Methamphetamine Sensitization.

As a rapid means of determining if GZ-11608 decreases the in vivo effects of methamphetamine, locomotor sensitization following repeated methamphetamine injection was the initial assay employed (Alvers et al., 2012; Lee et al., 2018). Repeated methamphetamine administration results in a robust and stable increase in locomotor activity from day-to-day, allowing for reliable evaluation of the ability of compound to reduce the effects of methamphetamine on behavior. Locomotor activity was measured in a locomotor chamber (24 × 24 × 30 cm) with clear acrylic walls and floor. A horizontal 16 × 16 grid of photo beams was located 7 cm above the chamber floor, with each beam 2.5 cm apart. Movement in the chamber resulted in beam breaks, which were recorded and transformed into distance traveled (centimeter) by Versamax and Digipro System software (AccuScan Instruments Inc., Columbus, OH). The effect of GZ-11608 on methamphetamine-sensitized activity was determined using a mixed factor design with methamphetamine as a between-subjects factor and GZ-11608 as within-subjects factor. Rats were assigned randomly to methamphetamine or saline groups. After a week of acclimation, rats were habituated to the apparatus by being placed in the chamber for 1 hour with no injection (day 0). On days 1–10, rats were injected (subcutaneous, s.c.) daily with either methamphetamine (1 mg/kg) or saline (1 ml/kg), immediately placed in the chamber, and activity measured for 1 hour. Methamphetamine dose and number of daily injections were chosen to provide stable, sensitized locomotor activity, on the basis of previous findings (Lee et al., 2018). On day 11, GZ-11608 (0, 1–30 mg/kg, s.c.) in a quasi-randomized dose order was injected 15 minutes prior to the daily methamphetamine or saline injection, and then, rats were placed immediately into the chamber for 1 hour. A washout period (2 to 3 days) intervened between testing of GZ-11608 doses to avoid potential drug accumulation. On washout days, methamphetamine or saline was injected and locomotor activity determined.

In a separate experiment employing a mixed factor design, the effect of GZ-11608 administration by oral gavage (p.o.) on methamphetamine locomotor sensitization was determined. Following repeated methamphetamine or saline for 5 days, rats were habituated to the oral gavage procedure. On days 5–10, rats received vehicle [15% (v/v) Kolliphor EL in saline, p.o., 3 ml] 15 minutes prior to methamphetamine or saline injection (subcutaneous) and were placed in the activity chamber for 1 hour (Wilmouth et al., 2013). On days 11–27, GZ-11608 (0, 17–300 mg/kg, p.o., ascending dose order) was administered, followed 15 minutes later by either methamphetamine or saline injection (1 ml/kg, s.c.), depending on group assignment, and immediate placement into the chamber for 1 hour. Between GZ-11608 doses, 2 to 3 days of washout occurred. On washout days, vehicle was administered by oral gavage and methamphetamine or saline was injected subcutaneously.