healthcare Article Potential Benefits of N-Acetylcysteine in Preventing Pregabalin-Induced Seeking-Like Behavior Atiah H. Almalki 1,2 , Hashem O. Alsaab 2,3, Walaa F. Alsanie 2,4, Ahmed Gaber 2,5 , Turki Alkhalifa 6, Ahmad Almalki 6, Omar Alzahrani 2, Ana Maria Gregio Hardy 7, Qasim Alhadidi 8, Zahoor A. Shah 9 and Yusuf S. Althobaiti 2,6,10,* 1 Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia; [email protected] 2 Addiction and Neuroscience Research Unit, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia; [email protected] (H.O.A.); [email protected] (W.F.A.); [email protected] (A.G.); [email protected] (O.A.) 3 Department of Pharmaceutics and Pharmaceutical Technology, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia 4 Department of Clinical Laboratories Sciences, The Faculty of Applied Medical Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia 5 Department of Biology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia 6 Ministry of Interior, General Directorate of Narcotics Control, General Administration for Precursors and Laboratories, Riyadh 11564, Saudi Arabia; [email protected] (T.A.); [email protected] (A.A.) 7 Department of Physiology and Pharmacology, College of Medicine and Life Sciences, University of Toledo, Toledo, OH 43614, USA; [email protected] 8 Citation: Almalki, A.H.; Alsaab, Department of Anesthesiology, Perioperative and Pain Medicine, Stanford Medical School, H.O.; Alsanie, W.F.; Gaber, A.; Stanford University, Stanford, CA 94305, USA; [email protected] 9 Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, Alkhalifa, T.; Almalki, A.; Alzahrani, University of Toledo, Toledo, OH 43614, USA; [email protected] O.; Hardy, A.M.G.; Alhadidi, Q.; 10 Department of Pharmacology and Toxicology, College of Pharmacy, Taif University, P.O. Box 11099, Shah, Z.A.; et al. Potential Benefits of Taif 21944, Saudi Arabia N-Acetylcysteine in Preventing * Correspondence: [email protected]; Tel.: +966-545736200 Pregabalin-Induced Seeking-Like Behavior. Healthcare 2021, 9, 376. Abstract: Substance-use disorder is globally prevalent and responsible for numerous social and https://doi.org/10.3390/ medical problems. Pregabalin (Lyrica), typically used to treat diabetic neuropathy, has recently healthcare9040376 emerged as a drug of abuse. Drug abuse is associated with several neuronal changes, including the downregulation of glutamate transporters such as glutamate transporter 1 and cystine/glutamate Academic Editor: Farhad Kamali antiporter. We investigated the effects of N-acetylcysteine, a glutamate transporter 1 and xCT Received: 18 February 2021 upregulator, on pregabalin addiction using a conditioned place preference paradigm. Pregabalin Accepted: 18 March 2021 (60 mg/kg) was found to induce conditioned place preference when compared to a vehicle. A Published: 29 March 2021 100 mg/kg dose of N-acetylcysteine was found to block pregabalin-seeking behaviors. These results support previous findings showing that glutamate transporters play an important role in pregabalin- Publisher’s Note: MDPI stays neutral induced seeking behaviors. N-acetylcysteine may represent a beneficial agent in preventing the abuse with regard to jurisdictional claims in potential of pregabalin. published maps and institutional affil- iations. Keywords: NAC; pregabalin; addiction; GLT-1; xCT; CPP Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. Pregabalin, a gamma-aminobutyric acid derivative, reduces the release of glutamate This article is an open access article by binding to a voltage-gated calcium channel subunit [1]. Pregabalin is predominantly distributed under the terms and used to treat neuropathy in diabetic patients, anxiety disorder, and partial epilepsy [2,3]. conditions of the Creative Commons Recently, pregabalin emerged as an illicit drug widely abused throughout the world [4–6], Attribution (CC BY) license (https:// especially among Saudi Arabian youth [7,8]. This may be due to the lack of regulations creativecommons.org/licenses/by/ and knowledge about pregabalin’s addictive properties. High doses and extended use of 4.0/). Healthcare 2021, 9, 376. https://doi.org/10.3390/healthcare9040376 https://www.mdpi.com/journal/healthcare Healthcare 2021, 9, 376 2 of 11 pregabalin were reported to be associated with euphoria and withdrawal symptoms [9–12]. Althobaiti et al. showed that pregabalin induced conditioned place preference (CPP) and might affect the glutamatergic mechanism when used at doses of 60–90 mg/kg [13]. It is essential that the mechanisms by which pregabalin induces rewarding effects are further understood. Glutamate transporters are significantly affected by drug abuse and addiction [14], and glutamate transporter 1 (GLT-1) is accountable for 90% of glutamate uptake [15]. GLT-1 expression is downregulated in individuals with a history of drug abuse and addiction, and this can provoke alcohol, cocaine, and methamphetamine addiction [16–18]. Conversely, GLT-1 upregulation attenuates drug-seeking behavior [17]. Drug abuse also downregulates the cystine/glutamate antiporter (xCT), which is responsible for glutamate and cysteine exchange in glial cells. Furthermore, GLT-1 and xCT downregulation increases glutamate concentration, which then causes the overactivation of postsynaptic receptors such as mGlu-1 [19] and mGlu-5 [20,21]. In contrast, blocking these receptors in the nucleus accumbens (NAcc), the reward center of the brain [22], is associated with a reduction in both drug-seeking behavior and addiction relapse [23–26]. N-acetylcysteine (NAC), an acetylated form of cysteine and a glutathione (GSH) precursor, is a source of scientific enthusiasm due to its efficacy in drug-addiction treat- ment [27,28]. In humans, NAC is known for its role in mitigating cocaine and nicotine dependence [29–31]. This drug was shown to normalize glutamatergic functions and reduce drug-seeking behavior [32,33]. Likewise, NAC was revealed to diminish heroin drug-seeking effects in rats [34]. NAC, therefore, emerged as a potential candidate for the treatment of nicotine, heroin, and cocaine drug-seeking effects [34–36]. NAC can upregu- late GLT-1 and xCT expression, which in turn regulate glutamate homeostasis by increasing the exchanging amount of glutamate by glial cells [27,37,38]. Therefore, in this study, we assessed the role of NAC on pregabalin induced-seeking behavior using the conditioned place preference (CPP) paradigm. The CPP paradigm is a model that was extensively used for studying the preference behavior of animals [39–42]. This model was used to study the effects of several enforcers such as drugs and food. The CPP strategy is characterized by two periods of investigation and two compartments, whereby a reinforcing stimulus during the first period is eval- uated during a second (test) period where animals are allowed to choose between the enforcer-paired and nonpaired compartments [41–43]. Even though the CPP experiment is a widely used strategy, there are many variables that may affect the results, such as flooring, apparatus design, experimental design, and cues [39,43]. Therefore, in this study, an unbiased CPP where animals showing strong initial preference were excluded from the study to examine the role of NAC on pregabalin induced-seeking like behavior. 2. Materials and Methods 2.1. Animals Thirty-six male mice (BALB/c) were purchased from King Fahd Medical Research Center (Jeddah, Saudi Arabia) (weight, 25–30 g) at the beginning of the study. The mice were housed in groups at optimal temperature (21 ◦C) and humidity (30%) levels, with ad libitum access to water and food; they were allowed to acclimate for 1 week before the experiments. All experiments were conducted during the light cycle of 8:00–10:00 a.m. 2.2. Drugs Both pregabalin (kindly provided by the research and development department at Jamjoom Pharma, Jeddah, Saudi Arabia) and NAC (Sigma-Aldrich, St. Louis, MO, USA) were reformulated in normal saline solution (0.9% NaCl) before use. 2.3. Experimental Design The experimental scheme is shown in Figure1. Mice were randomly assigned to 1 of 4 groups treated with different treatment regimens for 8 consecutive days throughout Healthcare 2021, 9, x FOR PEER REVIEW 3 of 11 Healthcare 2021, 9, 376 3 of 11 2.3. Experimental Design The experimental scheme is shown in Figure 1. Mice were randomly assigned to 1 of 4 groups treated with different treatment regimens for 8 consecutive days throughout the theconditioning conditioning period. period. Mice Mice in Group in Group 1 (V-V 1 (V-V group, group, n =n 10)= 10)were were given given a vehicle a vehicle (saline, (saline, 10 10mL/kg), mL/kg), followed followed by another by another injection injection of the of vehicle the vehicle after 30 after min. 30 Group min. Group2 (NAC-V 2 (NAC-V group, group,n = 9) receivedn = 9) received intraperitoneal intraperitoneal (i.p.) injections (i.p.) injections of NAC of (pretreatment, NAC (pretreatment, 100 mg/kg, 100 mg/kg,i.p. × 4) × i.p.followed4) followed by vehicle by injections vehicle injections 30 min after. 30 min Group after. 3 Group (V-Preg 3 (V-Preggroup, n group, = 9) receivedn = 9) received vehicle × vehiclepretreatment pretreatment 30 min before 30 min pregabalin before pregabalin injection injection (60 mg/kg, (60 i.p. mg/kg, × 4). This i.p. dose4). of This pregab- dose ofalin pregabalin was selected was because selected it becauseinduced itplace induced preference place preferencein a previous in astudy previous [13]. studyMoreover, [13]. Moreover, pregabalin at low doses (30 mg/kg) or less is less effective than high doses pregabalin at low doses (30 mg/kg) or less is less effective than high doses are, which were are, which were reported in numerous case studies in inducing rewarding effects [44–48]. reported in numerous case studies in inducing rewarding effects [44–48]. Lastly, Group 4 Lastly, Group 4 (NAC-Preg group, n = 8) received NAC pretreatment (100 mg/kg, i.p.