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. Author manuscript; available in PMC: 2024 Jan 1.
Published in final edited form as: Drug Alcohol Depend. 2022 Dec 5;242:109719. doi: 10.1016/j.drugalcdep.2022.109719

Troriluzole inhibits methamphetamine place preference in rats and normalizes methamphetamine-evoked glutamate carboxypeptidase II (GCPII) protein levels in the mesolimbic pathway

Sonita Wiah a, Abigail Roper a,b, Pingwei Zhao a, Aryan Shekarabi a, Mia N Watson a, Daniel J Farkas a, Raghava Potula c, Allen B Reitz d, Scott M Rawls a,e
PMCID: PMC9850846  NIHMSID: NIHMS1857979  PMID: 36521236

Abstract

Riluzole, approved to manage amyotrophic lateral sclerosis, is mechanistically unique among glutamate-based therapeutics because it reduces glutamate transmission through a dual mechanism (i.e., reduces glutamate release and enhances glutamate reuptake). The profile of riluzole is favorable for normalizing glutamatergic dysregulation that perpetuates methamphetamine (METH) dependence, but pharmacokinetic and metabolic liabilities hinder repurposing. To mitigate these limitations, we synthesized troriluzole (TRLZ), a third-generation prodrug of riluzole, and tested the hypothesis that TRLZ inhibits METH hyperlocomotion and conditioned place preference (CPP) and normalizes METH-induced changes in mesolimbic glutamate biomarkers. TRLZ (8, 16 mg/kg) reduced hyperlocomotion caused by METH (1 mg/kg) without affecting spontaneous activity. TRLZ (1, 4, 8, 16 mg/kg) administered during METH conditioning (0.5 mg/kg × 4 d) inhibited development of METH place preference, and TRLZ (16 mg/kg) administered after METH conditioning reduced expression of CPP. In rats with established METH place preference, TRLZ (16 mg/kg) accelerated extinction of CPP. In cellular studies, chronic METH enhanced mRNA levels of glutamate carboxypeptidase II (GCPII) in the ventral tegmental area (VTA) and prefrontal cortex (PFC). Repeated METH also caused enhancement of GCPII protein levels in the VTA that was prevented by TRLZ (16 mg/kg). TRLZ (16 mg/kg) administered during chronic METH did not affect brain or plasma levels of METH. These results indicate that TRLZ, already in clinical trials for cerebellar ataxia, reduces development, expression and maintenance of METH CPP. Moreover, normalization of METH-induced GCPII levels in mesolimbic substrates by TRLZ points toward studying GCPII as a therapeutic target of TRLZ.

Keywords: methamphetamine, riluzole, troriluzole, CPP, psychostimulant, glutamate, GCPII

1. Introduction

Riluzole (RLZ) inhibits glutamate transmission by enhancing astrocytic glutamate reuptake (Brothers et al., 2013; Carbone et al., 2012; Liu et al., 2011). It also inhibits neuronal glutamate release through calcium and sodium channel inhibition and potassium channel activation (Cheah et al., 2010; Machado-Vieira et al, 2009; Pittenger et al, 2008). Effectively, RLZ combines mechanistic features of β-lactam compounds (e.g., ceftriaxone and clavulanic acid), which clear extracellular glutamate by enhancing protein levels of glutamate transporter subtype 1 (GLT-1) (Rothstein et al., 2005), with a concurrent reduction in glutamate release. Pharmacological approaches targeting elements of the glutamate reuptake and receptor systems normalize psychostimulant-induced glutamatergic dysregulation and reduce psychostimulant reward, reinforcement and seeking behaviors (Koob and Volkow, 2016; Parsegian and See, 2014; Kalivas and Volkow, 2011; Knackstedt and Kalivas, 2009). The glutamate-targeted profile of RLZ is highly attractive for managing psychostimulant addiction, but studies are sparse and have yielded mixed outcomes. RLZ inhibits cocaine seeking in rat self-administration models but lacks efficacy against cocaine behavioral sensitization in mice (Sepulveda-Orengo et al., 2018; Itzhak and Martin, 2000; Brackett et al., 2000). RLZ reduces acute locomotor activation caused by METH or amphetamine, and reduces expression, but not development, of locomotor sensitization induced by repeated METH exposure (Itzhak and Martin, 2000; Lourenço Da Silva et al., 2003). In clinical studies, RLZ improved craving, withdrawal, and depression measures in METH-dependent patients but lacked efficacy against cocaine dependence (Farahzadi et al., 2019; Ciraulo et al., 2005).

The inconclusive results with RLZ may be related to pharmacokinetic (PK) and metabolic limitations (McDonnell et al., 2012). Indeed, even in ALS patients, RLZ displays extensive hepatic metabolism and high levels of patient-to-patient variability related to variable first-pass elimination effects caused by heterogeneous cytochrome P450 isoform Cyp1A2 expression (Greonveld et al., 2001). To mitigate these limitations, we designed, synthesized, and evaluated troriluzole (TRLZ) as a third-generation prodrug taken from >400 analogs, with optimized in vitro and in vivo features including enhanced gastrointestinal absorption, metabolic cleavage in plasma, minimized first-pass metabolism, active transport by the PepT1 transporter, and a favorable safety profile (Pelletier et al., 2018). We adopted a prodrug approach where the RLZ exocyclic nitrogen was masked as a functional group not recognized by Cyp1A2 as a substrate for oxidation (Fig. 1) (McDonnell et al., 2012). The first generation validated our desired in vitro profile, the second established a favorable in vivo profile in multiple species but had hERG activity, and the third contained the positive attributes of the previous two but without hERG activity (IC50 > 30 μM), leading ultimately to TRLZ (2) (Fig. 1).

Fig. 1. TRLZ (troriluzole) synthesis:

Fig. 1.

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Riluzole (1) and prodrug troriluzole (2) showing conversion of troriluzole to give riluzole after PepT1-mediated absorption of troriluzole.

In the present study, we assessed effects of TRLZ on METH place preference in rats, hypothesizing that TRLZ would reduce development and expression of CPP and counteract an established METH CPP. Effects of TRLZ on METH-induced hyperlocomotion and on brain and plasma levels of METH were investigated. We also tested the hypothesis that TRLZ would rescue dysregulation of glutamate biomarkers (glutamate carboxypeptidase II [GCPII], GLT-1 and glutaminase) in mesocorticolimbic substrates during repeated METH exposure.

2. Materials and Methods

2.1. Animals

Male Sprague-Dawley rats (250–275 g) obtained from Harlan/Envigo Laboratories (Indianapolis, IN) were used. Rats were housed in a controlled environment (21–23 °C) on a 12-h light/dark cycle. Rats were provided food and water ad libitum except during behavioral testing. Experimental procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and Temple University’s Guidelines for the Care of Animals. Rats were assigned randomly to experimental groups with appropriate sample sizes and used only once for a specific experiment.

2.2. Chemicals

Methamphetamine hydrochloride (METH) was purchased from Sigma-Aldrich (St. Louis, MO). Troriluzole mono hydrochloride monohydrate was designed and synthesized by Dr. Allen Reitz (Fox Chase Chemical Diversity Center, Doylestown, PA) as described (Pelletier et al., 2018), and shown in Fig. 1. All drugs were dissolved in physiological saline and injected intraperitoneally (IP) in a volume of 1 ml/kg. Doses of METH (0.5–1 mg/kg) were selected based on studies showing robust rewarding and locomotor stimulant effects in rats (Hofford et al., 2014). The dose range of TRLZ (1–16 mg/kg) was based on studies with RLZ (Sepulveda-Orengo et al., 2017), including adjustments for molecular weight differences between TRLZ (prodrug) and the parent compound (RLZ) (i.e., the molecular weight of TRLZ is approximately 2-fold greater than RLZ, so a dose of 8 mg/kg approximates a 4 mg/kg of RLZ).

2.3. Effects of TRLZ on METH locomotor activation

Locomotor activity was measured as described using a Digiscan DMicro system (Accuscan Inc.) consisting of transparent plastic chambers (45 cm × 20 cm × 20cm) set inside metal frames equipped with 16 infrared light emitters and detectors (Simmons et al., 2022; Nayak et al., 2020). Following a 60-min pre-drug habituation interval in the activity chambers, rats were injected with TRLZ (0, 4, 8 or 16 mg/kg). Fifteen min later, all rats were injected with METH (1 mg/kg) and activity was measured for 100 min. In separate experiments, effects of TRLZ itself on spontaneous locomotor activity were assessed. Following the standard 60-min habituation interval, rats were injected with TRLZ (16 mg/kg) or water, and locomotor activity was measured for 60 min.

2.4. Effects of TRLZ on development of METH CPP

CPP experiments were conducted as described using CPP chambers (45 cm × 20 cm × 20 cm) consisting of 2 compartments separated by a removable door (Philogene-Khalid et al., 2022; Nayak et al., 2020). A 30-min pre-test was conducted on day 1 to determine initial compartment preference. The compartment in which a rat spent less time was designated as the METH-paired side. The day after the pre-test, a 4-day conditioning paradigm with morning and afternoon sessions was initiated. In the morning, rats pretreated with TRLZ (0, 1, 4, 8 or 16 mg/kg) were injected 15 min later with METH (0.5 mg/kg) and confined to the METH-paired compartment for 30 min. In the afternoon session, rats pretreated with water were injected 15 min later with water and placed in the opposite compartment for 30 min. On day 6, a post-test was conducted in which rats were placed into the chamber and given free access to roam both compartments for 30 min. A difference score (difference in time spent on METH-paired side between post-test and pre-test) was then determined.

In separate experiments, TRLZ by itself was tested in the CPP paradigm to control for rewarding or aversive effects. Experiments proceeded as described above with pre-test (day 1) and post-test (day 6) sessions. The 4-d conditioning phase again consisted of morning and afternoon sessions. In the morning, rats treated with TRLZ (0, 16 mg/kg) were confined to the TRLZ-paired compartment for 30 min. In the afternoon, rats treated with water were confined to the opposite compartment for 30 min. The post-test followed on day 6.

2.5. Effects of TRLZ on expression of METH CPP

Experiments assessing impacts of a single injection of TRLZ on the expression of METH CPP proceeded as described above with a few exceptions. Following the standard pre-test on day 1, a 4-d conditioning paradigm ensued in which rats injected with METH (0.5 mg/kg) (morning session) were confined to the METH-paired compartment for 30 min. In the afternoon session, rats were injected with water and confined to the opposite compartment for 30 min. On day 6, the post-test was conducted in which rats injected with TRLZ (0, 8, 16 mg/kg) were placed into the chamber 15 min later and given free access to roam both compartments for 30 min.

2.6. Effects of TRLZ on maintenance of METH CPP

Experiments were next designed to determine if TRLZ impacted the maintenance of an established METH CPP. During the standard 6-d experiment described above (pre-test, conditioning phase, post-test), all rats were conditioned with METH (0.5 mg/kg) to establish an initial CPP (designated as baseline (BL) CPP). For the extinction phase, TRLZ (0, 16 mg/kg) was injected once daily for 11 d. TRLZ was always administered in the afternoon following CPP testing to avoid potential behavioral confounds due to acute exposure. Over the 11-d extinction phase, a series of intermittent 30-min post-tests were conducted in which rats were placed into the chamber and allowed access to roam both compartments for 30 min. A total of 4 different post-tests were conducted and designated as extinction sessions (1, 2, 3 and 4) (Session 1=2 d TRLZ; Session 2=5 d TRLZ; Session 3=8 d TRLZ; and Session 4=11 d TRLZ). For each CPP (post-test) test, a difference score (difference in time spent on METH-paired side between post-test and original pre-test) was determined.

2.7. Effects of chronic METH exposure on mRNA levels of glutamate biomarkers

Rats were injected for 7 d with METH (1 mg/kg) or water, with injections occurring 3x/d and spaced 60 min apart. Brains were extracted 60 min after the last METH (or water) injection, and gene expression experiments were conducted on harvested brain tissue as described (Nayak et al., 2020). The PFC, VTA, hippocampus (HPC), and NAC were dissected from frozen slices using 1- and 2-mm round punches, respectively (mRNA from the NAC was not analyzed because tissue was accidentally placed into a concentration of buffer that resulted in degradation). RNA was isolated using the Quick-RNA Miniprep kit (Zymo Research, Irvine, CA, USA), and cDNA was synthesized using RT^2 First Strand Kit (Qiagen, Germantown, MD, USA). Glutamate biomarkers selected for analysis were glutamate carboxypeptidase II (GPCII), GLT-1, glutaminase 1 (GLS), and glutaminase 2 (GLS2). Quantitative real-time PCR was carried out with TaqMan Fast Advanced Master Mix and the TaqMan Gene Expression Assays for GCPII (Rn00589333_m1), GLT-1 (Rn00691548_m1), Gls (Rn00561285_m1), and GLS2 (Rn01529404_m1), using the QuantStudio 3 Real-Time PCR System (Applied Biosystems). Relative gene expression was measured according to the 2−ΔΔCT method.

2.8. Effects of chronic TRLZ and METH on protein levels of glutamate biomarkers

Rats were again injected for 7 d with METH (1 mg/kg) or water, with injections occurring 3x/d and spaced 60 min apart. Rats were also injected with TRLZ (0, 16 mg/kg) for the entire 7-d interval, with TRLZ administration occurring once daily 15 min before the first METH injection. Brain tissue was collected 60 min after the last METH (or water) injection for protein analysis of glutamate biomarkers (GCPII, GLT-1, and GLS1) in the PFC, VTA, and NAC. Western-blot experiments were conducted as previously described (Philogene-Khalid et al., 2022). Tissue was homogenized and lysed with RIPA buffer. Samples were centrifuged for 15 min at 12,000 g, and supernatant was transferred to new tubes. Samples were then heated at 95°C for 5 min, resolved by SDS-PAGE gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with Licor buffer for 60 min, membranes were incubated at 4°C overnight with the corresponding primary antibodies. Membranes were then washed with TBS and incubated with secondary antibodies for 2 h at room temperature. Membranes were scanned using Licor Odssey-2014. Immunoreactive bands were quantified by Image Studio (Licor Ver 2.0) and normalized to bands of GAPDH. Data were then imported into GraphPad Prism 7 for graphing and statistical analysis. Antibodies used were: GCPII monoclonal antibody, ThermoFisher; GLS1 monoclonal antibody, ThermoFisher; GLT-1 polyclonal antibody, Sigma; and GAPDH polyclonal antibody, ThermoFisher. Blocking buffer, Licor; Secondary antibodies used were: IRDye800CW Donkey anti-Guinea pig; IRDye800CW Goat anti-Mouse; IRDye680RD Donkey anti-Rabbit; Licor.

2.8. Effects of TRLZ on plasma and brain levels of METH

Pharmacokinetic experiments were conducted to determine if TRLZ affected plasma and brain levels of METH. Rats were injected for 7 d with METH (1 mg/kg) or water. TRLZ (16 mg/kg) or water was injected 15 min prior to METH. Fifteen min after the last METH injection, brain tissue and blood were collected from rats following euthanization. Whole brains were extracted, placed into dry ice, and stored at −80 °C until shipping to Touchstone Biosciences for further preparation and analysis. Following decapitation blood from the carotid artery and jugular vein was drained into tubes that were placed into ice. Blood samples were then centrifuged (5000 RPM) for 15 min at 4 °C. Following centrifugation plasma samples were collected and stored at −80 °C until shipping to Touchstone Biosciences for further preparation and analysis. The experiment was repeated but with brain tissue and blood collected 60 min after the last METH injection.

Plasma sample preparation and analysis.

Three volumes of acetonitrile containing internal standard were added to one volume of plasma to precipitate remaining proteins. Samples were centrifuged (3000 g for 10 min) and supernatant removed for analysis by LC-MS/MS. Calibration standards and quality controls were made by preparation of a 1 mg/mL stock solution and then a series of working solutions in methanol: water (1/:1, v/v) which were spiked into blank plasma to yield a series of calibration standard samples in the range of 1 ng/mL to 10 μg/mL and quality control samples at three concentration levels (low, middle and high). All incurred PK/PD plasma samples were treated identically to the calibration standards. LC-MS/MS analysis was performed utilizing multiple reaction monitoring for detection of characteristic ions for each drug candidate, additional related analytes and internal standard.

Tissue sample preparation for analysis.

Three volumes of PBS buffer (pH 7.4) were added to one volume of each tissue sample, which was then homogenized to obtain each tissue homogenate sample. The remainder of the preparation and LC-MS/MS analysis proceeded as described above for plasma.

2.9. Statistical analysis

Two-way ANOVA was used to analyze locomotor data (TRLZ treatment × time) followed by a Dunnett’s test. One-way ANOVA was used to analyze CPP data assessing effects of TRLZ on the development and expression of METH CPP followed by a Dunnett’s test. A Student’s t-test was used to assess effects of TRLZ by itself in the CPP paradigm. For maintenance (extinction) studies, two-way ANOVA (TRLZ treatment × time) was used to analyze data (followed by a Dunnett’s test). For mRNA analysis, a Student’s t-test was used to analyze effects of METH exposure on glutamate biomarkers. For protein expression, two-way ANOVA (TRLZ × METH) was used followed by a Bonferroni test. And, for PK studies assessing effects of TRLZ on METH plasma and brain levels, two-way ANOVA (TRLZ × time) was used followed by a Bonferroni test. Statistical significance was set at P < 0.05 in all cases.

3. Results

3.1. TRLZ reduced locomotor activation caused by acute METH (Fig. 2)

Fig. 2. TRLZ reduced METH-evoked hyperlocomotion but did not affect spontaneous locomotor activity.

Fig. 2.

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(A) METH hyperlocomotion: Rats pretreated with TRLZ (0, 4, 8, or 16 mg/kg) were treated with METH 15 min later and activity counts were measured for 100 min. Data are expressed as mean locomotor activity counts + S.E.M. N= 8 rats/group. ***P < 0.001, **P < 0.01, or *P < 0.05 compared to TRLZ-naïve rats (TRLZ (0) + METH). (B) Spontaneous locomotor activity: Rats were treated with water or TRLZ (16 mg/kg) and activity counts were measured for 60 min. Data are expressed as mean locomotor activity counts + S.E.M. N= 4 rats/group.

For effects of different doses of TRLZ against hyperlocomotion evoked by a fixed dose of METH (1 mg/kg) (Fig. 2A), two-way ANOVA revealed significant effects of TRLZ [F(3,168) = 8.07, P < 0.001] and time [F(5, 168) = 22.73, P < 0.001]. Locomotor activity prior to METH injection and 15 min after TRLZ exposure (0 min time point) was not significantly different in any of the treatment groups (P > 0.05). Locomotor activity in rats injected with METH alone (TRLZ (0) + METH) was elevated compared to rats exposed to TRLZ (16 mg/kg) and METH at 20 min (P < 0.01) and 40 min (P < 0.01) following METH administration. Treatment with a lower dose of TRLZ (8 mg/kg) (TRLZ (8) + METH) also reduced locomotor activation caused by METH alone (TRLZ (0) + METH) at 20 min (P < 0.001) and 40 min (P < 0.01) post-METH exposure. Treatment with a lower dose of 1 mg/kg TRLZ did not affect METH locomotor activation (P > 0.05). For experiments with TRLZ by itself (Fig. 2B), locomotor activity in rats treated with TRLZ (16 mg/kg) was not significantly different compared to water treatment [F(1, 24) = 0.96, P < 0.05].

3.2. TRLZ reduced development and expression of METH place preference (Figs. 3–4)

Fig. 3. TRLZ reduced development of METH place preference but is not aversive or rewarding by itself.

Fig. 3.

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(A) TRLZ vs METH CPP: Rats were conditioned with METH (0.5 mg/kg) for 4 d and pretreated (15 min) before each conditioning session with TRLZ (0, 4, 8, or 16 mg/kg). CPP testing was conducted the following day. Data are presented as a difference score (difference in time spent on METH-paired side between post-test and pre-test). ***P < 0.001, **P < 0.01, or *P < 0.05 compared to TRLZ-naïve rats conditioned with METH (TRLZ (0) + METH). N= 7–22 rats/group (note: 7–13 rats/TRLZ treatment groups and 22 rats in METH alone control group). (B) TRLZ alone: Rats were conditioned with TRLZ (16 mg/kg) or water for 4 d and CPP testing was conducted the following day. Data are presented as a difference score (difference in time spent on TRLZ-paired side between post-test and pre-test). N= 8 rats/group.

Fig. 4. TRLZ reduced expression of METH place preference.

Fig. 4.

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Rats were conditioned with METH (0.5 mg/kg) for 4 d. CPP testing was conducted on the following day with rats being injected once with TRLZ (0, 8, 16 mg/kg) 15 min before testing. Data are presented as a difference score (difference in time spent on METH-paired side between post-test and pre-test). **P < 0.01 compared to TRLZ-naïve rats conditioned with METH. N= 8–15 rats/group.

For effects of different doses of TRLZ against development of METH (0.5 mg/kg) CPP (Fig. 3A), one-way ANOVA revealed a significant main effect [F(4, 55) = 6.95, P < 0.001]. Rats conditioned with METH by itself (first bar) displayed greater CPP than rats treated with TRLZ during METH conditioning. The reduction of METH-evoked CPP by TRLZ was observed across all doses of TRLZ that were tested (1 mg/kg, P < 0.001; 4 mg/kg, P < 0.05; 8 mg/kg, P < 0.01; 16 mg/kg, P < 0.01). Rats conditioned with TRLZ (16 mg/kg) by itself did not display place preference that was significantly different than rats conditioned with water (P > 0.05, Student’s t-test) (Fig. 3B).

For effects of TRLZ against expression of METH (0.5 mg/kg) CPP (Fig. 4), one-way ANOVA revealed a significant main effect [F(2, 28) = 6.89, P < 0.01]. Rats conditioned with METH and then injected once with TRLZ (16 mg/kg) before the post-test displayed significantly less CPP than TRLZ-naïve rats injected with water (P < 0.01). A lower dose of TRLZ (8 mg/kg) did not significantly affect expression of CPP produced by METH (P > 0.05).

3.3. TRLZ accelerated extinction of established METH place preference (Fig. 5)

Fig. 5. TRLZ facilitated extinction of established METH CPP.

Fig. 5.

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Rats were conditioned with METH (0.5 mg/kg) for 4 d, and CPP testing was conducted on the following day. Rats were then treated with TRLZ (16 mg/kg) or water (TRLZ (0) for 11 d. CPP testing was conducted intermittently across 4 extinction sessions (session 1 = 2 d TRLZ; session 2 = 5 d TRLZ; session 3 = 8 d TRLZ; session 4 = 11 d TRLZ). TRLZ was administered after conditioning sessions to minimize potential behavioral confounds from acute exposure. Data are presented as percentage of baseline CPP score (baseline CPP score determined prior to initiation of TRLZ treatment paradigm). **P < 0.05 compared to TRLZ-naïve rats. N= 7 rats/group.

For effects of TRLZ against maintenance of established METH (0.5 mg/kg) CPP (Fig. 5A), two-way ANOVA revealed a significant main effect [F(1, 60) = 14.51, P < 0.001]. Mean baseline CPP values for the two different groups (TRLZ-naïve (TRLZ (0) and TRLZ-treated (TRLZ (16)) following METH conditioning and prior to TRLZ treatment were not significantly different: TRLZ (0) group was 242 ± 86 s and TRLZ (16) group was 284 ± 51 s (P > 0.05, Student’s t-test). Post-hoc analysis revealed that the percentage of baseline CPP (place preference before TRLZ treatment) was significantly lower in rats treated with TRLZ than in TRLZ-naïve rats for the second extinction session (Extinction Session 2 equating to 6 d of TRLZ treatment) (P < 0.05). For extinction sessions 3 and 4, the percentage of baseline CPP values were still lower in the TRLZ treatment group relative to the TRLZ-naïve group, but the difference did not reach statistical significance (P > 0.05).

3.4. Repeated METH exposure increased mRNA levels of glutamate biomarkers (Fig. 6)

Fig. 6. Effects of METH on mRNA levels of glutamate biomarkers.

Fig. 6.

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Rats were treated for 7 d with METH (1 mg/kg, 3x/d) or water. Brains were extracted 60 min after the last METH (or water) treatment and gene expression studies were conducted to analyze mRNA levels of glutamate biomarkers (GCPII, GLT-1, GLS1 and GLS2) in the PFC, VTA, and NAC. Results are grouped by region: PFC, top panel (A); VTA, middle panel (B); and hippocampus (HPC), bottom panel (C). For each target, data are expressed as fold-change + S.E.M. N=6–8 rats/group. **P < 0.01 or ***P < 0.01 compared to METH-naïve control rats.

Effects of chronic binge METH exposure on mRNA levels of glutamate biomarkers (GCPII, GLT-1, GLS1 and GLS2) are presented in Fig. 6 (separated by brain region: (Fig. 6A, PFC; Fig 6B, VTA; and Fig. 6C, HPC). For the PFC (Fig. 6A), GCPII levels were significantly elevated compared to METH-naïve rats exposed to water (P < 0.01, Student’s t-test). For the VTA (Fig. 6B), rats treated with METH showed elevated mRNA levels of three glutamate biomarkers relative to METH-naïve rats (GCPII, P < 0.01; GLT-1, P < 001; and GLS1, P < 0.001). For the HPC (Fig. 6C), no significant differences in mRNA levels of GCPII, GLT-1, GLS2 or GLS2 between METH-treated and METH-naïve groups (P > 0.05).

3.5. TRLZ normalized increase in GCPII protein levels induced by repeated METH (Fig. 7)

Fig. 7. TRLZ normalized elevation of GCPII protein levels induced by chronic METH exposure.

Fig. 7.

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Rats were treated for 7 d with METH (1 mg/kg, 3x/d) or water. TRLZ (16 mg/kg) or water was administered 15 min before the first METH or water injection on each day. Brains were extracted 60 min after the last METH (or water) treatment for protein analysis (Western blot analysis) of glutamate biomarkers (GCPII, GLT-1 and GLS1) in the PFC, VTA, and NAC. Results are grouped by region: PFC, top panel (A); VTA, middle panel (B); and NAC, bottom panel (C). For each glutamate substrate, data are expressed as relative ratio (glutamate marker/GADPH) + S.E.M. N= 6–8 rats/group. **P < 0.01 or *P < 0.05 compared to drug-naïve control rats (Water + Water group). ++P < 0.01 or +P < 0.05 compared to rats treated with METH by itself (Water + METH group).

Effects of repeated TRLZ and METH exposure on protein levels of glutamate biomarkers (GCPII, GLT-1, GLS1 and GLS2) in mesocorticolimbic substrates are presented in Fig. 7 (separated by brain region: (Fig. 7A, PFC; Fig 7B, VTA; and Fig. 7C, NAC). For GLT-1 in the PFC (Fig. 7A), two-way ANOVA revealed a significant effect of TRLZ [F(1, 27) = 10.60, P < 0.01]. In rats treated with TRLZ by itself (TRLZ + Water), GCPII protein levels were significantly elevated compared to drug-naïve rats (Water + Water) (P < 0.05). In rats treated with TRLZ by itself (TRLZ + Water) or a combination of TRLZ and METH (TRLZ + METH), GCPII protein levels were significantly greater compared to rats treated with METH alone (Water + METH) (P < 0.05 and P < 0.01, respectively).

In the VTA (Fig. 7B), for GCPII, two-way ANOVA revealed a significant effect of TRLZ [F(1, 22) = 4.33, P < 0.05] and a significant interaction [F(1, 22) = 12.18, P < 0.01]. GCPII protein levels in rats treated with METH by itself (Water + METH) were significantly elevated compared to drug-naïve rats (Water + Water) (P < 0.01). For rats treated with a combination of TRLZ and METH (TRLZ + METH), GCPII levels were significantly reduced compared to TRLZ-naïve rats exposed to METH by itself (Water + METH) (P < 0.01). In the NAC (Fig. 7C), for GCPII, two-way ANOVA revealed a significant effect of TRLZ [F(1, 26) = 9.57, P < 0.05] but post-hoc analysis did not reveal any significant differences between individual groups.

3.6. TRLZ did not affect plasma or brain levels of METH (Fig. 8)

Fig. 8. TRLZ does not affect plasma or brain levels of METH.

Fig. 8.

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Rats were treated for 7 d with METH (1 mg/kg, 3x/d). TRLZ (16 mg/kg) was administered 15 min before the first METH injection on each day. Brains were extracted 15 min after the last METH treatment and brains were extracted for pharmacokinetic analysis of plasma and brain levels of METH by analysis by LC-MS/MS. The experiment was repeated and brains were extracted 60 min after the last METH injection. (A) Plasma METH levels: Data are expressed as plasma concentration of METH (mg/ml) + S.E.M. N= 7–8 rats/group. (B) Brain METH levels: Data are expressed as brain concentration of METH (ng/g) + S.E.M. N= 7–8 rats/group.

For plasma levels of METH (Fig. 8A), two-way ANOVA revealed a time effect [F(1, 26) = 57.21, P < 0.001] but not a significant effect of TRLZ [F(1, 26) = 0.28, P > 0.05] or a significant interaction [F(1, 26) = 2.88, P > 0.05]. For brain levels of METH (Fig. 8B), two-way ANOVA revealed a time effect [F(1, 26) = 87.67, P < 0.001] and a significant interaction [F(1, 26) = 5.53, P < 0.05] but not a significant effect of TRLZ [F(1, 26) = 0.70, P > 0.05].

4. Discussion

The present study was the first to investigate effects of TRLZ, a pharmacokinetically- and metabolically-enhanced prodrug of FDA-approved riluzole (Pelletier et al., 2018), in an animal model of a CNS disorder. The main behavioral finding was that TRLZ inhibited three different phases of METH place preference (i.e., development, expression and maintenance). TRLZ administered during METH conditioning prevented development of place preference. Inhibitory effects of TRLZ were robust, as METH CPP was reduced by approximately 75%. The dose-effect curve for TRLZ was flat, as 1 mg/kg was just as effective at reducing development of METH place preference as 16 mg/kg. In METH-naïve rats, TRLZ did not produce conditioned rewarding or aversive effects or affect spontaneous locomotor activity. Thus, the blocking effect of TRLZ on development of METH place preference could not be explained by conditioned aversion or non-specific locomotor effects. In expression experiments where TRLZ was administered once following METH conditioning, TRLZ was still effective in reducing METH place preference but only at the highest dose. The lower potency of TRLZ against expression may be due to a greater degree of difficulty in countering an established METH preference than disrupting conditioning reward. Alternatively, TRLZ may have acted through different mechanisms to reduce the different stages of METH CPP. Given that TRLZ treatment did not affect plasma or brain levels of METH, it is likely that pharmacodynamic, rather than pharmacokinetic, mechanisms were responsible for the efficacy of TRLZ.

The glutamate-based profile of TRLZ, and established role of glutamate neurotransmission in conditioned rewarding effects of METH, provide insight into mechanisms underlying TRLZ efficacy. Because TRLZ is catabolized to RLZ, inhibition of glutamate transmission after TRLZ administration is likely due to actions of RLZ, which inhibits neuronal glutamate release (Cheah et al., 2010; Machado-Vieira et al, 2009; Pittenger et al, 2008) and increases clearance of extracellular glutamate by increasing glutamate transporter subtype 1 (GLT-1) protein expression (Brothers et al., 2013; Carbone et al., 2012; Liu et al., 2011). Agents that disrupt glutamate transmission, including antagonists of NMDA, AMPA or mGlu5 receptors and enhancers of glutamate reuptake (e.g., MS-153) reduce METH CPP, indicating that enhanced glutamatergic neurotransmission facilitates development and/or expression of METH place preference (Miyatake et al., 2005; Nakagawa et al., 2005). Thus, it is plausible that TRLZ, by reducing extracellular glutamate originating from neuronal and astrocytic pools, prevented normal increases in glutamate transmission at NMDA, AMPA and metabotropic (e.g., mGluR5) that facilitated METH CPP.

Further clues about TRLZ mechanism and site of action are provided by our cellular studies, where chronic METH exposure caused enhancement of GCPII protein levels in the VTA that was prevented by concurrent TRLZ administration. GCPII protein levels in the NAC or PFC were not affected by chronic METH, indicating VTA-specific effects. Notably, mRNA levels of GCPII in the VTA were also enhanced by repeated METH exposure. GCPII, also known as N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), is primarily expressed on astrocytes and hydrolyzes synaptically released N-acetylaspartyl-glutamate (NAAG) into glutamate. NAAG is the third most prevalent neurotransmitter in the mammalian nervous system and is an endogenous agonist at mGlu3 receptors (Vornov et al., 2016), which are inhibitory GCPRs that, when activated, inhibit neuronal glutamate release and enhance astrocytic glutamate reuptake (Aronica et al., 2003; Sanabria et al., 2004). GCPII inhibitors cause an increase in extracellular NAAG levels that leads to mGlu3 activation (Slusher et al., 1999; Vornov et al., 1999), and inhibit cocaine seeking in rat self-administration studies and cocaine place preference in rats (Peng et al., 2011, Xi et al., 2010; Slusher et al., 2001). Hence, one explanation for our results is that repeated METH exposure enhanced glutamate transmission in the VTA through GCPII upregulation, leading to downstream enhancement of mesolimbic dopamine release that facilitated METH reward (Cai and Tong, 2022). In the presence of TRLZ, upregulation of GCPII in the VTA by METH was prevented, which (1) dampened the increase in extracellular glutamate in the VTA that occurs during repeated METH exposure and (2) normalized NAAG levels, leading to restoration of mGlu3 receptor tone that reduced neuronal glutamate release in the VTA (Manzoni and Williams, 1999). If so, the efficacy of TRLZ against METH CPP could be explained by a reduction in METH-evoked glutamate transmission in the VTA that led to reduced mesolimbic dopamine output. In support of a VTA-based glutamatergic mechanism for TRLZ, amphetamine produces a delayed, sustained increase in glutamate efflux in the VTA of drug-naive rats, as well as in rats treated for 5 d with amphetamine, that may contribute to the rewarding effects of METH (Giorgetti et al., 2002; Xue et al.,1996; Yamamoto and Zhu, 1998). Additionally, a VTA excitatory microcircuitry in which activation of local glutamate neurons in the VTA promotes glutamate receptor-mediated CPP may have been inhibited in the presence of TRLZ (Wang et al., 2015). TRLZ efficacy may have also involved PKC inhibition since RLZ is a direct inhibitor of PKC and a PKC inhibitor (NPC 15437) blocks place conditioning produced by amphetamine in rats (Noh et al., 2000; Aujla and Beninger, 2003).

It is worth noting that TRLZ by itself caused a non-significant increase in GCPII protein levels in the VTA compared to drug-naïve rats. The reason for this paradoxical effect (i.e., TRLZ normalizing the METH-evoked increase in GCPII levels while non-significantly increasing basal GCPII levels) is unclear but suggests that the effects of TRLZ on GCPII are not entirely the same in the absence and presence of METH. Under METH-naïve conditions, one possibility is that TRLZ caused changes in basal extracellular glutamate that led to downstream effects on GCPII levels. In this scenario, TRLZ may have reduced neuronal glutamate release and enhanced cellular glutamate uptake (Cheah et al., 2010; Machado-Vieira et al, 2009; Pittenger et al, 2008; Brothers et al., 2013; Carbone et al., 2012; Liu et al., 2011), leading to a reduction in basal extracellular glutamate that caused a compensatory upregulation of GCPII protein to enhance glutamate synthesis and mitigate deficits in basal glutamate levels. Although extracellular glutamate levels during TRLZ exposure were not assessed in our study, prior work shows that RLZ causes robust, prolonged reductions in basal glutamate release in the spinal cord of rats (Coderre et al., 2007). In contrast, under conditions of chronic METH exposure, where GCPII mRNA and protein levels were both elevated, TRLZ may have normalized GCPII levels through one or more of the mechanisms discussed previously, thereby masking the non-significant increase in GCPII protein levels caused by TRLZ under basal conditions. In addition, direct effects of METH or TRLZ on the activity of the GCPII enzyme cannot be excluded, and future studies will address this intriguing possibility.

TRLZ also accelerated extinction of an existing METH place preference. Our results are consistent with evidence that that RLZ enhances extinction of cocaine self-administration in rats (Sepulveda-Orengo et al., 2018), where active lever responding is extinguished within the first 3 d of TRLZ treatment. In our CPP experiments, the effect of TRLZ was more delayed and occurred after 5 d of administration but not after 2 d. In later extinction sessions corresponding to 8–11 d of TRLZ treatment, METH CPP was still reduced in the TRLZ versus vehicle group. However, the difference between the groups did not reach statistical significance, perhaps due to variability associated with the extended CPP paradigm or the development of tolerance to TRLZ. Identifying the neurochemical mechanisms by which TRLZ facilitated METH extinction is challenging because we analyzed glutamate biomarkers in brain regions immediately following repeated METH and TRLZ exposure, but not during abstinence. One potential mechanism involves GLT-1 transporters, which are downregulated in the NAC during cocaine (Knackstedt and Kalivas, 2009; Sari et al., 2009) or MDPV abstinence (Gregg et al., 2015). Evidence linking GLT-1 transporter deficits to METH abstinence is mixed, with one study detecting reduced GLT-1 protein levels in the NAC 2–3 d following chronic non-contingent METH exposure (Althobaiti et al., 2016) and another study demonstrating no significant changes in GLT-1 levels during abstinence from METH self-administration (Siemsen et al., 2019). Although prior work shows that RLZ treatment enhances GLT-1 expression (Brothers et al., 2013; Carbone et al., 2012; Liu et al., 2011), the PFC was the only region in which we detected a significant elevation of GLT-1 protein levels following a 7-d TRLZ regimen. Nonetheless, over the 11 d of METH abstinence in our extinction experiments, normalization of GLT-1 dysregulation by TRLZ may have occurred and disrupted the maintenance of METH place preference. It is unlikely that cognitive deficits resulting from inhibition of glutamate transmission contributed to TRLZ efficacy because RLZ improves memory performance in aged rats through anti-oxidative and anti-cholinersterase effects (Mokhtari et al., 2017). Considering that RLZ increases GABA transmission (He et al., 2002), and activation of GABA interneurons in the VTA enhances METH extinction through inhibition of VTA dopamine neurons and mesolimbic dopamine release (Chen and Chen, 2015), a role for GABA systems in TRLZ efficacy is also possible.

TRLZ reduced locomotor activation produced by acute METH in our experiments, which is consistent with RLZ reducing METH-evoked hyperlocomotion in mice (Itzhak and Martin, 2000; Lourenço Da Silva et al., 2003). Locomotor activity peaked 20–40 min after METH exposure (Fig. 2) whereas increased VTA glutamate efflux is not detected until 2–3 h after amphetamine exposure (Wolf and Xue, 1996). This temporal divergence does not support a major role for VTA glutamate efflux in acute METH locomotor activation, and suggests TRLZ acted through other mechanisms to reduce METH hyperlocomotion. One possibility is direct or indirect inhibition of dopamine transmission in the NAC. CTX, which like TRLZ enhances glutamate reuptake, reduces hyperlocomotion induced by acute amphetamine or cocaine administration in rats through mechanisms that may involve inhibition of both glutamate and dopamine transmission (Rasmussen et al., 2011; Barr et al., 2015). Another possibility is that TRLZ interacted directly with a cellular site of METH action, such as trace amine associated receptor 1 (TAAR1), which is activated by METH and potentially contributes to METH-induced behaviors (Jing et al., 2015). The TAAR 1 agonist RO5263397 reduces METH behavioral sensitization and self-administration in rats (Jing et al., 2015) and shares structural similarities with TRLZ, suggesting the potential for overlapping mechanisms of action. Future studies will investigate effects of TRLZ on METH sites of action such as TAAR1 and VMAT2.

In summary, TRLZ disrupted three different phases of METH place preference in rats and normalized METH-induced changes in mesolimbic GCPII levels. The identification of GCPII as a potential site of action of TRLZ points toward determining the nature of TRLZ interactions with GCPII, which is an attractive therapeutic target for psychostimulant addiction but lacks FDA-approved inhibitors. The pharmacodynamic diversity (i.e., glutamate release inhibitor and glutamate reuptake enhancer) of TRLZ, coupled with the pharmacokinetic and metabolic advantages of TRLZ versus RLZ, point toward studying it further for METH use disorder and determining if efficacy extends to other addictive drugs. One limitation in our study was the use of only males. Because the preclinical efficacy of glutamatergic pharmacotherapies to reduce psychostimulant reward is sex- and hormone-sensitive (Bechard et al., 2018; Becker and Hu, 2008), future studies will determine if TRLZ displays similar efficacy against METH behaviors in females, and if it is estrous-cycle dependent. Another limitation in our study was the use of a single dose of METH that produced robust locomotor activation and CPP. It will be important in future studies to test the effects of different doses of TRLZ against a range of METH doses to obtain full-dose effect data and determine how TRLZ impacts the METH dose-effect curve. Also, it will be important to expand on the findings presented here by testing effects of TRLZ on METH intake and seeking in rat self self-administration assays that better model aspects of METH use disorder in humans.

Highlights.

  • Troriluzole (TRLZ) is a riluzole prodrug and glutamate release inhibitor/transport activator.

  • TRLZ was tested for the first time in animal models of addiction.

  • TRLZ reduced development, expression and maintenance of methamphetamine place preference.

  • Methamphetamine enhanced VTA levels of glutamate carboxypeptidase II (GCPII).

  • TRLZ normalized methamphetamine-evoked increase in mesolimbic GCPII levels.

Acknowledgements

This work was supported by the following grants from the National Institute on Drug Abuse: R01 DA051205 and P30 DA013429. We acknowledge the support and contributions from Biohaven Pharmaceuticals who is developing troriluzole for a variety of therapeutic indications. We would also like to acknowledge Sean Peng, and Touchstone Biosciences, for conducting pharmacokinetic studies assessing brain and plasma levels of methamphetamine.

We acknowledge the support and helpful contributions from Biohaven Pharmaceuticals who is developing troriluzole for a variety of therapeutic indications. We would also like to acknowledge Sean Peng, and Touchstone Biosciences, for conducting pharmacokinetic studies assessing brain and plasma levels of methamphetamine.

Footnotes

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Conflict of Interest

Authors declare that they have no conflicts of interest.

Disclosures

Allen Reitz is a shareholder of Biohaven Pharmaceuticals (BHVN).

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