Effects of N-Alkyl-4-Methylamphetamine Optical Isomers on Plasma Membrane Monoamine Transporters and Abuse-Related Behavior

Abstract

4-Methylamphetamine (4-MA) is an emerging drug of abuse that acts as a substrate at plasma membrane transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT), thereby causing non-exocytotic release of monoamine transmitters via reverse transport. Prior studies by us showed that increasing the N-alkyl chain length of N-substituted 4-MA analogs converts 4-MA from a transportable substrate (i.e., releaser) at DAT and NET to a non-transported blocker at these sites. Here we studied the effects of the individual optical isomers of N-methyl-, N-ethyl-, and N-n-propyl 4-MA on monoamine transporters and abuse-related behavior in rats because action/function might be related to stereochemistry. Uptake inhibition and release assays were conducted in rat brain synaptosomes whereas electrophysiological assessments of drug-transporter interactions were examined using cell-based biosensors. Intracranial-self stimulation (ICSS) in rats was employed to assess abuse potential in vivo. The experimental evidence demonstrates that S(+)N-methyl 4-MA is a potent and efficacious releaser at DAT, NET, and SERT with the highest abuse potential among the test drugs whereas R(−)N-methyl 4-MA, is a less potent releaser with reduced abuse potential. The S(+)ethyl analog displays decreased efficacy as a releaser at DAT but retains full release activity at NET and SERT with a reduction in abuse-related effects; the R(−)ethyl analog has a similar profile but is less potent. S(+)N-Propyl 4-MA is a non-transported blocker at DAT and NET, but an efficacious releaser at SERT, whereas the R enantiomer is almost inactive. In conclusion, the S enantiomers of the N-alkyl 4-MA analogs are most potent. Lengthening the N-alkyl chain converts compounds from potent non-selective releasers showing abuse-related effects, to more selective SERT releasers with no apparent abuse potential.

Using Ca2+-channel biosensors to profile amphetamines and cathinones at monoamine transporters: electro-engineering cells to detect potential new psychoactive substances

“…In the Ca 2+ assay, N-methyl-4-MA produced Ca 2+ signals in cells expressing each of the transporters, indicating that N-methyl-4-MA is a releaser (or substrate) at the three MATs (Solis et al 2017). As expected, S(+) and racemic N-methyl-4-MA showed potent release activity at all three MATs as measured in synaptosomes and in the Ca 2+ assay (Battisti et al 2018). To assess the abuse-related effects of this drug in vivo, it was tested in ICSS studies.…”

Interactions between Cocaine and the Putative Allosteric Dopamine Transporter Ligand SRI-31142

“…In the present study, SRI-31142 effects on DAT function were further evaluated using imaging procedures to assess DAT-mediated fluorescent signals in live cells. Previous studies have shown a high correlation between drug effects in rat brain synaptosomes and in this assay of DAT-mediated fluorescent signals (Solis et al, 2017;Battisti et al, 2018); however, SRI-31142 effects were not consistent across assays. Specifically, in contrast to findings in rat brain synaptosomes, SRI-31142 failed to produce a cocaine-like blockade of DAT-mediated fluorescent signals.…”

“…Accordingly, SRI-31142 pharmacokinetics in rats were assessed to evaluate the adequacy of brain penetration. Additional studies on SRI-31142 pharmacodynamics were also conducted to examine possible off-target binding sites (Besnard et al, 2012) and the effects on DAT-mediated Ca 21 fluorescence and APP 1 uptake in live cells (Solis et al, 2017;Battisti et al, 2018). Overall, our results suggest that SRI-31142 does not produce abuse-related behavioral or neurochemical effects and that it attenuates cocaine effects by acting via a non-DAT mechanism of action.…”

“…The experiments were performed using an imaging solution consisting of the following (in millimolar): 130 NaCl, 4 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, and 10 glucose, at pH 7.3. As previously described, substrates of monoamine transporters (e.g., DA) generate Ca 21 signals in cells coexpressing these transporters and L-type voltage-gated channels, whereas monoamine transporter inhibitors (e.g., cocaine) fail to generate Ca 21 signals on their own but block substrate-induced signals (Ruchala et al, 2014;Cameron et al, 2015;Solis et al, 2017;Battisti et al, 2018). To test the effect of SRI-31142 in this system, cells were subjected to four phases of treatment over 110 seconds.…”

“…The behavioral effects of SRI-31142 administered alone or in combination with cocaine were evaluated using intracranial self-stimulation (ICSS) in rats, and GBR-12935 was included as a positive control. ICSS is one procedure that can be used in preclinical assessment of abuse potential (Wise, 1996;Carlezon and Chartoff, 2007;Negus and Miller, 2014), and it has been used extensively to evaluate both the abuse-related effects of DAT ligands (Bauer et al, 2013;Bonano et al, 2014a;Solis et al, 2017;Battisti et al, 2018) and the effectiveness of candidate medications to reduce the abuse potential of abused DAT ligands like cocaine (Bauer et al, 2014;Bonano et al, 2014b;Johnson et al, 2018). In ICSS procedures, subjects equipped with microelectrodes targeting a brain reward area are trained to emit a response (e.g., a lever press) to receive pulses of electrical brain stimulation.…”