Investigation of the Optical Isomers of Methcathinone, and Two Achiral Analogs, at Monoamine Transporters and in Intracranial Self-Stimulation Studies in Rats

Article abstract of DOI:10.1021/acschemneuro.9b00617

Methcathinone (MCAT; 1), the progenitor of numerous and widely abused synthetic cathinone central stimulants, exists as a pair of optical isomers. Although S(-)MCAT is several-fold more potent than R(+)MCAT in rodent locomotor stimulation and in stimulus generalization studies in rat drug discrimination assays, the individual optical isomers of MCAT have never been directly compared for their actions at monoamine transporters that seem to underlie their actions and have never been examined for their relative abuse potential. Here, we found that the isomers of MCAT are nearly equieffective at dopamine and norepinephrine transporters (DAT and NET, respectively) as transporter substrates (i.e., as releasing agents) and are ≥63-fold less potent at the serotonin transporter (SERT). In intracranial self-stimulation (ICSS) studies to evaluate abuse-related drug effects in rats, S(-)MCAT was approximately twice as potent as its R-enantiomer. Achiral analogs, α-methyl MCAT (3) and α-des-methyl MCAT (4), also were DAT/NET substrates and also produced abuse-related ICSS effects, indicating that they retain abuse potential and that they might be useful for the further study of the stereochemistry of synthetic cathinone analogs with chiral β- (or other) substituents.

Full text of DOI:10.1021/acschemneuro.9b00617

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Article
An investigation of the optical isomers of methcathinone
(MCAT), and two achiral analogs, at monoamine transporters
and in intracranial self- stimulation studies in rats
Rachel A. Davies, Tyson R. Baird, Vy T. Nguyen, Brian Ruiz, Farhana
Sakloth, Jose M. Eltit, S. Stevens Negus, and Richard A. Glennon
ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 01 May 2020
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An investigation of the optical isomers of methcathinone (MCAT), and two
achiral analogs, at monoamine transporters and in intracranial self-
stimulation studies in rats
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Rachel A. Davies,1 Tyson R. Baird,2 Vy T. Nguyen,3 Brian Ruiz,3 Farhana Sakloth,1
Jose M. Eltit,3 S. Stevens Negus,4 and Richard A. Glennon1
1Department of Medicinal Chemistry, School of Pharmacy, 2Integrative Life Sciences,
3Department of Physiology and Biophysics, School of Medicine, 4Department of
Pharmacology and Toxicology, School of Medicine, Virginia Commonwealth University;
Richmond, Virginia 23298, U.S.
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ABSTRACT
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Methcathinone (MCAT; 1), the progenitor of numerous and widely abused “synthetic
cathinone” central stimulants, exists as a pair of optical isomers. Although S(-)MCAT is
several-fold more potent than R(+)MCAT in rodent locomotor stimulation and in stimulus
generalization studies in rat drug discrimination assays, the individual optical isomers of
MCAT have never been directly compared for their actions at monoamine transporters
that seem to underlie their actions, and have never been examined for their relative
abuse potential. Here, we found that the isomers of MCAT are nearly equi-effective at
dopamine and norepinephrine transporters (DAT and NET, respectively) as transporter
substrates (i.e., as releasing agents), and are >63-fold less potent at the serotonin
transporter (SERT). In intracranial self-stimulation (ICSS) studies to evaluate abuse-
related drug effects in rats, S(-)MCAT was approximately twice as potent as its R-
enantiomer. Achiral analogs, -methyl MCAT (3) and -des-methyl MCAT (4), also were
DAT/NET substrates and also produced abuse-related ICSS effects, indicating that they
retain abuse potential and that they might be useful for the further study of the
stereochemistry of synthetic cathinone analogs with chiral - (or other) substituents.
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KEYWORDS
Synthetic cathinones, Methcathinone optical isomers, Monoamine transporters, DAT,
NET, SERT, ICSS, Drug abuse, Achiral methcathinone,
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INTRODUCTION
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Drug abuse and drug dependence are issues of concern, and central nervous system
(CNS) stimulants play an increasing role both domestically and worldwide.1 Increases in
CNS stimulant-related consequences and deaths have coincided with changes in
clandestine stimulant manufacturing;2 these changes include the emergence of a wide
array of novel psychoactive substances developed to subvert regulation,1 and the
increased stereochemical purity of illicitly-produced traditional stimulant drugs of abuse.
Chemically, many of the new stimulants are synthetic cathinones (often generically
referred to as “bath salts”) that produce unpredictable and frequently severe, even fatal,
adverse effects.3–9 Methcathinone (i.e., MCAT; 1, Figure 1), the β-keto analog (i.e., the
-carbonyl counterpart) of methamphetamine (2), is the prototypical synthetic cathinone
and a U.S. Schedule I synthetic stimulant initially scheduled in 1992.10
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NHCH3
CH3
NHCH3
CH3
O
1
2
NHCH3
CH3
NHCH3
O
O
CH3
3
4
Figure 1. Chemical structures of MCAT (1), methamphetamine (2), and achiral analogs
3 and 4.
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Stereochemistry can profoundly impact the pharmacology of CNS stimulants via
modulation of their actions at one or more monoamine transporters (MATs).e.g. 11–15
These stereochemical differences have implications for abuse potential7-9 and toxicity.16
However, the stereochemical effects of MCAT (1), the progenitor of all synthetic
cathinones, have received relatively little attention.
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In the 1990s, following the initial identification of racemic MCAT (1) as a drug of
abuse,17 our laboratory synthesized and evaluated the two optical isomers of MCAT in
assays of mouse locomotor activity and in tests of stimulus generalization in rat drug
discrimination studies.18,19 As a locomotor stimulant in mice, both isomers produced
similar maximal effects but S(-)MCAT (S(-)1) was nearly five times more potent than
R(+)MCAT (R(+)1).19 Others later found that S(-)MCAT was at least twice as potent as
its R(+)-enantiomer in locomotor studies using mice and rats.16 In drug discrimination
studies, both MCAT optical isomers substituted for cocaine in cocaine-trained rats, and
for S(+)amphetamine in S(+)amphetamine-trained rats.18,19 In both investigations, S(-
)MCAT was 2- to 3-fold more potent than its R(+)-enantiomer. We then expanded upon
these studies using S(-)MCAT as a training drug in rat drug discrimination studies, and
S(-)MCAT stimulus generalization occurred to a number of CNS stimulants, with S(-
)MCAT being about four times more potent than R(+)MCAT (ED50 = 0.11 and 0.43
mg/kg, respectively).20 Pretreatment with haloperidol antagonized these effects
suggesting dopaminergic involvement.20 All studies suggested that S(-)MCAT was at
least twice as potent as R(+)MCAT. Futhermore, modulation of monoamines, in
particular dopamine, was suggested as being important to the stimulant effects of S(-
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)MCAT. This was later supported by evaluation of S(-)MCAT at monoamine transporters
(MATs) in rat brain synaptosome assays where S(-)MCAT was a potent releaser of
monoamines with >100-fold selectivity for dopamine and norepinephrine over
serotonin.21
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Yet, despite the long history of MCAT abuse, its optical isomers have never been
directly compared at the three MATs, and the impact of MCAT stereochemistry on
potential abuse-related behavioral effects (other than as indirectly measured by rodent
locomotor activity and rat drug discrimination studies) remains uninvestigated.
Additionally, further modifications to the MCAT structure are prevalent in emerging
synthetic cathinones; these structural modifications can result in multiple stereogenic
centers at, for example, the -position and (if the carbonyl group is replaced with a
saturated substituent) the -position. In order to advance the development of structure-
activity relationships for synthetic cathinones, it would be preferable to eliminate chirality
at the α-carbon so that the stereochemistry of - (and other) substituents might be more
easily examined. Thus, in the current study, two such strategies were examined; we
prepared and evaluated two achiral MCAT analogs, 3 and 4 (Figure 1), for direct
comparison with racemic MCAT and its optical isomers both in vitro at the three hMATs
expressed in cultured cells, and in vivo in an assay of intracranial self-stimulation
(ICSS)22 in rats. In 3, an additional -methyl group was added to MCAT and in 4, the -
methyl group was deleted; both result in -achiral analogs.
Results
Synthesis. All synthetic targets have been previously reported in the literature. The
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hydrochloride salts of MCAT (1) and its individual optical isomers were prepared as we
earlier reported.19,23 Compound 3 is known,24 but was prepared here by a different
method; -bromoisobutyrophenone25 was heated with potassium carbonate, methanol,
and methylamine, and the isolated product was converted to its hydrochloride salt.
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Compound 4 also has been previously prepared,26 but repeated attempts to replicate
the literature method were unsuccessful and resulted only in nearly intractable tar-like
materials from which miniscule amounts of the target product were obtained. Perboni
and Giubellina27 reported on an “improved process” for the synthesis of a related series
of compounds. Application of this procedure provided the desired target, albeit in low
yield. Specifically, Friedel-Crafts acylation of benzene with N-trifluoromethylsarcosinyl
chloride28 afforded the N-trifluoromethyl-protected free base of 4 that was de-protected
with hydrochloric acid to afford the target compound as its hydrochloride salt.
Transporter studies. As shown in Table 1, all examined compounds were more potent
as substrates (i.e., as releasing agents) at hDAT and hNET than at hSERT.
Representative tracings for substrate action at DAT are shown in Figure 2. At hDAT,
there was little difference in potency between the isomers of 1 (EC50 = 240 and 315 nM
for the S and R isomers, respectively). Compounds 3 (EC50 = 590 nM) and 4 (EC50 =
1,860 nM) displayed somewhat lower potency than ()1 (EC50 = 248 nM) but were,
nevertheless, substrates. At hNET, the compounds showed little difference in potency
as substrates with EC50 values ranging from 121 to 182 nM. At hSERT, S(-)MCAT,
()MCAT, and -methyl MCAT (3) were weak substrates (EC50 = 15,300, 24,750, and
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12,860 nM, respectively) whereas R(+)MCAT and -des-methyl MCAT (4) were inactive
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at concentrations of up to 50,000 nM.
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Table 1. Potency of MCAT, MCAT optical isomers, and achiral analogs 3 and 4, as
substrates of hDAT, hNET, and hSERT in a calcium flux assay. Potency was estimated
by evaluating the depolarizing action of releasing agents when they interact with the
three MATs. Voltage-gated Ca2+ channels were used as voltage sensors and the
opening of these channels results in Ca2+ signals proportional to the transporter
depolarizing activity. Results are means ± SEM of n ≥ 23 cells analyzed per
concentration, total number of cells analyzed per dose-response curve is indicated in
parenthesis.
Agent
hDAT EC50, nM
±SEM
hNET EC50, nM
±SEM
hSERT EC50, nM
±SEM
S(-)MCAT (S(-)1)
R(+)MCAT (R(+)1)
240 ± 11 (504)
15,300 ± 1439 (346)
121  2 (331)
144  1 (227)
315 ± 14 (511)
>50,000 (147)
(±)MCAT ((±)1)
248 ± 15 (631)
590 ± 19 (832)
24,750 ± 5745 (231)
12,860 ± 1270 (443)
>60,000 (240)
182  3 (299)
153  15 (281)
148  13 (519)
α-Methyl MCAT (3)
α-des-Methyl-MCAT (4) 1,860 ± 90 (432)
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Figure 2. Concentration-response curves for MCAT and its optical isomers
(upper trace), and compounds 3 and 4 relative to racemic MCAT (lower trace), as
DAT substrates. The potency of these compounds was estimated by evaluating
the depolarizing action of releasing agents when they interact with DAT. Voltage-
gated Ca2+ channels were used as voltage sensors and the opening of these
channels results in Ca2+ signals proportional to the transporter depolarizing
activity. The experimental points correspond to mean ± SEM of n ≥ 23 cells
analyzed per concentration.
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Intracranial self-stimulation. Under baseline conditions, electrical brain stimulation
maintained a frequency-dependent increase in ICSS rates. Across all rats in the study,
the mean ± SEM MCR was 59.0 ± 2.6 stimulations per trial, and the mean ± SEM total
stimulations earned per baseline component was 249.6 ± 12.8 stimulations per
component.
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Figure 3 shows that all five compounds produced dose-dependent ICSS facilitation
manifested as leftward and upward shifts in ICSS frequency-rate curves. Two-way
ANOVA indicated significant frequency x dose interactions for racemic MCAT [F(3.083,
15.42) = 8.577, P=0.0013], S(-)MCAT [F(3.082, 15.41) = 9.105, P=0.0010], R(+)MCAT
[F(3.379, 16.90) = 7.849, P=0.0013], α-methyl MCAT (3) [F(2.528, 12.64) = 9.465,
P=0.0020], and α-des-methyl MCAT (4) [F(3.648, 18.24) = 9.864, P=0.0003]. A higher
32 mg/kg dose of α-des-methyl MCAT (4) was tested in two rats and produced robust
stereotypies (e.g. excessive sniffing) without a further increase in ICSS frequency-rate
curves (data not shown); therefore, further testing of this dose was discontinued for 4,
and data were not included in statistical analysis.
Table 2 compares the potencies (ED150 values) and efficacies (Emax values) of drugs to
facilitate ICSS. The S(-)-enantiomer of MCAT was significantly more potent than its
R(+)-enantiomer by 2.4-fold as indicated by non-overlapping 95% confidence intervals.
Both racemic MCAT and α-methyl MCAT (3) displayed potencies that were intermediate
between potencies of the MCAT isomers, and statistically indistinguishable either from
each other or from the potencies of the MCAT optical isomers. In contrast, α-des-methyl
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MCAT (4) was significantly less potent than all other compounds. Emax values were
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statistically indistinguishable across all compounds.
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Figure 3. Dose-dependent ICSS facilitation by racemic MCAT, S(-)MCAT, R(+)MCAT, α-methyl MCAT,
and α-des-methyl-MCAT. Panels A-E show drug effects by dose on full ICSS frequency-rate curves.
Abscissae: Frequency of electrical brain stimulation in Hz (log scale). Ordinates: Percent maximal control
reinforcement rate (%MCR). Legends indicate the drug name and doses. Filled points represent
frequencies at which ICSS rate was statistically different from vehicle as determined by two-way repeated-
measures ANOVA followed by a Holm-Sidak post hoc test, p < 0.05. Panel F shows summary ICSS data
expressed as percent baseline number of stimulations per component delivered across all brain-
stimulation frequencies for all drugs. Abscissa: Drug dose in mg/kg (log scale). Ordinate: Percent
baseline number of stimulations per component. Filled points represent significant difference compared to
vehicle using a repeated-measures one-way ANOVA for each drug followed by a Holm-Sidak post hoc
test, p < 0.05. All points show mean ± SEM for six rats (n=6).
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Figure 4. Time course of ICSS facilitation by racemic MCAT, S(-)MCAT, R(+)MCAT, α-methyl MCAT, and α-des-
methyl-MCAT. Panels A-E show full frequency-rate curves determined 10, 30, 100, and 300-minute after drug
injection. Abscissae: Frequency of electrical brain stimulation in Hz (log scale). Ordinates: Percent maximal control
reinforcement rate (%MCR). Filled points represent frequencies at which ICSS rate was statistically different from
baseline as determined by two-way repeated-measures ANOVA followed by a Holm-Sidak post hoc test, p < 0.05.
Panel F shows summary data for total stimulations per component compared to baseline for all drugs. Abscissa: Time
after injection in minutes (log scale). Ordinate: Percent baseline number of stimulations per component. Filled points
represent significant difference compared to baseline using a repeated-measures one-way ANOVA followed by a
Holm-Sidak post hoc test, p < 0.05. All points mean ± SEM for six rats (n=6) with the exception of S(-)MCAT, which
had five subjects (n=5).
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Table 2. Potency (ED150) and efficacy (Emax) of MCAT isomers and analogs to facilitate
ICSS in rats. 95% confidence limits (95%Cl) are shown in parentheses (n = 5-6 animals
per dose), and values across drugs are considered significantly different if 95%Cl
values do not overlap.
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ED (95%CI)
E
(95% CI)
max
150
Agent
(mg/kg)
(% BL)
S(-)MCAT (S(-)1)
R(+)MCAT (R(+)1)
0.25 (0.17-0.34)
189 (159-219)
0.60 (0.37-0.98)
207 (166-248)
0.38 (0.25-0.59)
0.36 (0.22-0.61)
6.3 (3.6-26.2)
180 (141-220)
189 (151-226)
164 (119-210)
(±)MCAT (()1)
α-Methyl MCAT (3)
α-des-Methyl MCAT (4)
Figure 4 shows the time course for the Emax dose of each compound. In general, all
agents produced peak ICSS facilitation after 10 to 30 min, and effects were no longer
significant after 300 min. Two-way ANOVA indicated significant frequency x time
interactions for racemic MCAT [1.0 mg/kg; F(3.168, 15.84) = 8.265, P=0.0014], S(-
)MCAT [1.0 mg/kg; F(2.742, 10.97) = 6.455, P=0.0098], R(+)MCAT [3.2 mg/kg; F(3.357,
16.78) = 11.72, P=0.0002], α-methyl MCAT [1.0 mg/kg; F(3.007, 15.04) = 4.363,
P=0.0212], and α-des-methyl-MCAT [10 mg/kg; F(3.035, 15.17) = 10.00, P=0.0007].
Discussion
MCAT is generally considered to produce its abuse-related effects primarily by acting as
a DAT substrate, promoting structural transitions in the transporter enabling inward
depolarizing current,29 and the release of dopamine (see Introduction). The present
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investigation represents the first comparative study of the optical isomers of MCAT at
the three human MATs, the racemate of which has been a substance of abuse for >25
years. Given that S(-)MCAT has been previously shown to be at least twice as potent as
R(+)MCAT in rodent locomotor and drug discrimination studies, it was expected that
similar findings might be reflected by the actions of the isomers at hDAT as substrates.
However, there was less than a 2-fold potency difference between the MCAT isomers.
The in vivo actions of MCAT isomers might be related to differences in their other
pharmacodynamic or pharmacokinetic properties.
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The potency of the MCAT isomers at hNET was, generally, twice their potency at hDAT
and here, too, there was little difference in the potency of the individual MCAT isomers.
At hSERT, S(-)MCAT displayed 50- to >100-fold lower potency than at hDAT or hNET,
and R(+)MCAT was essentially inactive.
Because there was little difference in the potency of the MCAT isomers at DAT, it was
expected that the  gem-dimethyl analog 3 would be no more potent that R(+)MCAT,
and this was found to be the case. That the  des-methyl analog 4 was several times
less potent than racemic MCAT suggests that the  methyl group of 1 might contribute
to its transporter interactions in a positive fashion. This did not appear to be the case at
hNET; that is, there is essentially no difference in the potency of MCAT isomers, the 
gem-dimethyl analog 3, and the  des-methyl analog 4. Nevertheless, 4 displayed >10-
fold selectivity at hNET relative to hDAT. At hSERT, all agents were only weakly active,
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at best; and, 4 displayed >32- and >450-fold selectivity for DAT and NET relative to
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In ICSS studies to evaluate abuse-related behavioral effects, all agents produced
facilitation in a dose-dependent manner; this is characteristic of abused cathinone
analogs and other monoamine transporter substrates with selectivity for
DAT>SERT.15,22 The present results replicate previous ICSS findings with racemic
MCAT30 and extend those findings by showing modest but significant stereoselectivity
with slightly higher potency for the S(-)-enantiomer. Consistent with earlier locomotor
and drug discrimination studies, S(-)MCAT was at least twice as potent as R(+)MCAT.
Eliminating chirality at the -carbon atom of MCAT by replacing the α-hydrogen atom
with an additional α-methyl group (i.e., 3) had no effect on the potency or efficacy for
ICSS facilitation as compared to racemic MCAT. Conversely, eliminating chirality at the
-carbon atom by replacing the α-methyl group with an α-hydrogen atom (i.e., 4)
resulted in a decrease in DAT potency. The reduced ICSS potency of 4 might reflect, to
some extent, its reduced lipophilicity relative to the other agents and, hence, it’s
reduced ability to penetrate the blood-brain barrier in the in vivo study. That is, whereas
4 displays 7.5-fold lower potency than MCAT at hDAT, it was 16-fold less potent in
ICSS.
Overall, i) the individual optical isomers of MCAT displayed little selectivity as substrates
at DAT (and NET), but were much less potent at SERT, ii) R(+)MCAT is more selective
than S(-)MCAT for DAT relative to SERT, iii) both achiral analogs (i.e., 3 and 4),
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although somewhat less potent than MCAT, retained DAT substrate action, and iv)
consistent with their potency profile (i.e., DAT>SERT), all agents produced ICSS.
Achiral analogs, especially gem-dimethyl analogs as exemplified by 3, might find utility
for examining the stereochemistry of -position (and other chiral) substituents in future
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DAT SAR studies, whereas -des-methyl analogs as exemplified by 4 might find
application in the investigation of NET SAR.
Experimental
Synthesis. MCAT (1) and its optical isomers, as their hydrochloride salts, were prepared
by methods we have previously reported.19,23
α-Methylaminoisobutyrophenone (-Methylmethcathionone) Hydrochloride (3).
In a sealed tube at room temperature, -bromoisobutyrophenone25 (3.00 g, 13.2 mmol)
was added to a stirred solution of potassium carbonate (5.53 g, 39.6 mmol) in MeOH
(75 mL). The reaction mixture was allowed to stir for 5 h, at which time methylamine
gas was bubbled in. The temperature was increased to 57 °C, and the mixture stirred
overnight. Upon cooling to room temperature, Et2O (50 mL) was added to dilute the
mixture, which was then filtered. Solvent was evaporated under reduced pressure, and
the resulting crude mixture was dissolved in EtOAc, then acidified and extracted with
HCl (1 M, 5 mL, 6x). The aqueous portion was basified with NaOH (1 M, 50 mL), and
extracted with EtOAc (30 mL). The combined organic portion was dried (Na2SO4), and
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solvent was removed under reduced pressure, to yield 1.40 g of the free base as a
yellow oil.
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The free base was dissolved in EtOAc (30 mL), a saturated solution of gaseous HCl in
Et2O (10 mL) was added, and the reaction mixture was allowed to stir at room
temperature for 2 h. The solvent was removed by filtration to yield a white powder that
was recrystallized from EtOH/Et2O to yield 0.60 g (21%) of 3 as a white solid: mp 218-
220 °C (lit.24 mp 212-214 °C); 1H NMR (DMSO-d6) δ 1.75 (s, 6H, 2 x CH3), 2.53 (s, 3H,
CH3), 7.57 (dd, J = 7.6, 7.9 Hz, 2H, ArH), 7.70 (t, J = 7.4, 1H, ArH), 7.95 (dd, J = 8.5,
1.2, 1H, ArH), 9.49 (br s, 2H, NH2+).
2-Methylamino-1-phenylethan-1-one (-Desmethylmethcathinone) Hydrochloride
(4).
Synthesis of compound 4 was attempted by a literature method26 but resulted in an
intracible tar from which little to no no product was obtained. Compond 4 was
subsequently prepared in several steps. Freshly sublimed AlCl3 (9.20 g, 69 mmol) was
added in a portionwise manner to a stirred solution of N-trifluoromethylsarcosinyl
chloride,28 (61 mmol) in anhydrous benzene (40 mL) under an N2 atmosphere. The
stirred reaction mixture was heated at reflux for 4 h, allowed to cool to room
temperature, quenched by the addition of conc. HCl (12 mL), and ice (10 g). The
organic layer was separated, washed with saturated NaHCO3 (3 x 15 mL), and brine (2
x 10 mL), dried (Na2SO4), and evaporated under reduced pressure. The resulting brown
oil was dissolved in conc. HCl (50 mL) and i-PrOH (75 mL) and stirred at 40 °C for 48 h.
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The solvent was evaporated to yield 0.50 g of residue, which was recrystallized twice
from i-PrOH to yield 0.56 g of 4 (5%) as off-white crystals: mp 205-208 °C (lit.26 mp 219
°C). 1H NMR (DMSO-d6) δ 2.63 (s, 3H, CH3), 4.77 (s, 2H, CH2), 7.60 (t, J = 7.7 Hz, 2H,
ArH), 7.74 (t, J = 7.8 Hz, 1H, ArH), 8.00 (d, J = 7.4 Hz, 2H, ArH), 9.48 (br s, 2H, NH+).
Because the observed mp was slightly lower than that previously reported, it was
subjected to elemental analysis. Elemental analysis for C9H11ON.HCl: Calcd C, 58.23,
H, 6.51, N, 7.54; Found 57.96, H, 6.57, N, 7.40.
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Transporter studies. Voltage-gated Ca2+ channels were used as biological reporters of
the monoamine transporter activity. The transport of substrates happens concomitant
with inward currents depolarizing the cell membrane. This depolarization activates the
voltage-gated Ca2+ channels producing an intracellular Ca2+ signal.31,32 The amplitude of
this Ca2+ signal is proportional to the concentration of monoamine transporter substrate
applied during the experiment and, by using several concentrations of substrate
together with intracellular Ca2+ determinations, dose-response curves can be
constructed to estimate the potencies of substrates at transporters. 15,30-33 This
methodology has been described in detail before;34 briefly, stable and inducible cell
lines expressing DAT, SERT, or NET, all of human origin, were previously made using
the targeted recombination system HEK293 Flp-In T-REx (Invitrogen) following the
manufacturer’s suggested procedure.31,32 Cells were plated in 96-well imaging plates
and were co-transfected with plasmids encoding voltage-gated Ca2+ channels (rabbit
α1.2, rat β3, rat α2δ) for cells expressing DAT and SERT, or (rat α1.3, rat β3, rat α2δ)
for cells expressing NET. Although using α1.2 or α1.3 does not alter the apparent
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potency of the tested compound in this functional assay,32 α1.3 facilitates signal
acquisition in cells expressing NET (empirical observation). In addition, an EGFP
encoding plasmid was used as transfection marker and three days before the
experiment the culturing media were supplemented with doxycycline (1µg/mL) to induce
the expression of monoamine transporters.15,31 The experimental day cells were loaded
with the fluorescent Ca2+ indicator Fura 2AM. Ca2+ signals were acquired using an
Olympus IX70 microscope equipped with fluorescence imaging attachment (Till
Photonics) and an EMCCD camera (Andor) that allows the acquisition of ratio images
(340 nm and 380 nm excitation). The experiments were performed under constant
perfusion using a pressurized system and automatic solution exchange was achieved
using electronic valves controlled by the acquisition computer (ValveLink 8.2 Automate
Scientific) and at 35oC using a solution heater (ThermoClamp-1, Automate Scientific).
For these experiments the imaging solution (IS) used consisted of: NaCl 140 mM, KCl
4mM, MgCl2 1 mM, CaCl2 2mM, glucose 10 mM and HEPES 10mM (pH adjusted to 7.3
using NaOH). The 340/380 images were acquired at 3 Hz continuously and recorded for
off-line analysis. The solution exchange sequence was: 10 s of IS (baseline), 5 s of
positive control (10µM DA for DAT or NET, and 10µM 5HT for SERT), after a 30 s IS
wash, cells were exposed for 5 s to the test compound at a single variable
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concentration, and finishing the protocol with 30 s IS wash as described
previously.15,33,34 Control experiments showed that the compounds under investigation
do not promote Ca2+ mobilization in cells expressing voltage-gated Ca2+ channels in the
absence of monoamine transporters (not shown). The ratio signal analysis was
performed exactly as described before;15,33 the response to the test compound was
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expressed as a fraction of the control signal for each cell. Each field of view in a well
normally has ~5 (range: 3-10) responding cells, in a regular experiment we tested
approximately 3-4 wells per concentration, and these was repeated up to 4 days for all
concentrations. The total number of cells analyzed per concentration varied from ~25
cells (at very low or very high concentrations where the variability of the data is very
small) to 50-100 cells at concentrations that are more variable (closer to the EC50).
Thus, each concentration point has variable numbers of cells analyzed. Then the mean,
SEM and n of each concentration is entered in GraphPad Prism 5.0 and a dose-
response curve is fitted from the experimental data, and “best - fit” values are used to
obtain EC50 and the SEM of the EC50. The number of total cells analyzed per dose-
response curve (including all concentrations) range from 227 to 832 per compound.
Since many cells are analyzed per concentration, the SEM of the mean response for
each concentration is small and sometimes is not visible in the graph.15,33 The EC50
values reported in this assay are a functional estimation of the potency of the tested
compounds to produce structural transition at transporters that promote inward current
associated to the uptake of substrates, association between depolarizing currents and
substrate uptake has been described previously.35-37
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ICSS. Subjects: Subjects consisted of 13 adult male Sprague-Dawley rats (Envigo,
Indianapolis, IN, USA) weighing from 317 to 365 g at the time of surgery. Only males
were used to permit direct comparison of the present results to previous results with
cathinone analogs and monoamine releasers.13,15,21,30,38 The rats were individually
housed in a facility accredited by the Association for the Assessment and Accreditation
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of Laboratory Animal Care (AAALAC) and maintained on a 24-hour light/dark cycle
(lights on from 6:00 am to 6:00 pm). Rats had free access to food and water except
during behavioral sessions. Animal care and research complied with the National
Institutes of Health guidelines for the care and use of animal subjects in research, and
all protocols were approved by the Virginia Commonwealth University Institutional
Animal Care and Use Committee.
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Surgical Procedure: Surgical and experimental procedures were similar to those
described previously.13,15,21,30,38 Anesthesia was induced by inhalation of 2.5-3.0%
isoflurane (Zoetis Inc., Kalamazoo, MI, USA) in oxygen until the rat was unresponsive to
toe pinch, and this level of anesthesia was maintained throughout surgery. A stainless
steel, bipolar electrode (Plastics One, Roanoke, VA, USA) was implanted via
stereotaxic surgery by inserting the cathode into the left medial forebrain bundle at the
lateral hypothalamus (2.8 mm posterior to bregma, 1.7 mm lateral to the midsagittal
suture, and 8.8 mm ventral to the exterior surface of the skull). The anode was
grounded by coiling around one of three screws anchored into the dorsal surface of the
skull. The electrode, screws, and grounding wire were permanently affixed to the skull
using dental acrylic resin. Ketoprofen (5 mg/kg) was administered for post-operative
analgesia immediately and 24 h after surgery, and rats were allowed to recover for at
least 7 days prior to commencing ICSS training.
Apparatus: Behavioral sessions were conducted in modular operant test chambers
(29.2 x 30.5 x 24.1 cm) (Med Associates, St. Albans, VT, USA) constructed of stainless
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steel and clear polycarbonate with a grid floor and housed inside ventilated sound-
attenuating cubicles. The test chamber contained a response lever located 3 cm above
the floor and 7.6 cm below three stimulation lights (red, yellow, and green), a 2 W house
light, and an ICSS stimulator. A bipolar cable and swivel commutator (Model SL2C;
Plastics One, Roanoke, VA, USA) connected the ICSS stimulator to the electrode. Data
collection and ICSS programming were mediated by a computer system running Med-
PC IV software (Med Associates, St. Albans, VT, USA).
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Training: Following initial shaping of lever-press responding, rats were trained under a
fixed-ratio 1 (FR 1) schedule of brain stimulation. During behavioral sessions, each
lever press resulted in the delivery of a 0.5 s train of square wave cathodal pulses (0.1
ms pulse duration) and illumination of the stimulus lights over the lever. Stimulation
intensity and frequency were set at 150 μA and 126 Hz, respectively, during initial 60-
min training sessions. Stimulation intensity was then individually increased for each rat
until it sustained ICSS rates > 30 stimulations/min. This intensity was then held
constant, and frequency manipulations were introduced. Sessions involving frequency
manipulations consisted of three sequential 10 min components. During each
component, a descending series of 10 frequencies (158 to 56 Hz in 0.05 log
increments) was presented, with each frequency available for a 1-min trial. Each
frequency trial consisted of a 10 s time-out, during which five non-contingent “priming”
stimulations were delivered at the frequency of stimulation that would be available
during that trial, followed by a 50 s “response” period, during which responding
produced electrical stimulation under a FR 1 schedule as described above. The
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stimulation intensity was adjusted up or down to yield baseline frequency-rate curves
that had high rates of reinforcement for the upper frequencies and minimal responding
at the remaining frequencies. This intensity (110-340 µA across rats) was then held
constant throughout the study. Training continued until frequency-rate curves were not
statistically different over three days of training as indicated by lack of a significant effect
of “day” in a two-way analysis of variance (ANOVA) with frequency and day as the two
variables (see data analysis).
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Testing: Each drug was evaluated with dose-effect and time-course testing procedures.
For dose-effect studies, test sessions consisted of three sequential “baseline”
components followed first by a time-out period and then by two sequential “test”
components. A single dose of test drug was administered at the beginning of the time-
out period, and dose-effects studies with all drugs were conducted with 10-min time-out
periods and associated 10-min pretreatment times. R(+)-Methcathinone was also
tested in dose-effect studies with 30-min time-out periods and 30-min pretreatment
times because time-course studies indicated maximal ICSS facilitation at 30 min (see
Results). Time-course test sessions consisted of three consecutive baseline
components followed first by drug injection and then by pairs of consecutive test
components beginning after 10, 30, 100 and 300 min. Five drugs were tested: racemic
MCAT (0.10 – 1.0 mg/kg), S(-)MCAT (0.10-1.0 mg/kg), R(+)MCAT (0.10-3.2 mg/kg), -
methyl MCAT (0.32-3.2 mg/kg), and -desmethyl-MCAT (1.0-32 mg/kg). For dose-effect
studies, drug doses were counterbalanced across rats using a Latin-Square design.
The maximally effective dose of each drug was tested in time-course studies. Rats were
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tested in groups of six (n=6) except for the time course on S(-)MCAT, which was tested
with five animals due to the spontaneous detachment of the electrode in one animal.
Testing was generally performed on Tuesdays and Fridays. All testing was performed
during the light phase of the light/dark cycle.
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Data Analysis: The primary dependent variable was reinforcement rate in stimulations
per minute during each frequency trial. To normalize these data, raw reinforcement
rates from each trial in each rat were converted to percent maximum control rate
(%MCR), with MCR defined as the mean of the maximal rates observed during the
second and third baseline components for that rat on that day. Thus, %MCR values for
each trial were calculated as (reinforcement rate during a frequency trial) / (MCR) x 100.
For each test session, data from the second and third baseline components were
averaged to yield a baseline frequency-rate curve, and data from each pair of test
components were averaged to generate test frequency-rate curves. Baseline and test
curves were then averaged across rats to yield mean baseline and test curves for each
manipulation. For statistical analyses, results were compared by repeated measures
two-way ANOVA with ICSS frequency as one factor and either dose or time as the
second factor. A significant ANOVA was followed by the Holm-Sidak post hoc test, and
the criterion for significance was set at p < 0.05.
To provide a secondary summary measure of drug effects, the total number of
stimulations delivered across all 10 frequency-trials of each component was determined
for each drug dose or time point. These summary data were normalized to individual
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baseline data using the equation % baseline total stimulations per component = (mean
total stimulations per test component) / (mean total stimulations per baseline
component) x 100. Data were then averaged across rats and plotted as a function of
drug dose or time. Dose-effect functions were used to calculate measures of drug
potency and efficacy. For potency, linear regression of logarithm-transformed doses
was used to identify the ED150 value, defined as the effective dose to reach 150% of
baseline total stimulations. For efficacy, the maximum effect (EMax) was defined as the
peak % baseline total stimulations per component at any drug dose. ED150 and Emax
values were considered to be significantly different across drugs if 95% confidence
limits did not overlap. Statistical analyses were performed using Prism 8.1.2 (GraphPad
Software, San Diego, CA, USA).
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Corresponding Author Information:
*E-mail- glennon@vcu.edu
ORCID
Richard A. Glennon: 0000-0002-3600-9045
Author Contributions:
RAD and FS performed the synthesis of the target compounds under the supervision of
RAG. Transporter studies were conducted by RAD, VTN, and BR under the supervision
of JME. ICSS studies were conducted by TRB in the laboratory of SSN. RAG proposed
the project and all co-authors had an opportunity to contribute to the final manuscript.
Funding:
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The work was supported in part by DA033930 (SSN, JME, RAG) and P30DA033934
(SSN).
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Notes:
The authors claim no conflicting financial interest.
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