4. Discussion
The necessity of scheduling NPS under the Controlled Substances Act (CSA) is supported by studies that investigate their potential for abuse and provide scientific evidence of its pharmacological and toxicological profiles in vitro and in vivo (for review see (Bonson et al., 2019)). Thus, the main goal of the present study was to characterize the pharmacological profile and the role of the amino substituent, as well as the psychostimulant and rewarding properties of five different α-aminovalerophenone derivatives, which structurally differ only in their amino substituent (Figure 1).
A first evidence of the direct blockade of DA uptake by five different α-aminovalerophenone derivatives was obtained in rat brain synaptosomes. Thereafter, pharmacological profiling of the five drugs from synaptosomal preparations was further confirmed by heterologous assays. The expression of cloned transporters in heterologous systems enabled us to investigate direct interactions of the synthetic cathinones to a single human transporter type. In this case, uptake inhibition in HEK293 cells demonstrated that all five compounds tested also potently inhibit DA uptake but with weak 5-HT uptake inhibition. However, the absolute IC50 values obtained from rat brain synaptosomes differ when compared to those obtained from HEK293 cells. In fact, frequent discrepancies between both preparations in absolute potency estimations have been reported (Baumann et al., 2014; Saha et al., 2015; Mayer et al., 2016a; Sandtner et al., 2016). Moreover, drug selectivity for DAT vs SERT is a key determinant of the abuse potential of drugs targeting monoamine transporters (Stevens Negus and Miller, 2014; Negus and Banks, 2017). High hDAT/hSERT ratios indicates a high abuse potential of these substances and, therefore, a threat to public health.
SAR and QSAR studies attempt to explain how a functional group of a molecule influences its action at its target (Glennon and Dukat, 2016). Kolanos and colleagues performed a SAR study with “deconstructed” MDPV analogues (Kolanos et al., 2013). They conclude that a tertiary amine is the major contributor to the potent effect of MDPV as a DAT blocker, compared to analogues with a secondary and a primary amine. These results agree with our molecular docking results in which the geometry defined by the amine induces a differential binding mechanism. Similarly, the shortening of the pyrrolidine ring of MDPV or the expansion of the α-PVP pyrrolidine ring to a piperidine ring resulted in a progressive decrease in the DAT potency (Kolanos et al., 2015; Glennon and Young, 2016). The five compounds tested in the present study only differ in their amino-substituent, including secondary and tertiary amine analogues. The in vitro data extend previous findings and reveal that the potency of these compounds to block hDAT reuptake increases when the amino group expands from a methyl to an ethyl but decreases from a pyrrolidine to a diethyl and piperidine ring. However, the fact that the N,N-diethyl-pentedrone showed a low affinity to hDAT suggests that carrying a primary, secondary or tertiary amine may not be the sole contributing factor to the activity at these transporters. Moreover, a significant correlation was observed between predicted binding affinities (docking analysis) and Ki values for hDAT. Additionally, a positive correlation was also observed between hDAT/hSERT ratio and the CLogP; the higher the lipophilicity of the substituent, the higher is its selectivity for hDAT vs hSERT. In parallel, it seems that the potency in inhibiting 5-HT uptake improves with decreasing bulk, surface and lipophilicity of the amino-substituent. Although these SAR on hSERT inhibition is extremely useful, we must point out that these studies have to be expanded and corroborated with other synthetic cathinones that have a higher affinity for SERT.
OCT-2 and OCT-3 are low-affinity/high-capacity transporters (Engel and Wang, 2005; Koepsell, 2020) that play a role in neurotransmitter uptake in the brain (Koepsell et al., 1999; Jonker and Schinkel, 2004; Koepsell and Endou, 2004). There is evidence both for and against a direct interaction of psychostimulants such as D-Amphetamine and MDMA to significantly inhibit OCTs (Amphoux et al., 2006; Mayer et al., 2018).In fact, amphetamine is known to promote non-exocytosis release of substrates in the presence of cocaine in an OCT-3-dependent manner (Sitte and Freissmuth, 2015).. Our findings demonstrated that all the five compounds tested inhibit hOCT-2 function with similar potencies. By contrast, and like cocaine (Amphoux et al., 2006), all the drugs tested do not produce any effect on hOCT-3 function.
To assess the psychostimulant and rewarding properties of the five compounds, we used a motor performance and CPP test, respectively. Particularly, a dose-response effect in locomotor activity was observed after synthetic cathinones injections and confirms previous reports about psychostimulant effects of α-PVP and pentedrone (Marusich et al., 2014, 2016; Gatch et al., 2015a, 2015b; Giannotti et al., 2017; Hwang et al., 2017; Javadi-Paydar et al., 2018; Wojcieszak et al., 2018). As expected, the substances that needed lower doses to produce the same behavioural effects (N-ethyl-pentedrone and α-PVP) are those with highest hDAT affinities and potencies. However, despite being one of the substances with the higher affinity for hDAT, a higher dose of α-PpVP was required to induce the same locomotor effect when compared to the other compounds. At this point, we can only hypothesize that some pharmacokinetic effect such as absorption and/or crossing blood-brain-barrier may be involved in such difference in in vitro vs in vivo effects.
Finally, our results also demonstrated that all the compounds tested induced rewarding effects and are consistent with previous studies (Gatch et al., 2015b; Hwang et al., 2017). Particularly, the lowest tested dose of α-PpVP, pentedrone and N-ethyl-pentedrone induced place-conditioning in spite of not producing hyperlocomotion, while the other two doses of N-ethyl-pentedrone induced both effects In contrast, the other two doses tested of α-PpVP produced an increase in the locomotor activity but do not exert rewarding properties. This fact might be related to the unpleasant and/or deleterious effects that animals may suffer at such high doses.
In summary, all of the α-aminovalerophenone compounds studied act as potent DA uptake inhibitors. Increasing the length of the amino group from a methyl to an ethyl group decreased hDAT IC50, while changing a pyrrolidine to a diethyl and piperidine ring increased the IC50 at hDAT. A positive correlation between the hDAT/hSERT ratio and the CLogP of the amino-substituent exists, pointing to a high abuse liability with increased lipophilicity of the substituent. Finally, our study also provides the first evidence that N-ethyl-pentedrone, N,N-diethyl-pentedrone and α-PpVP are able to induce psychostimulant and rewarding effects in mice, suggesting their abuse liability.