Pregabalin: Potential for Addiction and a Possible Glutamatergic Mechanism

Drug addiction remains a prevalent and fatal disease worldwide that carries significant social and economic impacts. Recent reports suggest illicit pregabalin (Lyrica) use may be increasing among youth, however the addictive potential of pregabalin has not been well established. Drug seeking behavior and chronic drug use are associated with deficits in glutamate clearance and activation of postsynaptic glutamatergic receptors. In the current study, we investigated the abuse potential of pregabalin using conditioned place preference (CPP) paradigm. Different doses of pregabalin (30, 60, 90, and 120 mg/kg) were used to assess the seeking behavior in mice. Glutamate homeostasis is maintained by glutamate transporter type-1 (GLT-1), which plays a vital role in clearing the released glutamate from synapses and drug seeking behavior. Therefore, we investigated the role of glutamate in pregabalin-seeking behavior with ceftriaxone (CEF), a potent GLT-1 upregulator. Mice treated with pregabalin 60 and 90 mg/kg doses demonstrated drug seeking-like behavior, which was significantly blocked by CEF pretreatment. These results suggest that pregabalin-induced CPP was successfully modulated by CEF which could serve as a lead compound for developing treatment for pregabalin abuse.

Drugs. Pregabalin was generously provided by Jamjoom Pharmaceuticals (Jeddah, KSA). Ceftriaxone was gifted by King Abdulaziz Hospital at Taif. All the drugs used in this study were reconstituted in sterile saline solution (0.9% NaCl).
Experimental design. The overall experimental design is presented in Fig. 1A.
Experiment 1: Mice were randomly assigned into five groups; Group 1: Control group (n = 8), mice in this group were treated with vehicle for 8 days. Group 2: Preg-30 group (n = 8), mice were injected with pregabalin (30 mg/kg, i.p.x 4) and vehicle for eight days during the acquisition phase. Group 3: Preg-60 group (n = 8), mice were injected with pregabalin (60 mg/kg, i.p.x 4) and vehicle for eight days during the acquisition phase. Group 4: Preg-90 group (n = 8), mice were injected with pregabalin (90 mg/kg, i.p.x 4) and vehicle for eight days during the acquisition phase. Group 5: Preg-120 group (n = 8), mice were injected with pregabalin (120 mg/kg, i.p.x 4) and vehicle for eight days during the acquisition phase. Mice were then examined for place preference following completing the conditioning training. Experiment 2: Mice were randomly assigned into four groups; Group 1: C-C group (n = 8) Mice in this group were treated with vehicle in home cage as a 30 minutes pretreatment and vehicle for eight days during the acquisition phase. Group 2: CEF-C group (n = 8) Mice were injected with CEF (200 mg/kg, i.p.) in home cage as a 30 minutes pretreatment and then vehicle for eight days during the acquisition phase. Group 3: C-Preg group www.nature.com/scientificreports www.nature.com/scientificreports/ (n = 8) Mice were treated with vehicle in home cage as a 30 minutes pretreatment and then Pregabalin (60 mg/ kg, i.p.x4) as well as vehicle for eight days during the acquisition phase. The dose of Pregabalin which induced CPP in the previous experiment was used in this study. Group 4: CEF-Preg group (n = 8) Mice were injected with CEF (200 mg/kg, i.p.) in home cage as a 30 minutes pretreatment and then Pregabalin (60 mg/kg, i.p.x4) as well as vehicle for eight days during the acquisition phase. Mice were then examined for place preference following completing the conditioning training.
Conditioned place preference paradigm. A custom made acrylic CPP apparatus (Fig. 1B) was used in this study as described in our previous work 46 . Briefly, this apparatus consists of two equal-sized conditioning chambers (35 cm × 35 cm × 50 cm), and one start box (10 cm × 15 cm × 10 cm) located outside of the CPP apparatus. The two conditioning chambers are distinguished by both tactile and visual cues. The interior walls of the first chamber are white in color with horizontal black stripes and textured walls (chamber 1). The interior walls of the other chamber (chamber 2) are black in color with vertical white stripes and smooth walls. The floor of chamber 1 is perforated with round holes. The floor of the other chamber is perforated with rectangle holes.
Habituation phase: The preconditioning day is considered as day one. On days one, two, and three each mouse was placed in the start box with door closed for 3 minutes. Then, the door was opened to let the mouse explore the conditioning chambers for 30 minutes. On day three, the animal exploration in both conditioning chambers was recorded by a digital camera fixed on the top of the apparatus. The time spent in both chambers was calculated using ANY-maze software (Stoelting, USA).
Conditioning phase: An un-biased CPP design has been used. Therefore, in each treatment group, half of the animals were randomly assigned to receive pregabalin and were placed in chamber 1, while the other half received this drug and were placed in chamber 2 during the conditioning phase (days four to eleven). Each mouse received intraperitoneal injection of specific treatment and were then located in the corresponding chamber with the door closed for 30 minutes session. On the following day, each mouse received vehicle and was located in the other chamber with the door closed for 30 minutes. The process was repeated until the completion of the eight conditioning sessions.
On day twelve, each mouse had free access to both chambers for 30 minutes. The time spent by the mice in both chambers was documented using digital camera (post-conditioning test) and counted by ANY-maze video tracking system. Statistical analysis. Two-way repeated measure ANOVA, (Phase × Treatment) was used to analyze time spent, at two different timepoints (pre-test and post-test), in the conditioning chambers in response to the selected dose of pregabalin or saline. This analysis was selected based on previous published work [46][47][48][49] . When significant main interactions or effects were found, Newman-Keuls multiple comparisons were performed. All data were statistically analyzed by GraphPad Prism, using a 0.05 level of significance.   www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
In the present study, we demonstrate for the first time, using a mice model of drug addiction, that pregabalin can induce CPP. These findings are in contrast to previous reports, however, the maximum tested dose of pregabalin was 30 mg/kg, which did not induce rewarding effects and did not change place preference 15,16 . Consistent with the previous findings from Andrews, et al., the dose of 30 mg/kg did not change place preference in our study. However, when the dose was increased to 60 mg/kg, a significant place preference was induced by pregabalin. This suggests a dose dependent effect of pregabalin's rewarding effects. Interestingly, this effect is supported by previous controlled clinical studies [17][18][19][20] showing that pregabalin can cause euphoric effects as a side effect in participants of these studies.
The drug seeking effects found in several drugs of abuse have been consistently reported to be mediated by glutamatergic mechanism. GLT-1, an astrocyte-specific excitatory amino acid transporter, which is responsible for glutamate homeostasis in the brain 37 . It had been previously demonstrated that downregulation of GLT-1  expression in the NAc was associated with continuous exposure to addicting drugs [50][51][52] . Interestingly, GLT-1 expression was found to be downregulated instantly in cocaine self-administration model 53 . Of note, the glutamatergic transmission is amplified as a result of increase in glutamate concentrations and decrease in glutamate uptake in the synapses 54 . Additionally, it has been observed that glutamate receptors such as mGlu-5 and N-Methyl-D-aspartate could be potentiated and activated by the spillover of glutamate which enhances drug seeking behavior 54 . In the present study, our results suggest that pregabalin at higher doses [60 mg, and 90 mg] may induce addiction partly by downregulating GLT-1 expression and thereby decreasing glutamate uptake at the synaptic cleft.
Treatment with CEF has been reported to prevent drug seeking behavior caused by, in part, decreased GLT-1 expression in methamphetamine, cocaine, ethanol, nicotine, and heroin dependence 26,[41][42][43][44][45] with the drug seeking associated with glutamate spillover secondary to GLT-1 downregulation 25,[55][56][57] . Additionally, the normalization of GLT-1 expression, by CEF treatment, was associated with a decrease in drug-seeking behavior 58,59 . Therefore, pregabalin seeking at the addictive doses of [60 mg, and 90 mg] might be mediated by altering GLT-1 expression as the drug seeking effects of pregabalin was eliminated by CEF pretreatment in the present study. CEF has been demonstrated to have neuroprotective efficacy in many neurological disorders 60,61 and can offer neuroprotective effects in drug addiction associated with glutamate excitotoxicity 60,62,63 . CEF can freely pass the blood brain barrier and enter the central nervous system to up-regulate GLT-1 making it an attractive potential therapeutic for future clinical use in antagonizing pregabalin-induced drug-seeking behavior 60,[62][63][64] .
One limitation of the present study is that we did not demonstrate a mechanisms of action for CEF in antagonism of pregabalin-induced drug-seeking behavior. GLT-1 plays a central role in inflammatory mechanisms in the brain, which has previously been demonstrated to be associated with drug addition [65][66][67] . It has been previously reported that central administration of some neurotoxicants causes significant impairement in motor functions, increased neuroinflammation and increased drug addiction [68][69][70] . Post-treatment with CEF (200 mg/ kg) significantly antagonized motor impairment, attenuated lipid peroxidation, restored endogenous antioxidant enzymes glutathione peroxidase and catalase, and decreased drug addiction 71,72 . Taken together, CEF-mediated antagonism of pregabalin-induced drug seeking like effects could promote restoration of glutamate homeostasis, and in this way modulate drug-seeking behavior. CEF could serve as a lead compound for developing treatment for pregabalin abuse as other pharmacological effects of this antibiotic could not be excluded.
Future studies are needed to investigate a mechanistic role for neuroinflamation in pregabalin abuse, as well as sex-and age-related mechanisms in pregabalin-induced neurochemical changes. Published: xx xx xxxx