Effects of Methylphenidate Analogues on Phenethylamine Substrates for the Striatal Dopamine Transporter

Methylphenidate (MPD) was found to inhibit competitively the striatal dopamine transporter (DAT) and bind at sites on the DAT in common with both cocaine (a non-substrate site ligand) and amphetamine (a substrate site ligand). Some methylphenidate analogues modified on the aromatic ring and/or at the nitrogen were tested to determine whether the profile of inhibition could be altered. None was found to stimulate the release of dopamine in the time frame (< or = 60 s) of the experiments conducted, and each of the analogues tested was found to noncompetitively inhibit the transport of dopamine. It was found that halogenating the aromatic ring with chlorine (threo-3,4-dichloromethylphenidate hydrochloride; compound 1) increased the affinity of MPD to inhibit the transport of dopamine. A derivative of MPD with simultaneous, single methyl group substitutions on the phenyl ring and at the nitrogen (threo-N-methyl-4-methylphenidate hydrochloride; compound 2) bound at a site in common with MPD. A benzyl group positioned at the nitrogen (threo-N-benzylmethylphenidate hydrochloride; compound 3) imparted properties to the inhibitor in which binding at substrate and non-substrate sites could be distinguished. This analogue bound at a mutually interacting site with that of methylphenidate and had a K(int) value of 4.29 microM. Furthermore, the N-substituted analogues (compounds 2 and 3), although clearly inhibitors of dopamine transport, were found to attenuate dramatically the inhibition of dopamine transport by amphetamine, suggesting that the development of an antagonist for substrate analogue drugs of abuse may be possible.     

The human nucleus accumbens is highly susceptible to G protein down-regulation by methamphetamine and heroin

Although the nucleus accumbens is assumed to be a critical brain "pleasure center," its function in humans is unknown. As animal data suggest that a unique feature of this small brain area is its high sensitivity to down-regulation of an inhibitory G protein by drugs of abuse, we compared G protein levels in postmortem nucleus accumbens with those in seven other brain regions of chronic users of cocaine, methamphetamine, and heroin, and of matched controls. Biochemical changes were restricted to the nucleus accumbens in which concentrations of G(alpha)1 and/or G(alpha)2 were reduced by 32-49% in the methamphetamine and heroin users. This selective responsiveness to these abused drugs implies a special role for the human nucleus accumbens in mechanisms of drug reinforcement and suggests that some features of the drug-dependent state (e.g., tolerance) might be related to inhibition of G(alpha)1-linked receptor activity.     

Voltammetric Studies on Mechanisms of Dopamine Efflux in the Presence of Substrates and Cocaine from Cells Expressing Human Norepinephrine Transporter

Abstract: The effects of substrates m -tyramine and β-phenethylamine, as well as cocaine, on the DA efflux from a cell line stably expressing the human norepinephrine transporter (hNET) were investigated by using rotating disk electrode voltammetry. Both the substrates and cocaine induced apparent DA efflux in a concentration-dependent manner. Their EC₅₀ values for inducing DA efflux were similar to their IC₅₀ values for inhibiting DA uptake. The substrate-induced DA efflux was inhibited by various NET blockers, enhanced by raising the internal [Na⁺] with Na⁺,K⁺-ATPase inhibition, but was insensitive to membrane potential-altering agents valinomycin, veratridine, and high [K⁺]. The initial rate of m -tyramine-induced DA efflux was related to preloaded [DA] in a manner defined by a Michaelis-Menten expression. In contrast, DA efflux in the presence of cocaine displayed a much slower efflux rate, lower efficacy, was not stimulated by elevated internal [Na⁺], and was nonsaturable with preloaded [DA]. Single exponential kinetic analysis of the entire time course of the DA efflux showed that the apparent first-order rate constant for m -tyramine-induced DA efflux declined with increased preloaded [DA], whereas that for the DA efflux in the presence of cocaine was unchanged with varying preloaded [DA]. These results suggest that the substrates stimulate the NET-dependent DA efflux by increasing the accessibility of the NET to internal DA, whereas cocaine "uncovers" NET-independent DA efflux by reducing the accessibility of diffused/leaked external DA to the NET.     

Repeated Cocaine Alters Glutamate Receptor Subunit Levels in the Nucleus Accumbens and Ventral Tegmental Area of Rats that Develop Behavioral Sensitization

Increased glutamate transmission in the nucleus accumbens and ventral tegmental area has been proposed as a mechanism underlying sensitized behavioral responses to repeated cocaine administration. GluR1, GluR2/3, and NMDAR1 subunits of glutamate receptors were quantified from immunoblots in these brain nuclei in rats at 24 h and 3 weeks after discontinuing 1 week of daily cocaine injections. Motor behavior was monitored after the first and last injections of daily cocaine, and those rats that showed >20% increase in motor activity after the last compared with the first injection were considered to have developed behavioral sensitization. The subjects that developed behavioral sensitization showed a significant increase in GluR1 levels in the nucleus accumbens at 3 weeks but not at 24 h of withdrawal. Conversely, sensitized animals showed a significant increase in NMDAR1 and GluR1 levels in the ventral tegmental area at 1 day but not at 3 weeks of withdrawal. None of these increases occurred in the rats exposed to daily cocaine that did not develop behavioral sensitization (<20% increase in motor activity), and no changes were measured in the level of GluR2/3 in any treatment group. The functional importance of the increases in glutamate receptor subunit levels is suggested by the fact that the changes were present only in rats that developed behavioral sensitization to repeated cocaine administration.     

High-performance liquid chromatographic determination of cocaine and its metabolites in serum microsamples with fluorimetric detection and its application to pharmacokinetics in rats

A sensitive, selective and simple HPLC method with fluorimetric detection is described for quantitating cocaine and its three metabolites in rat serum microsamples (50 μl). Chromatographic separation is achieved on a Hypersil BDS C₁₈ column (100×2.1 mm, 5 μm) with an isocratic mobile phase consisting of methanol–acetonitrile–25.8 mM sodium acetate buffer, pH 2.6, containing 1.0·10⁻⁴ M tetrabutylammonium phosphate (14:10:76, v/v/v). The detection limit (0.5 ng/ml) for all the compounds, using direct fluorometric detection operated at excitation and emission wavelengths of 230 and 315 nm, respectively, was approximately five-times lower than that of using a UV detector operated at 235 nm. The effects of ratio of 2-propanol to chloroform in extraction solvents on the recovery and precision for cocaine and its metabolites were systematically examined. The method was used to study the pharmacokinetics of cocaine after administration of intravenous 2 mg/kg and oral 20 mg/kg doses.     

Cocaine withdrawal and neuro-adaptations in ion channel function

Chronic exposure to psychostimulants induces neuro-adaptations in ion channel function of dopamine (DA)-innervated cells localized within the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). Although neuroplasticity in ion channel function is initially found in drug-sensitized animals, it has recently been believed to underlie the withdrawal effects of cocaine, including craving that leads to relapse in human addicts. Recent studies have also revealed remarkable differences in altered ion channel activities between mPFC pyramidal neurons and medium spiny NAc neurons in cocaine-withdrawn animals. In response to psychostimulant or certain “excitatory” stimuli, increased intrinsic excitability is found in mPFC pyramidal neurons, whereas decreased excitability is observed in medium spiny NAc cells in drug-withdrawn animals compared to drug-free control animals. These changes in ion channel function are modulated by interrupted DA/Ca²⁺ signaling with decreased DA D2 receptor function but increased D1 receptor signaling. More importantly, they are correlated to behavioral changes in cocaine-withdrawn human addicts and sensitized animals. Based on growing evidence, researchers have proposed that cocaine-induced neuro-adaptations in ion channel activity and DA/Ca²⁺ signaling in mPFC pyramidal neurons and medium spiny NAc cells may be the fundamental cellular mechanism underlying the cocaine withdrawal effects observed in human addicts.     

Chronic cocaine exposure in Drosophila: life, cell death and oogenesis

Developmental signaling cascades that can be perturbed by cocaine and other drugs of abuse have been difficult to study in humans and vertebrate models. Although numerous direct neural targets of cocaine have been elucidated at the molecular level, little is known about the specific cellular events that are impacted indirectly as a result of the drug's perturbation of neural circuits. We have developed oogenesis in Drosophila melanogaster as a model in which to identify downstream biochemical and/or cellular processes that are disrupted by chronic cocaine exposure. In this model, cocaine feeding resulted not only in expected reductions in viability, but also in unanticipated developmental defects during oogenesis, including aberrant follicle morphogenesis and vitellogenic follicle degeneration. To identify mechanisms through which cocaine exerted its deleterious effects on oogenesis, we examined candidate components of neural and hormonal signaling pathways. Cocaine-induced disruptions in follicle formation were enhanced by juvenile hormone exposure and phenocopied by serotonin feeding, while cocaine-activated follicle apoptosis was enhanced by concomitant dopamine feeding. HPLC analysis of dopamine and serotonin in the ovary suggests that these neurotransmitters could variably mediate cocaine's effects on oogenesis indirectly in the brain and/or directly in the ovary itself. We confirmed the involvement of hormone signaling by measuring ecdysteroids, which increase following cocaine exposure, and by demonstrating suppression of cocaine-induced follicle loss by hormone receptor mutants. Cocaine-induced ovarian follicle apoptosis and adult lethality appear to be caused by modulation of dopamine levels, while morphological defects during follicle formation likely result from perturbing serotonin signaling during cocaine exposure. Our work suggests not only a new role for juvenile hormone and/or serotonin in Drosophila ovarian follicle formation, but also a cocaine-sensitive role for dopamine in modulating hormone levels in the female fly.     

Extracellular Dopamine, Norepinephrine, and Serotonin in the Nucleus Accumbens of Freely Moving Rats During Intracerebral Dialysis with Cocaine and Other Monoamine Uptake Blockers

Abstract: Monoamine-uptake blockers were applied focally (0.1–1,000 µ M ) through a dialysis probe in the nucleus accumbens of freely moving rats, and the extracellular concentrations of dopamine, norepinephrine, and serotonin were measured. The selective dopamine-uptake blocker GBR 12935 increased dopamine preferentially with only a small effect on norepinephrine, whereas the selective serotonin-uptake blocker fluoxetine increased serotonin output preferentially. In contrast, the selective norepinephrine-uptake blockers desipramine and nisoxetine enhanced not only norepinephrine, but also serotonin and dopamine appreciably. Cocaine increased all three amines with the greatest effects on dopamine and serotonin. As in our previous study on the ventral tegmental area, there was a positive association between dopamine and norepinephrine output when all blocker data were taken together. The present results suggest a contribution of the increase in norepinephrine, but not serotonin, to the enhancement of dopamine after cocaine applied focally in the nucleus accumbens.     

Rapid Stereoselective Hydrolysis of (+)-Cocaine in Baboon Plasma Prevents Its Uptake in the Brain: Implications for Behavioral Studies

The naturally occurring enantiomer of cocaine, (–)-cocaine, has been previously labeled with ¹¹C on the N-methyl group and used in conjunction with positron emission tomography to show that cocaine is rapidly taken up in the striata of human and baboon brain. In the present study, the behaviorally inactive (+)-cocaine was similarly labeled, with a view to its use for measuring the nonspecific binding of cocaine. No brain uptake was seen, although transport of cocaine into the brain is not expected to be stereoselective. The explanation for the lack of uptake was determined to be very rapid metabolism of (+)-cocaine in the blood. By 30s after administration of labeled (+)-cocaine, it was undetectable in plasma. In vitro studies demonstrated that (+)-cocaine is 50% debenzoylated to (+)-ecgonine methyl ester within 5 s of exposure to baboon plasma but not to washed erythrocytes. The hydrolysis of (–)-cocaine is at least 1,000 times slower. Serum butyrylcholinesterase (EC 3.1.1.8) appears to be responsible for this hydrolysis, as evidenced by its inhibition by physostigmine and catalysis by commercially available pseudocholinesterase from horse and human blood.     

Sensitive and specific high-performance liquid chromatographic assay with ultraviolet detection for the determination of cocaine and its metabolites in rat plasma

A sensitive, specific and precise HPLC–UV assay was developed to quantitate cocaine (COC) and its metabolites benzoylecgonine (BE), norcocaine (NC) and cocaethylene (CE) in rat plasma. After adding 50 μl of the internal standard solution (bupivacaine, 8 μg/ml) and 500 μl of Sørensen's buffer (pH 6) to 100 μl of rat plasma sample, the mixture was extracted with 10 ml of chloroform. The organic layer was transferred to a clean test tube and was evaporated under nitrogen. The residue was reconstituted in 100 μl of mobile phase and 35 μl was injected onto the HPLC column. The mobile phase consisted of methanol–acetonitrile–50 mM monobasic ammonium phosphate (5:7:63, v/v/v) and was maintained at a flow-rate of 0.4 ml/min. Separation of COC and its metabolites was achieved using a Supelcosil ABZ+plus deactivated reversed-phase column (250×2.1 mm I.D., 5 μm). Calibration curves were linear over the range of 25–5000 ng/ml for COC and its three metabolites. The absolute extraction efficiencies for BE, COC, NC, CE and bupivacaine were 56.6%, 78.6%, 61.1%, 76.4% and 67.0%, respectively. COC and its metabolites were stable in mobile phase for 24 h at room temperature and in rat plasma for 2 weeks at −20°C. The limits of detection for BE, COC, NC and CE were 20, 24, 15 and 12.9 ng/ml, respectively. These values correspond to 0.70, 0.84, 0.525 and 0.452 ng of the according compound being injected on column. The within-day coefficient of variation for the four compounds ranged from 3.0% to 9.9% while the between-day precision varied from 3.6% to 14%. This method was used to analyze rat plasma samples after administration of COC alone and in combination with alcohol. The pharmacokinetic profiles of COC and its metabolites in these rats are also described.