Main Research Findings

1) Chronic treatment with JJC8-088 resulted in decreased choosing of psychostimulant drugs when compared to the choice of food indicating potential therapeutic benefits for disuse disorders.

2) Administration of JJC8-088 resulted in increased dynamic dopamine activity in the nucleus accumbens shell in both male and female mice.

 

Selected Data
1) The study completed by the research team of Rahimi et al was designed to evaluate the pharmacological and behavioral effects of two experimental dopamine transporter (DAT) ligands, JJC8-088 and JJC8-091, in rhesus monkeys with extensive cocaine self-administration histories. The investigators sought to determine whether these modafinil analogs could reduce cocaine-seeking behavior while maintaining acceptable pharmacokinetic properties and minimizing adverse effects. Three complementary experiments were conducted to characterize the compounds: assessment of dopamine transporter binding affinity, pharmacokinetic analysis following intravenous administration, and behavioral testing using a cocaine-versus-food choice paradigm. Together, these experiments provided information on the compounds’ receptor interactions, systemic exposure, and ability to alter cocaine-reinforced behavior [1].
The first experiment examined the affinity of JJC8-088 and JJC8-091 for dopamine transporters in nonhuman primate brain tissue. Frozen caudate nucleus samples were obtained from cocaine-naive rhesus monkeys and processed to isolate membrane preparations containing dopamine transporters. Radioligand binding assays were performed using tritiated WIN 35,428, a selective dopamine transporter ligand. Seven concentrations of each experimental compound were tested to determine their ability to displace the radioligand from dopamine transporters. Following incubation, binding curves were generated, and inhibitory concentration values were converted into equilibrium binding affinity values using established equations. Each experiment was repeated three times to ensure reproducibility. These measurements allowed direct comparison of DAT affinity between the two compounds in primate tissue and provided an important translational comparison with previous rodent studies.
The second experiment evaluated the pharmacokinetic profiles of both compounds in cocaine-experienced rhesus monkeys. Three monkeys received intravenous JJC8-088 at 0.7 mg/kg, while another three received JJC8-091 at 1.9 mg/kg. Blood samples were collected before dosing and repeatedly over a 24-hour period at intervals ranging from five minutes to twenty-four hours. Plasma was isolated through centrifugation and analyzed using liquid chromatography coupled with tandem mass spectrometry, a highly sensitive analytical technique capable of accurately quantifying low concentrations of drug molecules. Calibration curves were prepared using plasma obtained from drug-naïve monkeys, and internal standards were incorporated to ensure measurement accuracy. Pharmacokinetic parameters including peak plasma concentration, elimination half-life, area under the plasma concentration-time curve, and volume of distribution were calculated using noncompartmental analysis software. These data established dosing regimens for the subsequent behavioral experiments and confirmed that both compounds achieved sufficient systemic exposure for evaluating pharmacological efficacy [1].
Behavioral testing constituted the primary component of the investigation. Four adult male rhesus macaques with more than two years of cocaine self-administration experience were surgically implanted with chronic intravenous catheters connected to subcutaneous vascular access ports. Animals were housed individually under standardized laboratory conditions with environmental enrichment, unrestricted access to water, and carefully regulated food intake to maintain healthy body weights. Behavioral testing occurred inside sound-attenuated operant chambers equipped with two response switches, stimulus lights, automated pellet dispensers, and infusion pumps capable of delivering intravenous cocaine. All procedures complied with institutional animal care guidelines and federal regulations governing the ethical use of laboratory animals.
Monkeys were trained under a concurrent choice procedure in which one response option delivered banana-flavored food pellets while the other delivered intravenous cocaine. Each daily session consisted of five sequential components during which cocaine doses increased from no cocaine to 0.003, 0.01, 0.03, and 0.1 mg/kg per injection. Each component ended after ten reinforcers had been earned or twenty minutes had elapsed. Individual fixed-ratio response requirements were adjusted for each monkey to produce stable baseline dose-response curves characterized by predominant food choice at low cocaine doses and cocaine preference at higher doses. Stability criteria required consistent behavioral performance across multiple sessions before any drug testing commenced [1].
Acute dose-response studies were first conducted for each compound to determine effective pretreatment doses. Monkeys then underwent chronic treatment using five-day dosing cycles. During the first four treatment days only food reinforcement was available, allowing investigators to monitor effects on normal operant responding and detect adverse behavioral changes. On the fifth day, the complete cocaine-versus-food choice procedure was reinstated, permitting construction of an entire cocaine dose-response curve. Drug doses were subsequently adjusted according to predefined criteria based on changes in cocaine preference, food-maintained responding, and behavioral tolerability. Primary outcome measures included percent cocaine choice, cocaine ED50 values, numbers of cocaine injections, food reinforcers earned, total reinforcers completed, and food-only responding during chronic treatment. Statistical analyses primarily consisted of one-way analyses of variance comparing treatment effects with baseline performance, allowing investigators to quantify changes in cocaine preference and determine whether chronic administration produced beneficial or adverse behavioral effects [1].
2) The study completed by Hersey et al was designed to investigate how typical and atypical dopamine transporter (DAT) inhibitors influence dopamine neurotransmission in the nucleus accumbens shell (NAS) of male and female mice, with particular emphasis on identifying potential sex differences that could affect the development of treatments for psychostimulant use disorder (PSUD). The researchers compared the neurochemical effects of cocaine, a prototypical DAT inhibitor, with R-modafinil and two structurally related modafinil analogs, JJC8-088 and JJC8-091. Fast-scan cyclic voltammetry (FSCV), a technique capable of measuring dopamine release and clearance with subsecond temporal resolution, was used to quantify changes in extracellular dopamine signaling following acute administration of each compound. The study was designed to determine whether atypical DAT inhibitors produced dopamine responses that differed from cocaine while also evaluating whether these responses varied between male and female animals.
Adult male and female C57BL/6 mice between eight and twelve weeks of age served as experimental subjects. Animals were housed under controlled environmental conditions with regulated temperature, humidity, and a 12-hour light-dark cycle while receiving unrestricted access to food and water. All mice were experimentally naïve prior to testing, ensuring that previous drug exposure did not influence dopamine signaling. Female mice underwent vaginal cytology following completion of the experiments to determine estrous cycle stage, although these data were not included in the reported analyses.
Neurochemical recordings were performed using in vivo fast-scan cyclic voltammetry following stereotaxic surgery under urethane anesthesia. Each mouse was implanted with a carbon fiber microelectrode positioned within the nucleus accumbens shell to detect extracellular dopamine concentrations and a bipolar stimulating electrode placed into the medial forebrain bundle to evoke dopamine release electrically. A silver/silver chloride reference electrode was implanted in the contralateral hemisphere to complete the recording circuit. Dopamine release was elicited using standardized electrical stimulation parameters consisting of twenty-four pulses delivered at 180 μA and 60 Hz. The carbon fiber electrode continuously scanned between −0.4 and +1.3 volts at 400 volts per second, allowing oxidation and reduction currents generated by dopamine to be measured in real time. Specialized software was used to identify dopamine-specific oxidation peaks, calculate maximum evoked dopamine release, and determine dopamine clearance rates using the half-life required for dopamine concentrations to decline by 50% following stimulation. At the conclusion of each experiment, electrolytic lesions were created at recording sites to verify electrode placement histologically within the nucleus accumbens shell.
Drug preparation and administration were standardized across all treatment groups. Cocaine hydrochloride was dissolved in sterile saline, whereas the modafinil analogs JJC8-088 and JJC8-091 were dissolved in a solution containing dimethyl sulfoxide, Tween-80, and sterile water before sonication to ensure complete dissolution. Drug doses were selected according to previous behavioral and neurochemical investigations evaluating these compounds. Mice received only one pharmacological treatment to eliminate crossover effects between drugs. Four treatment conditions were examined independently: cocaine, R-modafinil, JJC8-088, and JJC8-091. Cocaine was administered at doses of 3 or 10 mg/kg, while R-modafinil, JJC8-088, and JJC8-091 were evaluated at doses of 10 or 32 mg/kg. All drugs were administered intraperitoneally after stable baseline dopamine recordings had been established.
For each experiment, four stable baseline recordings demonstrating less than 15% variability in dopamine release were obtained before drug administration. Dopamine measurements were subsequently collected every five minutes for a total of two hours following treatment, allowing investigators to characterize both the magnitude and duration of each compound’s effects on dopamine release and clearance. Individual animals contributed data to only one treatment condition, and recordings were excluded if signal stability deteriorated, electrode placement could not be confirmed histologically, or animals failed to survive the procedure. A small number of statistical outliers were identified using Grubb’s test and replaced with normalized group averages according to the investigators’ predefined analytical procedures.
Data analysis focused on two principal outcome measures: maximum evoked dopamine release, representing the amount of dopamine released following electrical stimulation, and dopamine clearance half-life, reflecting the efficiency of dopamine transporter-mediated reuptake. Dopamine values were normalized relative to each animal’s baseline recordings and expressed as percentages of baseline activity. Statistical analyses included repeated-measures three-way analyses of variance examining the effects of treatment dose, biological sex, and time following drug administration, as well as their interactions. When significant overall effects were detected, post hoc comparisons were conducted to identify differences between baseline and treatment conditions. Through this experimental design, the investigators systematically compared how cocaine, R-modafinil, JJC8-088, and JJC8-091 altered dopamine neurotransmission in male and female mice while assessing whether sex influenced the pharmacological actions of typical versus atypical dopamine transporter inhibitors.

 

Discussion
1) The study by Rahimi et al demonstrated clear pharmacological and behavioral differences between the two dopamine transporter (DAT) ligands, JJC8-088 and JJC8-091, although both compounds showed some capacity to alter cocaine choice in rhesus monkeys. Initial receptor-binding experiments confirmed that JJC8-088 possessed substantially greater affinity for the dopamine transporter than JJC8-091 in nonhuman primate tissue. Specifically, JJC8-088 exhibited a DAT binding affinity of approximately 14.4 nM, whereas JJC8-091 displayed a much weaker affinity of approximately 2,730 nM. While JJC8-088 maintained a binding profile similar to that previously reported in rodents, the affinity of JJC8-091 for monkey DAT was considerably lower than values reported in rat studies. This species difference suggested that JJC8-091 might be less pharmacologically potent in primates than anticipated from earlier rodent experiments and provided a potential explanation for later behavioral findings [1].
Pharmacokinetic analyses demonstrated that both compounds achieved plasma concentrations sufficient for behavioral testing following intravenous administration. JJC8-088 produced high plasma exposure but was eliminated relatively quickly, exhibiting an average half-life of approximately 1.1 hours and becoming undetectable by 24 hours after administration. In contrast, JJC8-091 exhibited a substantially longer half-life of approximately 3.5 hours, remained measurable throughout the 24-hour sampling period, and displayed a larger apparent volume of distribution despite being administered at a higher dose. Overall systemic exposure, measured by area under the concentration-time curve, was comparable between the compounds, indicating that differences in behavioral efficacy were unlikely to result from inadequate drug exposure. Instead, the investigators concluded that differences in dopamine transporter affinity probably accounted for the divergent behavioral outcomes observed during chronic treatment.
Under baseline behavioral conditions, all monkeys consistently chose food when either no cocaine or very low cocaine doses were available and shifted their preference almost exclusively to cocaine as higher unit doses became available. This stable dose-dependent pattern provided a reliable baseline against which treatment effects could be evaluated. Acute administration of both JJC8-088 and JJC8-091 generally shifted the cocaine dose-response curves to the left, indicating that cocaine became more reinforcing at lower doses immediately after treatment. Because of these acute effects, the investigators focused primarily on chronic administration to determine whether repeated exposure would ultimately decrease cocaine choice through adaptation or tolerance [1].

 

Figure 1: Effects of acute treatment of JJC8-088 on cocaine versus food choice in animal test subjects.
During chronic treatment, JJC8-088 produced the strongest reductions in cocaine choice but also generated the greatest behavioral side effects. Initially, all monkeys displayed increased cocaine preference during the first weeks of treatment, reflecting the same leftward shift observed after acute administration. However, continued dosing gradually produced rightward shifts in the cocaine dose-response curves in two of the three monkeys, indicating that higher cocaine doses were required before cocaine was selected over food. These shifts reflected a reduction in cocaine’s reinforcing effectiveness and increased allocation of behavior toward food reinforcement. One monkey exhibited five consecutive weeks of reduced cocaine choice, while another showed a marked reduction only after higher JJC8-088 doses were administered. The third monkey failed to demonstrate sustained therapeutic benefit despite prolonged treatment.
Chronic administration resulted in reductions in cocaine choice in only one or two monkeys, and these effects were generally smaller and less consistent than those observed with JJC8-088. Some treatment weeks produced rightward shifts in cocaine choice, indicating reduced cocaine preference, whereas other weeks demonstrated transient leftward shifts or little change from baseline. Importantly, two of the three monkeys tolerated JJC8-091 without significant disruption of food-maintained responding, indicating fewer adverse behavioral effects than those associated with JJC8-088. One monkey developed temporary decreases in food responding at higher doses, but tolerance to these effects emerged during continued treatment. Overall session completion and food reinforcement remained relatively stable throughout most JJC8-091 treatment periods, suggesting that the compound produced fewer nonspecific behavioral impairments while still demonstrating limited efficacy in reducing cocaine preference [1].

 

Figure 2: Effects of chronic treatment of JJC8-088 on cocaine versus food choice in animal test subjects.
Taken together, the findings indicated that both DAT ligands produced pharmacological effects consistent with modulation of cocaine reinforcement, but neither compound provided an ideal therapeutic profile. JJC8-088 demonstrated greater efficacy in decreasing cocaine choice but exhibited cocaine-like behavioral effects and unacceptable adverse events that could limit clinical usefulness. Conversely, JJC8-091 displayed lower abuse-related behavioral effects and better tolerability but reduced cocaine choice less consistently, likely because of its substantially weaker affinity for the primate dopamine transporter. The investigators concluded that atypical dopamine transporter inhibitors remain promising candidates for cocaine use disorder treatment, but future compounds should combine the safety advantages of JJC8-091 with greater DAT potency. They further suggested that such agents may be most effective when combined with behavioral interventions to enhance reductions in cocaine choice and improve long-term treatment outcomes [1].
2) The study by Hersey et al demonstrated that cocaine, R-modafinil, JJC8-088, and JJC8-091 each produced distinct effects on dopamine neurotransmission within the nucleus accumbens shell, with cocaine exhibiting the most robust and sex-dependent responses. Before drug administration, male and female C57BL/6 mice displayed nearly identical baseline dopamine dynamics, with no significant differences in either maximum evoked dopamine release or dopamine clearance half-life. These findings established comparable baseline neurochemical function between sexes, allowing subsequent treatment effects to be attributed to pharmacological intervention rather than preexisting physiological differences [2].
Following administration of cocaine, however, dopamine transporter inhibition produced marked increases in extracellular dopamine signaling. Cocaine significantly slowed dopamine clearance and increased electrically evoked dopamine release in both male and female mice, but females consistently exhibited larger and more rapid increases in dopamine release, particularly during the early phase following drug administration. Although dopamine clearance was similarly slowed in both sexes, the enhanced dopamine release observed in females suggested a sex-dependent amplification of cocaine’s neurochemical effects that was consistent with previous reports linking estradiol and altered dopaminergic signaling to increased vulnerability to psychostimulant effects.
Time-course analyses further demonstrated that cocaine produced dose-dependent increases in both dopamine release and dopamine transporter inhibition throughout the two-hour recording period. Administration of either 3 or 10 mg/kg cocaine significantly elevated normalized DAmax while prolonging dopamine clearance half-life, with the higher dose producing more sustained effects. Statistical analyses identified significant effects of treatment time and dose for both dopamine release and clearance, as well as significant interactions between sex and time for dopamine release. Female mice exhibited a faster onset and greater peak increase in evoked dopamine release than males during the first fifteen minutes after cocaine administration, although these differences gradually diminished over time as dopamine levels returned toward baseline. The investigators discussed several mechanisms that could account for these observations, including hormonal regulation by estradiol, altered dopamine transporter phosphorylation, differences in dopamine autoreceptor function, and potential variations in cocaine metabolism. However, because the estrous cycle stage was not analyzed in the present study, these mechanisms remained speculative and were proposed as directions for future investigation rather than definitive explanations [2].
In contrast to cocaine, R-modafinil produced considerably milder effects on dopamine neurotransmission while exhibiting virtually no sex-dependent differences. Acute administration of R-modafinil increased evoked dopamine release and slowed dopamine clearance in a dose-dependent manner across both male and female mice. Although dopamine concentrations increased significantly following treatment, the magnitude of these changes remained substantially smaller than those produced by cocaine, and the effects developed more gradually while persisting for a longer duration. Repeated-measures analyses confirmed significant effects of treatment dose and time on both DAmax and dopamine clearance but found no significant influence of biological sex or interactions involving sex. The investigators interpreted these findings as evidence that R-modafinil, an atypical dopamine transporter inhibitor, alters dopamine neurotransmission without producing the exaggerated sex-dependent dopaminergic responses characteristic of cocaine. This pharmacological profile supports previous observations that R-modafinil exhibits lower abuse liability while maintaining therapeutic potential for psychostimulant use disorder [2].
The modafinil analog JJC8-088 demonstrated neurochemical properties that more closely resembled those of classical psychostimulants. Administration of JJC8-088 significantly increased maximum evoked dopamine release and prolonged dopamine clearance in a dose-dependent manner, confirming effective inhibition of dopamine transporter function. However, unlike cocaine, these responses did not differ significantly between male and female mice. Statistical analyses revealed strong effects of treatment time and dose for both dopamine release and clearance but no significant main effect of sex or interactions involving sex. The investigators noted that JJC8-088 has previously been shown to produce cocaine-like locomotor stimulation and to maintain self-administration behavior in rodents, indicating reinforcing properties similar to those of cocaine. Nevertheless, the absence of sex-dependent neurochemical differences suggested that not all typical dopamine transporter inhibitors produce the same sexually dimorphic dopamine responses observed following cocaine administration. These findings implied that cocaine may possess unique pharmacological characteristics beyond simple dopamine transporter inhibition that contribute to its enhanced effects in females.

 

Figure 3: Changes in maximum dopamine evoked and half-life following administration of JJC8-088.
JJC8-091, the atypical modafinil analog, produced the weakest neurochemical effects among the compounds examined. Treatment resulted in only modest increases in evoked dopamine release while slowing dopamine clearance in both male and female mice. Unlike cocaine, R-modafinil, or JJC8-088, JJC8-091 did not exhibit significant dose-dependent increases in dopamine release or transporter inhibition under the experimental conditions used. Although post hoc analyses confirmed that dopamine release and clearance differed from baseline following administration, repeated-measures analyses detected no significant effects of dose or sex. The investigators noted that these findings differed somewhat from previous studies conducted in Swiss Webster mice and Sprague-Dawley rats, suggesting that C57BL/6 mice may be less sensitive to JJC8-091 or that strain-specific pharmacological differences may exist [2].
Overall, the results demonstrated that both atypical dopamine transporter inhibitors, R-modafinil and JJC8-091, produced relatively modest and highly consistent dopaminergic responses across sexes, whereas cocaine generated pronounced sex-dependent neurochemical effects and JJC8-088 produced robust but sex-independent dopamine elevations. The authors concluded that atypical dopamine transporter inhibitors may represent promising therapeutic candidates for psychostimulant use disorder because they provide stable modulation of dopamine signaling without the exaggerated sex-dependent responses associated with cocaine, thereby supporting their potential effectiveness in both males and females [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Rahimi O, Cao J, Lam J, et al. The Effects of the Dopamine Transporter Ligands JJC8-088 and JJC8-091 on Cocaine versus Food Choice in Rhesus Monkeys. J Pharmacol Exp Ther. 2023;384(3):372-381. doi:10.1124/jpet.122.001363

[2] Hersey M, Chen AY, Bartole MK, Anand J, Newman AH, Tanda G. An FSCV Study on the Effects of Targeted Typical and Atypical DAT Inhibition on Dopamine Dynamics in the Nucleus Accumbens Shell of Male and Female Mice. ACS Chem Neurosci. 2023;14(15):2802-2810. doi:10.1021/acschemneuro.3c00354

 

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