DPAT, in DLB compared with controls. A slight, significant decrease in D3 binding in the caudal striatum of Parkinson's disease (13%) patients and an increase in Alzheimer's disease (20%) in the dorsal striatum at the level of the nucleus accumbens were found. The concentration and distribution of dopamine were disrupted in both DLB and Parkinson's disease, although in the caudate nucleus the loss of dopamine in DLB was uniform whereas in Parkinson's disease the loss was greater caudally. In the caudal putamen, dopamine was reduced by 72% in DLB and by 90% in Parkinson's disease. The homovanillic acid : dopamine ratio, a metabolic index, indicated compensatory increased turnover in Parkinson's disease, which was absent in DLB despite the loss of substantia nigra neurons (49%), dopamine and uptake sites. These differences between DLB, Parkinson's disease and Alzheimer's disease may explain some characteristics of the extrapyramidal features of DLB and its limited response to L-dopa and severe neuroleptic sensitivity. The distinct changes in the rostrocaudal pattern of expression of dopaminergic parameters are relevant to the interpretation of the in vivo imaging and diagnosis of DLB.

Damage to dopamine systems differs between Parkinson's disease and Alzheimer's disease with parkinsonism.

Parkinsonism occurs in approximately 35 to 40% of patients with Alzheimer's disease (AD) even with little or no neuronal degeneration in the substantia nigra, which in idiopathic Parkinson's disease (PD) results in the severe loss of striatal dopamine transporter sites. It is not known if there is a loss of striatal dopamine transporter sites in AD with coexistent parkinsonism (AD/parkinsonism). We quantified the pattern of these sites in the striatum and midbrain of patients with the clinical diagnosis of PD, AD, and AD/parkinsonism in comparison with a group of age-matched control subjects. We also quantified the number of D2 receptors and the levels of tyrosine hydroxylase in the substantia nigra and ventral tegmental area of the same groups. The results showed that in AD the loss of dopamine transporter sites was restricted to the nucleus accumbens. The loss of these sites in the AD/parkinsonism group was more extensive than in the AD group, with the most severe losses in the rostral caudate and putamen and least in the caudal caudate and putamen. While the PD group showed an equally severe reduction in numbers of sites, the caudal to rostral gradient of loss differed from that in the AD/parkinsonism group. The PD group also showed a marked loss of dopamine transporter sites, tyrosine hydroxylase, and D2 autoreceptors (located on dopamine neurons) in the substantia nigra and ventral tegmental area. In contrast, no reductions in dopamine transporter sites, tyrosine hydroxylase, and D2 autoreceptors were observed in the substantia nigra and ventral tegmental area of the AD or AD/parkinsonism groups. Thus, the loss of striatal dopamine transporter sites in AD/parkinsonism may be related to the clinical parkinsonian symptoms. However, the loss is not simply the result of neuronal degeneration in the substantia nigra, but must derive from other processes.

Cocaine accumulates in dopamine-rich regions of primate brain after i.v. administration: comparison with mazindol distribution.

Pharmacological and neurochemical evidence suggest that brain dopamine systems, and the dopamine transporter in particular, contribute significantly to the behavioral effects and reinforcing properties of cocaine. The first objective of this study was to determine whether the brain distribution of cocaine supports these conclusions. A high resolution neuroanatomical map of cocaine disposition in brain after i.v. administration was developed. [3H]Cocaine ([3H](-)-cocaine) was administered to squirrel monkeys (Saimiri sciureus) at a trace dose (0.001 mg/kg) and at doses at or above the threshold for producing behavioral effects (0.1 mg/kg, 0.3 mg/kg). After 15 min, ex vivo autoradiography revealed the highest accumulation of [3H]cocaine in dopamine-rich brain regions, including the caudate nucleus, putamen, and nucleus accumbens/olfactory tubercle. The norepinephrine-rich locus coeruleus, the hippocampus, and amygdala also accumulated large quantities of [3H]cocaine. Moderately high levels were found in the stria terminalis, medial septum, substantia nigra, and other regions. Lowest levels were found in the cerebellum. A high and positive correlation was established for the brain distribution of [3H]cocaine administered at trace or at behaviorally relevant doses (r: 0.94; P < 0.001). To determine whether radioactivity represented [3H]cocaine or its metabolic products, tissue extracts from brain regions with high levels of cocaine were subjected to thin layer chromatography using two solvent systems. In caudate-putamen, nucleus accumbens, cortex, and hippocampus, radioactivity comigrated with standard [3H]cocaine. In substantia nigra, less than 70% of the radioactivity comigrated with [3H]cocaine, suggesting that cocaine metabolites are generated more rapidly in the substantia nigra than in other brain regions. The second objective was to determine the brain distribution of mazindol, a potent norepinephrine and dopamine transport inhibitor with low abuse liability in humans. The disposition of intravenously administered [3H]mazindol in brain (0.001 mg/kg, 0.007 mg/kg) was surveyed by ex vivo autoradiography. In sharp contrast to [3H]cocaine distribution, the highest accumulation of [3H]mazindol was localized in the norepinephrine-rich pineal gland, discrete regions of the hypothalamus (paraventricular nucleus, supraoptic nucleus), and the locus coeruleus. Moderately high levels were detected in the caudate-putamen, nucleus accumbens, and other regions. The following conclusions were drawn: (1) Although dopamine-rich brain regions are principal targets of cocaine after i.v. administration to the nonhuman primate, other prominent targets of cocaine (locus coeruleus, hippocampus, and amygdala) may contribute to the acute and chronic effects of cocaine.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased glutamate decarboxylase mRNA levels in the striatum and pallidum of MPTP-treated primates.

The mRNA levels encoding for the enzyme glutamate decarboxylase (GAD67) were measured by computerized image analysis after in situ hybridization histochemistry and radioautography in the striatum and pallidum of normal squirrel monkeys (Saimiri sciureus), or after treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). All MPTP-injected monkeys exhibited profound motor deficits including akinesia. The dopaminergic innervation, as visualized and quantified on x-ray films after 3H-mazindol binding on tissue sections, was uniformly lost throughout the striatum of MPTP-treated monkeys. Brain sections processed with a probe synthesized from a feline or human GAD67 cDNA exhibited intense radioautographic labeling throughout the striatum. When measured on x-ray films, the intensity of GAD67 mRNA labeling was increased in the striatum of MPTP-treated versus control monkeys. Increased labeling reached statistical significance in the dorsolateral sector of the rostral putamen and throughout the putamen and the caudate at the caudal, postcommissural, level. Analysis of emulsion radioautographs demonstrated that the increase in GAD67 mRNA labeling in MPTP-treated monkeys occurred in individual neurons of the striatum. In the external and internal segments of the pallidum, numerous neurons labeled with the GAD67 cRNA probe were visualized on emulsion radioautographs. The intensity of GAD67 mRNA labeling in single neurons of both pallidal segments was increased in MPTP-treated versus control monkeys. Construction of the histograms of frequency distribution of labeling indicated that this increase occurred in a majority of labeled neurons. The present study demonstrates that GAD67 mRNA levels are significantly altered in the striatum and pallidum of parkinsonian monkeys. The preferential increase of GAD67 mRNA labeling in the dorsolateral putamen, which receives afferents from the sensorimotor cortex, provides further evidence of the involvement of GABAergic transmission in the expression of the motor deficits elicited after MPTP. In addition, increased GAD67 mRNA levels in the internal segment of the pallidum support the hypothesis of an increased activity of GABAergic neurons in the output structures of the basal ganglia in parkinsonism.

The effects of chronic continuous versus intermittent levodopa treatments on striatal and extrastriatal D1 and D2 dopamine receptors and dopamine uptake sites in the 6-hydroxydopamine lesioned rat--an autoradiographic study.

The effects of chronic 'continuous' infusion and 'intermittent' modes of levodopa/carbidopa administration on apomorphine induced circling behaviour, DA uptake sites (labelled with [3H]mazindol) and D1 and D2 DA receptor binding (labelled with [3H]SCH 23390 and [3H]sulpiride, respectively) were investigated in rats with unilateral 6-OHDA lesions of the medial forebrain bundle. The circling behaviour in response to apomorphine was greatly enhanced following chronic 'intermittent' but not 'continuous' levodopa treatments. Following the 'intermittent' regime, the lower dose of apomorphine induced a period of intense circling with delayed onset and rapid offset, than in rats given either 'continuous' infusion of levodopa or saline. The 6-OHDA lesion itself induced gross depletion of [3H]mazindol binding in all striatal subregions, NAc and OT, but not frontal cortex. [3H]Sulpiride binding in the ventrolateral striatal quadrant was increased on the denervated side and this correlated with the peak contralateral turns in response to 0.5 mg/kg apomorphine challenge. This asymmetry in striatal [3H]sulpiride binding was reduced in both groups of rats receiving levodopa. [3H]sulpiride binding in the NAc and OT and [3H]SCH 23390 binding in the striatum, NAc, OT and SNr were unaffected by DA denervation or either regime of levodopa treatments. 'Continuous' infusion and not 'intermittent' injections of levodopa reduced [3H]mazindol binding in the striatal subregions and the frontal cortex on both the denervated and intact sides. The potentiation of the behavioural response to apomorphine by chronic 'intermittent' levodopa treatment does not correspond with the levodopa induced alterations in striatal or extrastriatal DA receptors. In the same group of animals the narrowing of the duration of response to the lower dose of apomorphine may mimic the fluctuations in response to levodopa, seen clinically in long-term levodopa treated parkinsonian patients.

Spermine interacts with cocaine binding sites on dopamine transporters.

These studies were designed to assess the potential interaction of the polyamine spermine with cocaine binding to dopamine and serotonin transporters. The results of the experiments presented here indicate that spermine inhibits binding of the cocaine congener [3H] CFT to striatal synaptosomal membranes. Further, although [3H] CFT is known to interact with both dopamine and serotonin transporters, our results indicate that the observed inhibition of [3H] CFT binding is likely to reflect a specific inhibition of binding to dopamine transporters. Spermine significantly inhibited the binding of both [3H] CFT and [3H] mazindol to dopamine transporters, while it had no apparent effects on the binding of the potent serotonin uptake inhibitor [3H] paroxetine. Finally, saturation experiments show that the inhibition of ligand binding to the cocaine binding site on dopamine transporters appears not to be due to a modification of ligand affinity for the transporter, but to a decrease in the apparent density of ligand binding sites. The results of these experiments indicate that endogenously produced polyamines can alter cocaine binding to the dopamine transporter. The results are discussed in terms of possible impact on novel approaches for pharmacologically manipulating cocaine reinforcement and craving in clinical treatments for cocaine addiction, as well as for emergency treatment of cocaine overdose.