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.