Investigation On The Behavior And Related Neuro-Electrochemistry Of Potential Medications For The Treatment Of Substance Use Disorders.

National Institute On Drug Abuse

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Abstract

The dopamine (DA) transporter (DAT) is the main pharmacologic target of abused psychostimulants like cocaine but also of prescribed medications like modafinil (MOD), which has shown little, if any, propensity for abuse in clinical or preclinical studies. Recently, we have explored the neurochemical and behavioral actions of MOD to better characterize its atypical psychostimulant profile. We found that MOD had a lower potency and efficacy than cocaine in stimulating nucleus accumbens shell (NAS) and core (NAC) DA levels, but, at variance with abused psychostimulants there were no statistically significant regional differences between accumbens subregions. In drug-discrimination studies MOD showed cocaine-like subjective effects at lower doses and earlier onset times than expected based on its DA effects. Those results suggest that non-DA-dependent actions may be playing a role in its unique pharmacological profile. In order to discover a potential non-dopaminergic mechanism for these MOD actions, we compared its behavioral and neurochemical effects with those of methylphenidate, another clinically approved medication that inhibits the neuronal reuptake of DA. We showed that methylphenidate, but not MOD, maintained intravenous self-administration in Sprague-Dawley rats similarly to cocaine. Both MOD and methylphenidate pretreatments potentiated cocaine self-administration. Cocaine, at self-administered doses, stimulated mesolimbic DA levels. This effect was potentiated by methylphenidate, but not by MOD pretreatments, indicating DA-dependent actions for methylphenidate, but not MOD. MOD is known to facilitate electrotonic neuronal coupling by actions on gap junctions. Carbenoxolone, a gap junction inhibitor, antagonized MOD, but not methylphenidate potentiation of cocaine self-administration. Our results indicate that MOD shares mechanisms with cocaine and methylphenidate but has a unique pharmacological profile that includes facilitation of electrotonic coupling and lower abuse liability, which may be exploited in future therapeutic drug design for cocaine use disorder. Though MOD might prove useful as a treatment for specific addicted populations (e.g. heavy crack-cocaine users, or cocaine and methamphetamine addicts without alcohol abuse comorbidity), broader effective medications for psychostimulant use disorders are still an unmet medical need. To this end, several newly synthesized analogs of MOD have been tested in our preclinical models as potential medications for methamphetamine (METH) and cocaine use disorder. Under intravenous (i.v.) METH self-administration procedures in rats allowed short access (1 hour; ShA) or long access (6 hours; LgA) to the drug, ShA rats exhibited stable METH intake over sessions, whereas LgA rats exhibited an escalation of drug intake. R-MOD decreased METH self-administration in ShA rats. JJC8-016, JJC8-089 and JJC8-091, the MOD analogs that show an atypical DAT-blocker profile, decreased METH self-administration in both LgA and ShA rats. JJC8-088, which shows a typical (cocaine-like) DAT-blocker profile, did not have an effect on METH self-administration in either ShA or LgA rats. These findings support the potential of atypical DAT inhibitors for the treatment of METH use disorder. JJC8-091 and JJC8-088, were also assessed alone and in combination with cocaine to elucidate neurochemical correlates to their divergent behavioral profiles. Despite sharing significant structural similarity, JJC8-088 was more cocaine-like, increasing extracellular DA concentrations in the NAS efficaciously and more potently than JJC8-091. In contrast, JJC8-091 was not self-administered and was effective in blocking cocaine-induced reinstatement to drug seeking. Electrophysiology experiments confirmed that JJC8-091 was more effective than JJC8-088 at inhibiting cocaine-mediated enhancement of DA neurotransmission. Further, when VTA DA neurons in DAT-cre mice were optically stimulated, JJC8-088 produced a significant leftward shift in the stimulation-response curve, similar to cocaine, while JJC8-091 shifted the curve downward, suggesting attenuation of DA-mediated brain reward. Computational models predicted that while JJC8-088 prefers or stabilizes an outward facing conformation of DAT, like cocaine, JJC8-091 stabilizes DAT towards a more occluded conformation. Though further development of JJC8-091 is ongoing, diastereomeric separation, as well as improvements in potency and pharmacokinetics were desirable for discovering pipeline drug candidates. In recent studies, a series of bis(4-fluorophenyl)methyl)sulfinyl)alkyl alicyclic amines, where the piperazine-2-propanol scaffold was modified, were designed, synthesized, and evaluated for binding affinities at DAT, as well as the serotonin transporter and 1 receptors. Within the series, RDS3-094 showed improved DAT affinity (Ki = 23 nM) over JJC8-091 (Ki = 230 nM), moderate metabolic stability in human liver microsomes, and a hERG/DAT affinity ratio = 28. While RDS3-094 increased locomotor activity relative to vehicle, it was significantly lower than activity produced by cocaine. These results support further investigation of RDS3-094 as a potential treatment for psychostimulant use disorders. We have recently collaborated on a project related to the potential effects of cocaine on neural autophagy in vitro and in vivo. Autophagy inhibitors, i.e. hydroxychloroquine and SBI-0206965, reduce the locomotor stimulant effect of systemic administration of cocaine and its related stimulation of extracellular NAS DA levels in mice. Cocaine-induced autophagy degrades transporters for DA but not serotonin in the NAS. Autophagy inhibition impairs cocaine conditioned place preference in mice. These findings indicate that autophagic degradation of DAT modulates behavioral and neurochemical actions of cocaine. Recent collaborations also include an investigation on the role of the microRNA system on neuronal signaling. Mice lacking the translin/trax microRNA-degrading enzyme display an altered behavioral and neurochemical response to psychostimulants. These mice display robust adiposity in the context of normal body weight, suggesting that this phenotype might reflect elevated brain levels of the psychostimulants tested. To assess this hypothesis, plasma and brain amphetamine levels of wild type and Tsn KO mice were compared. Furthermore, we checked the effect of diet-induced increases in adiposity on plasma and brain amphetamine levels in wild type mice. Brain amphetamine levels were higher in Tsn KO mice than in wild type littermates and correlated with adiposity. Analysis of the effect of diet-induced increases in adiposity in wild type mice on brain amphetamine levels also demonstrated that brain amphetamine levels correlate with adiposity. Increased adiposity displayed by Tsn KO mice or by wild type mice fed a high-fat diet correlates with elevated brain amphetamine levels. As amphetamine and its analogues are widely used to treat attention deficit disorder, which is associated with obesity, further studies are warranted to assess the impact of adiposity on amphetamine levels in these patients. Collectively, these data reveal the underlying molecular mechanism at DAT that may be leveraged to rationally optimize leads for the treatment of psychostimulant use disorders.