REL-1017 (esmethadone; D-methadone) does not cause reinforcing effect, physical dependence and withdrawal signs in Sprague Dawley rats

Abstract

REL-1017 (esmethadone, D-methadone) is the opioid-inactive d-isomer of racemic D,L-methadone. REL-1017 may exert antidepressant effects via uncompetitive N-methyl-D-aspartate receptor (NMDAR) channel block. As REL-1017 is expected to exert central nervous system activity, full characterization of its abuse potential is warranted. We evaluated lack of reinforcing effect, physical dependence, and withdrawal of REL-1017 in Sprague Dawley rats. (1) Self-administration Study Rats were trained to self-administer oxycodone intravenously (IV) and then were subjected to 3-day substitution tests where saline, oxycodone, and REL-1017 were self-delivered IV by a fixed number of lever presses; (2) Drug Discontinuation Study Rats were treated for 30 days by oral gavage with vehicle, REL-1017, ketamine or morphine and evaluated for withdrawal with functional observational batteries (FOBs). In the self-administration study, rats treated with saline, vehicle, and all REL-1017 doses showed the typical "extinction burst" pattern of response, characterized by an initial rapid increase of lever-pressing followed by a rapid decrease over 3 days. Rats treated with oxycodone maintained stable self-injection, as expected for reinforcing stimuli. In the withdrawal study, REL-1017 did not engender either morphine or ketamine withdrawal signs over 9 days following abrupt discontinuation of drug exposure. REL-1017 showed no evidence of abuse potential and did not engender withdrawal symptomatology.

Figure 1

Self-administration study design (Study 1). The drawing illustrates Study 1 experimental protocol. During the first 7 days, rats were trained to respond by foot pressing on a lever, located within an operant response chamber, to receive a single food pellet reinforcement (45 mg). In this initial training session, rats were trained to respond under a fixed ratio (FR) 1:1 (1 lever press = 1 pellet delivery) schedule of reinforcement. When rats consistently responded to food rewards, the response requirements were increased until rats consistently responded under an FR4 (4 lever presses = 1 pellet delivery) schedule of reinforcement (Stage 1). Then, rats were trained to respond on the lever to self-administer cocaine (0.56 mg/kg/injection) paired with the delivery of a single 45 mg food pellet/injection for 1 training session; thereafter, food was discontinued, and the rats continued with self-administration of cocaine only. The progressive establishment of cocaine as a reinforcer was obtained with a final FR7 (7 lever presses = 1 cocaine delivery) (Stage 2). At this point, the conditioning session started, the cocaine was replaced, and the rats were given access to 0.18 mg/kg/injection of oxycodone. During the oxycodone only training sessions, the response requirements were increased until rats achieved a FR10 (10 lever presses = 1 oxycodone injection) (Stage 3). Stable operant responding (Stage 4) was defined as responding under an FR10 schedule for oxycodone injections with ≤ 20% day to day variations over 3 consecutive sessions. Stable responding rats started the Stage 5 operant testing sessions with saline or oxycodone, (“test sessions”), and with the test article or its vehicle (“substitution sessions”). Once a day for three consecutive sessions, lever pressing resulted in self-administration of saline or oxycodone at 0.01, 0.018, 0.032, 0.056, 0.10, 0.18, and 0.32 mg/kg/injection (Test Sessions), and REL 1017 at 0 (vehicle), 0.032, 0.056, 0.10, and 0.18 mg/kg/injection (Substitution Sessions).

Figure 2

Chronic drug exposure and withdrawal study design (Study 2). All drugs (morphine, ketamine, REL-1017, and vehicle control) were administered daily by oral gavage for 30 consecutive days. In the afternoon of Day 30, all rats received vehicle (sham) doses as their last delivered dose on study to test for withdrawal over 9 consecutive days. A total of 96 rats were tested, 16 rats for each treatment group. During the 9 days of discontinuation to assess physical dependence and withdrawal effects, we assessed neurobehavioral screening battery consisting of a set of functional observational batteries (FOBs), which included measures of activity/arousal, autonomic and physiological domains, and measures of neuromuscular activity (see “Methods” section).

Figure 3

Self-administration of REL-1017 in rats previously trained to administer oxycodone in 3-day substitution study. (A) Drugs were self-administered via intravenous (IV) bolus injections utilizing the surgically implanted venous catheters connected to a Med-PC computer system. Injection requirement was FR10 (10 lever presses = 1 injection). Trained rats were tested with saline control (n = 29, gray bars) or with the 0.18 mg/kg/injection training/maintenance dose of oxycodone (n = 29). Following the establishment of oxycodone as a reinforcer, 6 rats per each treatment were tested with the positive control substance (0.01, 0.018, 0.032, 0.056, 0.10, and 0.32 mg/kg/injection oxycodone; red bars), and/or REL-1017 (0, 0.032, 0.056, 0.10, and 0.18 mg/kg/injection; green bars). Each group of 3 bars represents the results for Days 1, 2, and 3. Each bar represents the mean of ≥ 6 rats. All positive control groups exposed to any oxycodone dose maintained a stable number of infusions over 3 days (p = ns between injections at day 1 and 3 for all dosing groups). As expected for non-reinforcing stimuli in this experiment, saline, vehicle, and all REL-1017 doses showed a typical “extinction burst” pattern of responding, characterized by an initial rapid increase of lever-pressing on the first day followed by a downward staircase pattern of responses on the second and third day (*p < 0.05, **p < 0.01 between injections at day 1 and 3). (B) Comparison of the number of injections at day 1. (C) Comparison of the number of injections at day 3. (D) Delta changes between the injection number at day 1–3. (E) Percent of delta changes between the injection number at day 1–3. REL-1017 at all tested doses showed a pattern comparable to vehicle and saline. For each condition, the differences in the day-to-day pattern of injection during the test session and the linear regression functions (slopes) fitted to the total number of injections during each three-day interval calculated for each study item is shown in (F). The calculated slopes for saline (gray dots), vehicle (black dots), and REL-1017 at all doses (green dots) were not statistically different between each other. The calculated slopes for saline, vehicle, and REL-1017 at all doses were all statistically significantly different when compared to oxycodone (red dots; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). All 4 doses of REL-1017 tested in this study demonstrated a downward staircase pattern resulting in negative sloped linear functions (saline = − 13.83; vehicle = − 9.667; RE-1017 0.032 mg/kg/injection = − 8.833; REL-1017 0.056 mg/kg/injection = − 11.67; REL-1017 0.1 mg/kg/injection = − 7.333; REL-1017 0.18 mg/kg/injection = − 9.083). All seven tested doses of oxycodone demonstrated a stable pattern resulting in neutral sloped functions (oxycodone 0.01 mg/kg/injection = − 1.083; oxycodone 0.018 mg/kg/injection = − 2.917; oxycodone 0.032 mg/kg/injection = − 1.667; oxycodone 0.056 mg/kg/injection = − 0.5833; oxycodone 0.1 mg/kg/injection = − 0.3333; oxycodone 0.18 mg/kg/injection = 0.569; oxycodone 0.32 mg/kg/injection = 0.5). (G) High rates of responding throughout the test sessions (responses/second) were observed in rats treated with saline (gray dots) and all doses of REL-1017 (green dots). The response rates for REL-21017 tested were significantly different from oxycodone (0.18 mg/kg/injection, red dots). (H) In particular, compared to the response rates for oxycodone 0.18 mg/kg/injection, saline had a 4.6-fold increase (p < 0.0001), REL-1017 vehicle had a 3.5-fold increase (p < 0.05), REL-1017 (0.032 mg/kg/injection) had a 3.8-fold increase (p < 0.001), REL-1017 (0.056 mg/kg/injection) had a 5.9-fold increase (p < 0.05), REL-1017 (0.1 mg/kg/injection) had a 5.6-fold increase (p < 0.05), and REL-1017 (0.18 mg/kg/injection) had a 4.4-fold increase (p < 0.05).

Figure 4

Discontinuation of chronic REL-1017 (esmethadone) administration did not cause modification in measures of excitability. Following cessation after 30 consecutive days of drug administration, measures of excitability, including ease of removal (A), handling reactivity (C), arousal (E), and rearing counts (G) were significantly different in morphine-treated rats (dark blue line) compared to vehicle (black line) and REL-1017-treated rats (yellow and red lines; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Area under the curve (AUC) values of ease of removal (B), handling reactivity (D), arousal (F), and rearing counts (H) were measured over the time interval 31–39 days. AUCs of morphine-treated rats (blue dot) were statistically different compared to AUCs of vehicle (black dot) and REL-1017- treated rats (yellow and red dots; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 5

Discontinuation of chronic REL-1017 (esmethadone) administration did not cause modification in sensorimotor, autonomic and physiological measures. Following abrupt cessation after 30 consecutive days of drug administration, measures related to sensorimotor functions performed during the following 9 consecutive days, including non-threatening approach response (A), tail pinch (C), and simple body-touch (E) were significantly different in morphine-treated rats (dark blue line) compared to vehicle (black line) and REL-1017-treated rats (yellow and red lines *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). AUC values of non-threatening approach response (B), tail pinch (D), and simple body-touch (F) were measured over the time interval 31–39 days. No differences were observed between AUCs of morphine-treated rats (blue dot) to AUCs of vehicle (black dot) and REL-1017-treated rats (yellow and red dots). (G) Defecation (autonomic) and (I) body weight (physiological) measures obtained during the 9 days following abrupt cessation after 30 consecutive days of vehicle (black line) and REL-1017 (yellow and red lines) administration differed significantly from those obtained following abrupt cessation after 30 consecutive days of morphine (dark blue line; *p < 0.05 at day 5; ***p < 0.001, at day 1–9). The AUC value of defecation (H) measures over the time interval 31–39 days of morphine-treated rats (blue dot) showed a trend of increase compared to the AUC of vehicle (black dot, p = 0.058). The AUC value of body weight (J) measures over the time interval 31–39 days of morphine-treated rats (blue dot) were statistically decreased compared to the AUC of vehicle (black dot) and REL-1017-treated rats (yellow and red dots, ****p < 0.0001).

Figure 6

Discontinuation of chronic REL-1017 (esmethadone) administration did not cause modification in neuromuscular measures. Measurements of neuromuscular functions evaluated during the 9 days of discontinuation in rats treated for 30 consecutive days with morphine (dark blue line) demonstrated statistically significant changes in (A) hindlimb and (C) forelimb grip strength compared to vehicle (black line) and REL-1017 (yellow and red lines **p < 0.01; ***p < 0.001; at days 7 and 8). Ketamine-treated rats (light blue) showed significant changes in (A) hindlimb grip strength compared to vehicle and REL-1017 (*p < 0.05 at day 7). No differences were observed in the AUC values of hindlimb (B) and forelimb (D) grip strength measured over the time interval 31–39 days between morphine-treated rats (blue dot), vehicle (black dot) and REL-1017-treated rats (yellow and red dots).