After saline (vehicle) administration, brain-stimulation reinforcement maintained a frequency-dependent increase in ICSS rates (Fig. nucleus accumbens dopamine levels and facilitated electrical brain stimulation reinforcement within 10?min in rats, providing evidence supporting PDM pharmacological activity. These results demonstrate that PDM functions as a DAT inhibitor that may also interact with the pharmacological effects of its metabolite PM. Overall, these results suggest a novel mechanism for PDM therapeutic effects via initial PDM DAT inhibition followed by PM DAT substrate-induced dopamine release. Phendimetrazine (PDM) is a clinically available and widely prescribed anti-obesity medication1. Recently, 14C21 day PDM treatment was shown to decrease cocaine self-administration in preclinical cocaine addiction models2,3, and it is currently being evaluated as a candidate pharmacotherapy in human laboratory cocaine self-administration studies (“type”:”clinical-trial”,”attrs”:”text”:”NCT02233647″,”term_id”:”NCT02233647″NCT02233647 and NCT0252235). Structurally, PDM is an studies found that PDM possessed measurable but very low potency to block the dopamine transporter (DAT) in rat brain synaptosomes (IC50?=?19?M)5. By contrast, PM was approximately 30-fold more potent to block DAT (IC50?=?0.6?M) and 100-fold more potent than PDM Ivacaftor hydrate to induce release at DAT (EC50?=?0.13?M). Second, microdialysis studies found that PDM failed to significantly increase extracellular nucleus accumbens (NAc) dopamine (DA) levels when either retrodialyzed at a 5-fold larger concentration than its IC50 value or administered intravenously at a dose of 10?mg/kg5. Rabbit Polyclonal to CD3EAP Conversely, PM increased NAc DA by retrodialysis and after intravenous doses as low as 1?mg/kg. Overall, this literature body suggests that PDM functions as an inactive prodrug for the active metabolite PM. However, more recent data suggest that PDM may be behaviorally active oocytes overexpressing hDAT12. Furthermore, given recent evidence demonstrating stereospecificity as a determinant of monoamine transporter inhibitor and substrate pharmacology13,14,15, we also determined the electrophysiological activity of two PDM and PM enantiomers at DAT. Lastly, PDM neurochemical and behavioral effects were determined in rats using microdialysis and intracranial self-stimulation methodologies. These two procedures allow for assessment of the potency and time course of drug effects involving the mesolimbic dopamine pathway16,17. We hypothesized that PDM would produce electrophysiological effects at DAT and rapid onset of neurochemical and behavioral effects consistent with pharmacological activity independent of its metabolism to PM. Results Electrophysiological effects of phenmetrazine and phendimetrazine In recent studies, we have established the two-electrode voltage-clamp technique in oocytes as a suitable tool to study the interaction of compounds with monoamine transporters12,18,19. Thus, we measured currents in voltage-clamped (?60?mV) oocytes overexpressing hDAT in response to PM and PDM enantiomers. Figure 2 shows PM and PDM enantiomer dose-response functions at hDAT. Both PM enantiomers elicited concentration-dependent inward currents indicating that PM functions as a hDAT substrate (Fig. 2A,B). In contrast, both PDM enantiomers produced concentration-dependent outward currents (representing the block of the endogenous transporter leak current) indicating that PDM functions as an hDAT inhibitor (Fig. 2D,E). The (+)- and oocytes. Panels C,F show fits of the dose-response function for PM and PDM enantiomers, respectively. Amplitude values are expressed Ivacaftor hydrate as a % of the amplitude of an initial 5?M dopamine (DA) effect. All points in Panels C,F represent mean SEM data from 5C10?oocytes. Data shown in panels A,B,D,E are from representative current traces. Open in a separate window Figure 3 (+)-Phendimetrazine (PDM) effects on either dopamine- (DA) or (+)-phenmetrazine (PM) -induced inward currents at hDAT in oocytes voltage-clamped to ?60?mV.Panel A shows application of 1 1?M -CIT (positive control) blocked the hDAT current induced by 5?M (+)-phenmetrazine. Panel B shows application Ivacaftor hydrate of 100?M (+)-PM attenuated the current induced by 2?M (+)-PM. Panel C shows application of 100?M (+)-PDM also attenuated the current induced by 2?M DA. For all panels, the initial DA-induced response was 5?M DA. Table 1 Mean EC50 values for phendimetrazine (PDM) and phenmetrazine (PM) enantiomers to produce either a hyperpolarizing (PDM) or inward (PM) current in oocytes expressing hDAT. neurochemical effects of phendimetrazine Baseline (mean??SEM) NAc extracellular DA and 5-HT levels.