Methoxetamine, a Ketamine Derivative, Produced Conditioned Place Preference and Was Self-Administered by Rats: Evidence of Its Abuse Potential

Pharmacology, Biochemistry and Behavior 133 (2015) 31–36

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Pharmacology, Biochemistry and Behavior

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Methoxetamine, a ketamine derivative, produced conditioned place preference and was self-administered by rats: Evidence of its abuse potential

Chrislean Jun Botanas a,1, June Bryan de la Peña a,1, Irene Joy dela Peña a, Reinholdgher Tampus a,RobinYoona, Hee Jin Kim a, Yong Sup Lee b,ChoonGonJangc, Jae Hoon Cheong a,⁎ a Uimyung Research Institute for Neuroscience, School of Pharmacy, Sahmyook University, 26-21 Kongreung-2-dong, Hwarangro-815 Nowon-gu, Seoul 139-742, South Korea b Laboratory of Medicinal Chemistry, School of Pharmacy, Kyunghee University, Seoul 130-701, South Korea c Department of Pharmacology, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, South Korea article info abstract

Article history: Methoxetamine (MXE) is an N-methyl-D-aspartate (NMDA) receptor antagonist that is chemically and Received 28 January 2015 pharmacologically similar to ketamine. Recently, there have been many reports regarding its use/misuse in Received in revised form 4 March 2015 humans which have resulted in serious or even fatal outcomes. Despite these reports, MXE is not controlled or Accepted 10 March 2015 regulated in many countries which may be partly due to the lack of scientiﬁc evidence regarding its abuse Available online 17 March 2015 potential. Thus, in the present study we evaluated the abuse potential (rewarding and reinforcing effects) of MXE through the conditioned place preference (CPP) and self-administration (SA) tests in Sprague–Dawley Keywords: Methoxetamine rats. In addition, locomotor activity during the conditioning phase of the CPP was also analyzed. Ketamine was Ketamine used as a reference drug. MXE (2.5 and 5 mg/kg) induced signiﬁcant CPP in rats, an effect comparable to that Conditioned place preference of ketamine (5 mg/kg). Interestingly, MXE did not produce any locomotor alterations while ketamine decreased Self-administration the locomotor activity of rats. In the SA test, rats showed modest self-administration of MXE (0.25, 0.5, Designer drugs 1.0 mg/kg/infusion), while ketamine (0.5 mg/kg/infusion) was robustly self-administered. These results Addiction demonstrate that MXE, similar to ketamine, has rewarding and reinforcing effects in rats. The present study strongly suggests that MXE has a potential for human abuse. In addition, the discrepant effects of MXE and ketamine on locomotor activity and rate of self-administration propose that the psychopharmacological effects of these drugs may diverge in some aspects. More importantly, this study advocates the careful monitoring and prompt regulation of MXE and its related substances. © 2015 Elsevier Inc. All rights reserved.

1. Introduction MXE in humans, which resulted in serious or even fatal outcomes (Zawilska, 2014). Accordingly, MXE was included in the list of new Methoxetamine (MXE) (2-(3-methoxyphenyl)-2-(ethylamino) psychoactive substances of the European Monitoring Centre for Drugs cyclohexane) is a new, synthetic, psychoactive drug derived from and Drug Addiction (EMCDDA), categorized as a ketamine derivative ketamine (Corazza et al., 2012; Meyer et al., 2013). Similar to ketamine (EMCDDA, 2014). It is believed that MXE is being used as a ketamine and phencyclidine, it is pharmacologically classiﬁed as an N-methyl-D- substitute, owing to its ability to produce comparable hallucinogenic aspartate (NMDA) receptor antagonist (Corazza et al., 2013; Roth and dissociative effects (EMCDDA, 2014; Kjellgren and Jonsson, 2013). et al., 2013). Initially, MXE was designed in part to prevent the Drug substitution is a common practice among drug users/abusers. urotoxicity associated with ketamine and to be tested as an antidepres- An important factor behind this practice is drug availability (de la sant (EMCDDA, 2014; Meyer et al., 2013). However, since its debut on Pena et al., 2013; EMCDDA, 2009). Obtaining a regulated psychoactive the internet in 2010, it has become a popular recreational drug especial- drug can be arduous; thus, an addicted individual would seek for alter- ly among adolescents (Morris and Wallach, 2014). Indeed, there native ways to obtain their “high” (de la Peña et al., 2014). An emerging has been an increase in the number of reports regarding the abuse of trend is the slight modiﬁcation of the molecular structure of a controlled drug — making a “new” drug that retains/mimics the psychoactive properties of the original drug, but circumvents any existing drug law (Corazza et al., 2012; Fattore and Fratta, 2011). These drugs, aptly called ⁎ Corresponding author. Tel.: +82 2 2339 1605; fax: +82 2 2339 1619. “designer drugs” or “legal highs”, are then sold on the internet or black E-mail address: [email protected] (J.H. Cheong). 1 These authors have equal contribution in doing the experiments and writing the markets (Morris and Wallach, 2014). This practice might also be the manuscript. case for ketamine and its analog, MXE.

Despite reports of human misuse, MXE is still not a controlled drug were injected intraperitoneally with MXE (1.25, 2.5 or 5 mg/kg) or ke- in many countries (EMCDDA, 2014). This may be related to the lack of tamine (5 mg/kg) and confined to their non-preferred compartment. supporting scientific evidence regarding the abuse potential of this On alternate days, they received saline and were placed on their drug. Abuse potential assessment is an integral part in the screening of preferred compartment. These treatments were repeated for three cycles psychoactive drugs. Thus, the goal of the present study was to character- (6 days). The control group received saline every day. Locomotor activity ize the abuse potential of MXE through animal models of drug addiction. was also recorded during this phase. The post-conditioning followed Assessment of abuse potential in animals is advantageous because it by- where the guillotine door was opened and, as in the pre-conditioning passes the ethical, methodological, and economic constraints associated phase, drug-free rats were allowed access to both compartments. with human studies. Two of the widely used animal models of drug addiction were employed: the conditioned place preference (CPP) and 2.4. Self-administration test the self-administration (SA) test. The CPP test evaluates the hedonic value (rewarding or aversive) of a drug, while the SA test measures the 2.4.1. Apparatus motivational/reinforcing effects of the drug (Shippenberg and Koob, SA tests were performed in standard operant chambers (Coulbourn 2002). Additionally, locomotor activity (during the conditioning phase Instruments, Allentown, Pennsylvania, USA) placed inside sound- of the CPP) was analyzed, based on the findings that euphorigenic effects attenuating boxes with ventilation fans, to further mask external activate the same neural processes as locomotor activation (Valjentetal., noise. Each operant chamber has a food pellet dispenser, two 4.5 cm 2010). Ketamine was used as a reference drug. wide response levers (left and right), a stimulus light located 6 cm above the left lever, and a centrally positioned house light (2.5 W, 2. Materials and methods 24 V) at the top of the chamber. A downward pressure (approximately 25 g) on the levers would result in a programmed consequence. Located 2.1. Subjects beside the operant chamber was a motor-driven syringe pump that de- livered solutions at a rate of 0.01 ml/s. Solutions flowed through Teflon Male, Sprague–Dawley rats (6 weeks) obtained from Hanlim Animal tubing connected to a swivel, which was mounted on a counterbalanced Laboratory (Korea) were the subjects of this study. The rats were arm at the top of the chamber that allowed free movement of the ani- housed in groups of 4 per cage (CPP) or individually (SA) in an animal mals. The tubing was connected to the animal's intravenous catheter room with controlled temperature (22 ± 2 °C) and humidity (55 ± system. The Graphic State Notation software (Coulbourn Instruments) 5%) under a 12/12-h light/dark cycle (0700 h–1900 h). The animals controlled experimental parameters and collected data. were acclimatized to the laboratory setting for five days, before the commencement of any experiments. Water and food were freely 2.4.2. Procedure available, except during lever training and the SA sessions. All tests After acclimatization, food access was limited until the rats were were performed in accordance with the Principles of Laboratory Animal reduced to 85% of their free-feeding body weight. Then, they were Care (NIH Publication No. 85-23, revised 1985) and the Animal Care and trained to press a lever (30 min/day for 3 days) for a contingent food Use of Guidelines of Sahmyook University, Korea. pellet reward on a continuous schedule of reinforcement. Only rats that earned at least 80 pellets on the last session of training were 2.2. Drugs prepared for surgery. Detailed description of the surgical and post- surgical procedures was outlined in our previous studies (de la Peña Methoxetamine in crystallized white powder was provided by the et al., 2012). After recovery, the rats were subjected to a two-hour Laboratory of Medicinal Chemistry of Kyunghee University (Seoul, daily SA session under a fixed-ratio (FR) 1 schedule for 7 days. During Korea). Ketamine was purchased from Bayer Animal Health Co. the SA sessions, both levers were present and a press on the left lever (Suwon, Korea). All drugs were diluted in normal saline (0.9% w/v of (active lever) would result in an infusion of 0.1 ml of MXE (0.25, 0.5, NaCl) and administered either intraperitoneally (CPP) or intravenously or 1.0 mg/kg/infusion), ketamine (0.5 mg/kg/infusion), or saline. Simul- (SA). The dosages used in the present study were based on previous CPP taneously, the house light was switched off, while the stimulus light was and SA studies with NMDA receptor antagonists (de la Peña et al., 2012; illuminated which remained lit for another 20 s after the end of the De Luca and Badiani, 2011; Trujillo et al., 2011). infusion (time-out period). Lever presses during time-out periods were recorded but did not have any corresponding effects. As a control 2.3. Conditioned place preference test for general activity, presses on the right lever (inactive lever) were recorded but not reinforced. A significant difference between active and 2.3.1. Apparatus inactive lever responses was considered to reflect self-administration. The CPP apparatus was a two-compartment, polyvinylchloride, To prevent possible intoxication, the rats were only allowed 30 drug infu- boxes measuring 47 × 47 × 47 cm. Each compartment had unique sions per session, although lever presses were still recorded until the end visual and tactile cues: one section had black walls with smooth black of the session. A day before and on the last day of the SA test, catheters floor while the other section had white painted dotted walls with were injected with 0.1 ml of thiopental sodium (10 mg/kg) to assess rough black floor. A guillotine door separated the compartments during catheter patency. The rats that did not lose muscle tone within 3–5s the conditioning phase. A software package (Ethovision, Noldus IT BV, were excluded from the experiment. Wageningen, Netherlands) was used to record animal behaviors. 2.5. Data analysis 2.3.2. Procedure The CPP test was performed as previously described (de la Peña All data were presented as means and standard error of the mean et al., 2012). Each test was composed of three phases: (1) habituation (SEM). CPP results were expressed as the difference in time spent in and preconditioning, (2) conditioning, and (3) post-conditioning. For the drug-paired compartment during the pre- and post-conditioning the first two days, the rats were allowed to access both compartments phases. One-way analysis of variance (ANOVA) was used to determine for 15 min, once a day (habituation). On the third day, preconditioning the variation between groups, followed by a Dunnett's posttest to com- followed where the time spent (seconds) on each compartment was pare each group to the control group. Locomotor activity was presented measured (Ethovision), to determine the preferred and non-preferred as the distance moved (cm) and movement duration (s) during the con- compartment of each rat. Then, the guillotine door was closed in ditioning phase of CPP. Two-way ANOVA was employed to determine preparation for the conditioning phase. During this period, the rats the effects of drug, day, or interaction between these factors, followed C.J. Botanas et al. / Pharmacology, Biochemistry and Behavior 133 (2015) 31–36 33 by Bonferroni's post hoc test to compare each group to the control doses of MXE [1.25 (q = 0.2039, p N 0.05), 2.50 (q = 1.031, p N 0.05) group. In the SA test (Fig. 3A), two-way ANOVA was used to determine and 5 mg/kg (q = 0.8137, p N 0.05)] were not statistically different variations in lever type, day, or interactions between these factors, also from those evoked by ketamine. followed by a Bonferroni's test. Similarly, two-way ANOVA with Bonferroni's posttest was used to analyze the data in Fig. 3B(drug, 3.2. Locomotor activity day, or drug × days). Fig. 3C was analyzed using one-way ANOVA followed by a Dunnett's posttest. The accepted level of significance was Fig. 2 demonstrates the effects of MXE and ketamine on the locomo- set at p b 0.05. GraphPad Prism software v. 4.01 (San Diego, California, tor activity (distance moved and movement duration) of rats during USA) was used for all statistical analyses. the conditioning phase of the CPP. In the distance moved, two-way ANOVA showed significant effect of drug [F (4, 93) = 9.122, p b 0.001] 3. Results and day [F (2, 93) = 3.595, p b 0.05], but no drug × day interaction. Sim- ilar effects were observed in movement duration: significant effects of 3.1. Conditioned place preference test drug [F (4, 93) = 13.49, p b 0.001] and day [F (2, 93) = 7.070, p b 0.01], but no drug × day interaction. Bonferroni's posttest showed Fig. 1A shows the time spent on the initially non-preferred area that ketamine significantly decreased the distanced moved [day 1 (drug-paired compartment) of the apparatus during the pre- and (t = 2.755, p b 0.05), day 2 (t = 3.269, p b 0.01), day 3 (t = 2.521, post-conditioning phases of the CPP. Fig. 1B represents the difference p b 0.05)] and movement duration [day 2 (t =4.364,p b 0.001), day 3 between the post and pre-conditioning scores of the saline-, MXE- and (t = 3.107, p b 0.01)] of rats. Interestingly, MXE, across all dosages, ketamine-treated rats. One-way ANOVA revealed a significant differ- did not produce any significant change in the distance moved and ence between the experimental groups [F (4, 29) = 4.475, p b 0.01]. movement duration of rats. The distance moved and movement Dunnett's posttest showed that rats conditioned with MXE at dosages duration of MXE-treated rats were different to that of the ketamine- of 2.5 (q = 3.763, p b 0.01) and 5 mg/kg (q = 3.537, p b 0.01) or treated rats. ketamine at 5 mg/kg (q =2.584,p b 0.05) significantly produced CPP. Although rats treated with 1.25 mg/kg MXE showed some increase in 3.3. Self-administration test the time spent on the drug-paired side, it failed to reach statistical significance (q =2.478,p N 0.05). Effects produced by the three tested Fig. 3A shows the number of lever responses during the 2 h/day, 7-day SA sessions under the FR1 schedule. Two-way ANOVA revealed that the saline group showed significant variations in lever type [F (1, 98) = 4.260, p b 0.05] and day [F (6, 98) = 4.746, p b 0.001], and interaction between the two factors [F (6, 98) = 3.403, p b 0.01]. However, post hoc analysis indicated that the variations in lever type were only evident on the first day of SA (Bonferroni's posttest). The rats self-administering MXE at 0.25 mg/kg/infusion displayed significant variations in lever type [F (1, 98) = 83.83, p b 0.001] and day [F (6, 98) = 2.223, p b 0.05], but no interaction between the two factors [F (6, 98) = 1.408, p N 0.05]. Bonferroni's posttest showed that these variations were present on the 1st, 2nd, 3rd, 4th, and 7th day of SA. Similarly, the 0.5 mg/kg/infusion dose of MXE induced significant difference in lever type [F (1, 98) = 68.54, p b 0.001] and day [F (6, 98) = 4.035, p b 0.01], but no interaction between the two factors [F (6, 98) = 2.167, p N 0.05]. Post hoc analysis revealed significant variations in lever responses on the 1st, 2nd, and 4th day of SA. The MXE 1.0 mg/kg/infusion group significantly demonstrated variations in lever type [F (1, 98) = 94.41, p b 0.001] and day [F (6, 98) = 3.215, p b 0.01], and interaction between the two factors [F (6, 98) = 2.298, p b 0.05]. These variations were identified at the 1st, 2nd, 3rd, 4th, and 7th day of SA. Ketamine (0.5 mg/kg/infusion) significantly induced variations in lever type [F (1, 98) = 55.28, p b 0.001], but failed to produce variations in day [F (6, 98) = 0.2968, p N 0.05] and interaction between the two factors [F (6, 98) = 0.2476, p N 0.05]. Significant variations in lever responses were evident during the 1st, 4th, 5th, 6th, and 7th day of SA test. Fig. 3B shows the number of infusions obtained by the rats during the daily SA sessions. Two-way ANOVA showed significant variations between drug [F (4, 238) = 22.17, p b 0.001] and day [F (6, 238) = 7.345, p b 0.001], but no interaction between these factors [F (24, 238) = 1.094, p N 0.05]. As compared to the saline group, the 0.25 mg/kg/infusion MXE group showed a significant difference on the 2nd and 4th SA days. The 0.5 mg/kg/infusion MXE group only exhib- ited a significant difference on the 4th SA day. The 1.0 mg/kg/infusion MXE group showed a significant difference on the 2nd and 3rd SA days. Meanwhile, the ketamine (0.5 mg/kg/infusion) group demonstrated a significant difference on the 3rd, 4th, 5th, 6th, and 7th SA days. Fig. 1. The effects of methoxetamine and ketamine on the conditioned place preference As can be observed in Fig. 3A and B, the first 3–4 days of self- test in rats. Each bar represents the mean ± SEM of the time spent in the drug-paired compartment (A), or the difference in the time spent in the drug-paired or saline-paired side administration is usually considered a transition period from the lever during the post- and preconditioning phases (B). n =7–8 animals per group. *p b 0.05, training to the actual drug-SA. Consequently, results from this period **p b 0.01 significantly different from the SAL/control group (Dunnett's posttest). may be confounded by the earlier food pellet training experienced by 34 C.J. Botanas et al. / Pharmacology, Biochemistry and Behavior 133 (2015) 31–36

Fig. 2. The effects of methoxetamine and ketamine injection on the locomotor activity of rats during the conditioning phase of the CPP. Each point represents the mean ± S.E.M. of the distanced moved (cm) and movement duration (s) during the MXE, ketamine or saline (for the control group) conditioning phase of the CPP. n =7–8 animals per group. *p b 0.05, **p b 0.01, ***p b 0.001 significantly different from the control group; #p b 0.05, ##p b 0.01, ###p b 0.001 significantly different from the ketamine group (Bonferroni's posttest). each subject. Thus, a separate analysis was performed where data 4. Discussion from the transition period were excluded and only data from the stable period of SA (days 5–7) were included (Fig. 3C). One-way ANOVA The use of methoxetamine as a recreational drug is on the rise, bear- showed significant variations in the mean number of infusions between ing significant health risks to users/abusers (EMCDDA, 2014). However, each group [F (4, 10) = 75.15, p b 0.001]. As compared to the saline relatively little is known regarding MXE and its effects, and to our group, rats self-administering MXE [0.25 (q = 5.831, p b 0.001), 0.5 knowledge, no earlier studies have characterized its potential for (q = 6.450, p b 0.001), and 1 mg/kg/infusion (q = 7.018, p b 0.001)] abuse (EMCDDA, 2014). Therefore, in the present study we assessed and ketamine [0.5 mg/kg/infusion (q = 17.00, p b 0.001)] acquired the abuse potential (rewarding and reinforcing effects) of MXE through more infusions (Dunnett's posttest). animal models of drug addiction, the CPP and SA tests. We found that

Fig. 3. The effects of methoxetamine and ketamine on the self-administration test in rats. Lever responses made (A) and infusions obtained (B) during the 2-h, 7-day SA sessions under the FR 1 schedule. Open symbols indicate lever responses on the inactive lever and ﬁlled symbols show responses for the active lever. The mean number of infusions during the stable periods (5th to 7th day) of SA was also presented (C). *p b 0.05, **p b 0.01, ***p b 0.001 relative to inactive lever responses [(A) Bonferroni's posttest] or to the saline group [(B) Bonferroni's posttest] [(C) Dunnett's posttest]. Values are mean ± S.E.M. n =7–8 animals per group. C.J. Botanas et al. / Pharmacology, Biochemistry and Behavior 133 (2015) 31–36 35

MXE produced significant conditioned place preference and was self- and MXE. The above-discussed distinct affinity of MXE to SERT and its administered by rats. resultant increase in serotonin levels may have also influenced the SA As aforementioned, MXE is derived from ketamine and, in fact, the result. This speculation is based on previous studies demonstrating name “methoxetamine” was reported to have been created as a contrac- that serotonin concentration in the brain may inversely affect the tion of methoxy-ketamine (EMCDDA, 2014). It is chemically and phar- reinforcing effects of certain drugs of abuse (Loh and Roberts, 1990; macologically analogous to ketamine; both are arylcyclohexylamine Müller and Homberg, 2014; Smith et al., 1986). Notably, a recent and considered NMDA receptor antagonists (Corazza et al., 2013; study has shown that serotonin may limit the initial sensitivity to the EMCDDA, 2014). In an in vitro examination, it was found that MXE positive reinforcing effects and delays the acquisition of reliable SA of has a submicromolar affinity to the NMDA glutamate receptor, an effect MDMA, an abused drug (Bradbury et al., 2014). equipotential to that of ketamine (Roth et al., 2013). As would be Another plausible explanation for this discrepant effect might be re- expected, MXE was also reported to induce ketamine-like mood lated to the slower onset of action of MXE, as compared to ketamine enhancement with hallucinogenic and dissociative effects (Bey and (Corazza et al., 2013). Previous studies have shown that a drug's onset Patel, 2007; EMCDDA, 2014; Kjellgren and Jonsson, 2013; Morgan and of action may influence its ability as a reinforcer, indicating that drugs Curran, 2012). Accordingly, in the present study we have found that with rapid onset of action have stronger reinforcing effects than drugs MXE produced significant CPP in rats, which was comparable to that having similar pharmacological properties but with a slower onset of produced by ketamine, indicating that this drug has rewarding effects action (Ko et al., 2002; Panlilio and Schindler, 2000; Winger et al., (Fig. 1). This result corroborates with earlier studies demonstrating 2002). Thus, the slower onset of action associated with MXE may have that NMDA receptor antagonists are capable of producing reward (de limited its reinforcing effects, resulting in lower response rates when la Peña et al., 2012; Morris and Wallach, 2014). The rewarding effects compared to ketamine. However, the report of Corazza et al., 2013 of this type of drugs are believed to be facilitated through their interac- concerns a study on humans where MXE was taken via the oral route, tion with the mesolimbic dopamine reward system (Marcus et al., 2001; and thus the differences between MXE and ketamine reported in that Noda et al., 1998; Xu et al., 2008). Substantial evidences have implicated study may be influenced by differences in the kinetics of absorption. that the rewarding properties of many, if not all, drugs of abuse are This and other factors that might influence the reinforcing effects of attributable to their ability to interact with the mesolimbic dopamine re- MXE (e.g. previous drug experience) are interesting subjects for future ward system and its efferent (Di Chiara, 2002; Di Chiara and Bassareo, studies. Regardless, the result of the SA test strongly demonstrated 2007; Jones and Bonci, 2005). Drugs that increase dopamine levels, that MXE was self-administered by rats, indicating that this drug has such as cocaine and amphetamine, are known to have rewarding effects reinforcing properties. and produce place preference (Brebner et al., 2005; Liao et al., 2000; In summary, we have found that the ketamine derivative, MXE, Mueller and Stewart, 2000; Pettit and Justice, 1989). NMDA receptor produced significant CPP and was self-administered by rats. These antagonists can inhibit dopamine reuptake, thereby increasing extracel- results strongly indicate that this new, synthetic, psychoactive drug lular dopamine levels (Morgan and Curran, 2012; Morris and Wallach, has rewarding and reinforcing effects. We have also found that MXE 2014; Noda et al., 1998). Indeed, MXE was reported to inhibit the reup- and ketamine have discrepant effects on locomotor activity and rates take of dopamine in the nucleus accumbens of rats (Zawilska, 2014). of self-administration, suggesting that the psychopharmacological Taken together, these findings suggest that MXE is capable of producing effects of these drugs may diverge in some aspects. Importantly, the CPP in rats, probably due to its ability to increase dopamine levels. present study has provided the much-needed scientificevidenceon As expected from an anesthetic and consistent with previous studies the abuse potential of MXE. More importantly, the present study advo- (Becker et al., 2003; Trujillo et al., 2011), ketamine injection decreased cates the careful monitoring and prompt regulation of methoxetamine the locomotor activity of rats during the conditioning phase of the and its related substances. CPP. On the other hand, MXE treatment did not induce any significant changes in the locomotor activity of the rats. This result was surprising Acknowledgment given that MXE was derived from ketamine. The reason behind this oc- currence is rather unclear, but this result highlights a disparity in the The authors would like to thank the Ministry of Food and Drug Safety psychopharmacological effects of these drugs. Indeed, a study by the (MFDS) of Korea (14182MFDS979) for the financial assistance. Advisory Council on the Misuse of Drugs (ACMD) has found that, unlike ketamine, MXE has appreciable affinity for serotonin transporter (SERT) References where it inhibits the reuptake of serotonin resulting to increase seroto- ninlevelsinthebrain(ACMD, 2012; EMCDDA, 2014). This unique Advisory Council on the Misuse of Drugs (ACMD). Methoxetamine report. https://www. feature of MXE may contribute to its psychopharmacological profile gov.uk/government/publications/advisory-council-on-the-misuse-of-drugs-acmd- methoxetamine-report-2012, 2012. [Accessed January 8, 2015]. (ACMD, 2012). Also, the effect of MXE on the serotonergic system may Becker A, Peters B, Schroeder H, Mann T, Huether G, Grecksch G. Ketamine-induced underlie the observed discrepant effects on locomotor activity observed changes in rat behaviour: a possible animal model of schizophrenia. Prog herein. Indeed, inhibition of SERT or increase in serotonin levels has Neuropsychopharmacol Biol Psychiatry 2003;27:687–700. Bey T, Patel A. Phencyclidine intoxication and adverse effects: a clinical and pharmacolog- been previously reported to alter (increase) locomotor activity in ical review of an illicit drug. Calif J Emerg Med 2007;8:9. rodents (Brocco et al., 2002). In any case, this result suggests that the Bradbury S, Bird J, Colussi‐Mas J, Mueller M, Ricaurte G, Schenk S. Acquisition of MDMA rewarding effects of these NMDA receptor antagonists (ketamine and self‐administration: pharmacokinetic factors and MDMA‐induced serotonin release. Addict Biol 2014;19:874–84. MXE) are independent of increases in locomotor activity. Brebner K, Ahn S, Phillips AG. Attenuation of D-amphetamine self-administration by In connection with the outcome of the CPP test, we have also found Baclofen in the rat: behavioral and neurochemical correlates. Psychopharmacology that MXE was self-administered by rats. Although a downward trend (Berl) 2005;177:409–17. was observed during the transition period (1st to 4th day of SA), rats Brocco M, Dekeyne A, Veiga S, Girardon S, Millan MJ. Induction of hyperlocomotion in mice exposed to a novel environment by inhibition of serotonin reuptake. A pharma- acquired and maintained modest self-administration of MXE during cological characterization of diverse classes of antidepressant agents. Pharmacol the stable periods (5th to 7th day) of SA. Fig. 3C evidently shows that Biochem Behav 2002;71:667–80. rats self-administering MXE, in all dosages, acquired more infusions Corazza O, Schifano F, Simonato P, Fergus S, Assi S, Stair J, et al. Phenomenon of new drugs on the Internet: the case of ketamine derivative methoxetamine. Hum than the saline group. This result indicates that MXE, although modest, Psychopharmacol 2012;27:145–9. has reinforcing effects in rats. Corazza O, Assi S, Schifano F. From “Special K” to “Special M”: the evolution of the On the other hand, ketamine showed robust acquisition and recreational use of ketamine and methoxetamine. CNS Neurosci Ther 2013;19: 454–60. maintenance of self-administration. This result, again, highlights de la Peña JBI, Lee HC, de la Peña IC, Woo TS, Yoon SY, Lee HL, et al. Rewarding and the dissimilarity in the psychopharmacological effects of ketamine reinforcing effects of the NMDA receptor antagonist–benzodiazepine combination, 36 C.J. Botanas et al. / Pharmacology, Biochemistry and Behavior 133 (2015) 31–36

Zoletil®: difference between acute and repeated exposure. Behav Brain Res 2012; Meyer MR, Bach M, Welter J, Bovens M, Turcant A, Maurer HH. Ketamine-derived design- 233:434–42. er drug methoxetamine: metabolism including isoenzyme kinetics and toxicological de la Peña JB, Yoon SY, de la Peña IC, Lee HL, IdlP IJ, Cheong JH. Pre-exposure to detectability using GC–MS and LC-(HR-)MSn. Anal Bioanal Chem 2013;405:6307–21. related substances induced place preference and self-administration of the NMDA Morgan CJ, Curran HV. Ketamine use: a review. Addiction 2012;107:27–38. receptor antagonist–benzodiazepine combination, Zoletil. Behav Pharmacol 2013; Morris H, Wallach J. From PCP to MXE: a comprehensive review of the non‐medical use of 24:20–8. dissociative drugs. Drug Test Anal 2014;6:614–32. de la Peña JB, Ahsan HM, dela Peña IJ, Park HB, Kim HJ, Sohn A, et al. Propofol pretreat- Mueller D, Stewart J. Cocaine-induced conditioned place preference: reinstatement by ment induced place preference and self-administration of the tiletamine–zolazepam priming injections of cocaine after extinction. Behav Brain Res 2000;115:39–47. combination: implication on drug of abuse substitution. Am J Drug Alcohol Abuse Müller CP, Homberg JR. The role of serotonin in drug use and addiction. Behav Brain Res 2014;40:321–6. 2015;277:146–92. De Luca MT, Badiani A. Ketamine self-administration in the rat: evidence for a critical role Noda Y, Miyamoto Y, Mamiya T, Kamei H, Furukawa H, Nabeshima T. Involvement of of setting. Psychopharmacology (Berl) 2011;214:549–56. dopaminergic system in phencyclidine-induced place preference in mice pretreated Di Chiara G. Nucleus accumbens shell and core dopamine: differential role in behavior with phencyclidine repeatedly. J Pharmacol Exp Ther 1998;286:44–51. and addiction. Behav Brain Res 2002;137:75–114. Panlilio LV, Schindler CW. Self-administration of remifentanil, an ultra-short acting Di Chiara G, Bassareo V. Reward system and addiction: what dopamine does and doesn't opioid, under continuous and progressive-ratio schedules of reinforcement in rats. do. Curr Opin Pharmacol 2007;7:69–76. Psychopharmacology (Berl) 2000;150:61–6. European Monitoring for Drugs and Drug Addiction (EMCDDA). Polydrug use: patterns Pettit HO, Justice Jr JB. Dopamine in the nucleus accumbens during cocaine self- and responses. Lisbon: Publications Office of the European Union; 2009 [http:// administration as studied by in vivo microdialysis. Pharmacol Biochem Behav 1989; www.emcdda.europa.eu/publications/selected-issues/polydrug-use. Accessed 34:899–904. January 13, 2015]. Roth BL, Gibbons S, Arunotayanun W, Huang X-P, Setola V, Treble R, et al. The ketamine European Monitoring for Drugs and Drug Addiction (EMCDDA). Report on the risk assess- analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are ment of 2-(3-methoxyphenyl)-2-(ethylamino)cyclohexanone (methoxetamine) high affinity and selective ligands for the glutamate NMDA receptor. PLoS One in the framework of the council decision on new psychoactive substances. 2013;8:e59334. Luxembourg: Publication Office of the European Union; 2014 [http://www.emcdda. Shippenberg TS, Koob G. Recent advances in animal models of drug addiction and europa.eu/publications/risk-assessment/methoxetamine. Accessed January 13, alcoholism. Neuropsychopharmacology 2002:1381–97. 2015]. Smith FL, Yu DS, Smith DG, Leccese AP, Lyness WH. Dietary tryptophan supplements Fattore L, Fratta W. Beyond THC: the new generation of cannabinoid designer drugs. Front attenuate amphetamine self-administration in the rat. Pharmacol Biochem Behav Behav Neurosci 2011;5. 1986;25:849–55. Jones S, Bonci A. Synaptic plasticity and drug addiction. Curr Opin Pharmacol 2005;5: Trujillo KA, Smith ML, Sullivan B, Heller CY, Garcia C, Bates M. The neurobehavioral 20–5. pharmacology of ketamine: implications for drug abuse, addiction, and psychiatric Kjellgren A, Jonsson K. Methoxetamine (MXE)—a phenomenological study of experiences disorders. ILAR J 2011;52:366–78. induced by a “legal high” from the internet. J Psychoactive Drugs 2013;45:276–86. Valjent E, Bertran-Gonzalez J, Aubier B, Greengard P, Hervé D, Girault J-A. Mecha- Ko M, Terner J, Hursh S, Woods J, Winger G. Relative reinforcing effects of three opioids nisms of locomotor sensitization to drugs of abuse in a two-injection protocol. with different durations of action. J Pharmacol Exp Ther 2002;301:698–704. Neuropsychopharmacology 2010;35:401. Liao R-M, Chang Y-H, Wang S-H, Lan C-H. Distinct accumbal subareas are involved in Winger G, Hursh S, Casey K, Woods J. Relative reinforcing strength of three N-methyl-D- place conditioning of amphetamine and cocaine. Life Sci 2000;67:2033–43. aspartate antagonists with different onsets of action. J Pharmacol Exp Ther 2002;301: Loh EA, Roberts DC. Break-points on a progressive ratio schedule reinforced by intrave- 690–7. nous cocaine increase following depletion of forebrain serotonin. Psychopharmacolo- Xu D, Mo Z, Yung K, Yang Y, Leung A. Individual and combined effects of methamphet- gy (Berl) 1990;101:262–6. amine and ketamine on conditioned place preference and NR1 receptor phosphory- Marcus M, Mathe J, Nomikos G, Svensson T. Effects of competitive and non-competitive lation in rats. Neurosignals 2008;15:322–31. NMDA receptor antagonists on dopamine output in the shell and core subdivisions Zawilska JB. Methoxetamine—a novel recreational drug with potent hallucinogenic of the nucleus accumbens. Neuropharmacology 2001;40:482–90. properties. Toxicol Lett 2014;230:402–7.