Direct and Systemic Administration of a CNS-Permeant Tamoxifen Analog Reduces Amphetamine-Induced Dopamine Release and Reinforcing Effects

Abstract

Amphetamines (AMPHs) are globally abused. With no effective treatment for AMPH addiction to date, there is urgent need for the identification of druggable targets that mediate the reinforcing action of this stimulant class. AMPH-stimulated dopamine efflux is modulated by protein kinase C (PKC) activation. Inhibition of PKC reduces AMPH-stimulated dopamine efflux and locomotor activity. The only known CNS-permeant PKC inhibitor is the selective estrogen receptor modulator tamoxifen. In this study, we demonstrate that a tamoxifen analog, 6c, which more potently inhibits PKC than tamoxifen but lacks affinity for the estrogen receptor, reduces AMPH-stimulated increases in extracellular dopamine and reinforcement-related behavior. In rat striatal synaptosomes, 6c was almost fivefold more potent at inhibiting AMPH-stimulated dopamine efflux than [³H]dopamine uptake through the dopamine transporter (DAT). The compound did not compete with [³H]WIN 35,428 binding or affect surface DAT levels. Using microdialysis, direct accumbal administration of 1 μM 6c reduced dopamine overflow in freely moving rats. Using LC-MS, we demonstrate that 6c is CNS-permeant. Systemic treatment of rats with 6 mg/kg 6c either simultaneously or 18 h prior to systemic AMPH administration reduced both AMPH-stimulated dopamine overflow and AMPH-induced locomotor effects. Finally, 18 h pretreatment of rats with 6 mg/kg 6c s.c. reduces AMPH-self administration but not food self-administration. These results demonstrate the utility of tamoxifen analogs in reducing AMPH effects on dopamine and reinforcement-related behaviors and suggest a new avenue of development for therapeutics to reduce AMPH abuse.

Figure 1

Structure of tamoxifen analog, 6c, and its effect on PMA-induced PKC activity in synaptosomes. (a) Structures of tamoxifen and its analog, 6c. (b, c). Rat striatal synaptosomes were incubated in the presence or absence of 6c for 1 h at 37 °C. In all, 100 nM PMA was added for 15 min to stimulate PKC, and the samples were lysed and probed for phosphoser⁴¹-GAP-43 (n=4) and phosphoser^152/156-MARCKS (n=5). GAPDH served as the loading control. (b) Data are represented as the percentage of vehicle optical density and each data set represents mean±SEM. (c) Representative western blots. V1, V2: vehicle; P1, P2: PMA control; Rub: 500 nM ruboxistaurin, a control PKC inhibitor.

Figure 2

6c Modulation of DAT efflux and uptake processes. (a) Rat striatal synaptosomes were incubated in the presence or absence of 6c for 1 h at 37 °C; efflux was stimulated with 10 μM AMPH (n=4). Post hoc Dunnett multiple comparison test, *p⩽0.05. (b) Synaptosomes were incubated with vehicle or 6c for 1 h at 37 °C and [³H]dopamine uptake was quantified (n=5). Post hoc Dunnett multiple comparison test, *p<0.01. (c) Efflux and uptake results represented as the percentage of vehicle. All points are mean±SEM.

Figure 3

The action of 6c on DAT surface expression. (a) Rat striatal synaptosomes were incubated in the presence or absence of 3 μM 6c for 1 h at 37 °C, followed by the biotinylation of surface DAT as previously described in Methods section. (b) Representative western blots. Data shown as mean±SEM (n=5).

Figure 4

The effect of 6c on in vivo AMPH-induced dopamine overflow and locomotion. (a, b) In all, 1 μM 6c or vehicle was perfused into the nucleus accumbens using retrodialysis 30 min prior to the administration of 2 mg/kg i.p. AMPH. (a) Dopamine overflow; vehicle (n=7) and 6c (n=8). Post hoc Sidak’s multiple comparison test, *p<0.05. (b) Locomotion; in post hoc Sidak’s multiple comparison test, *p<0.05; vehicle (n=8), 6c (n=8). (c, d) In all, 6 mg/kg 6c or vehicle were given to rats s.c. simultaneously with 1 mg/kg i.p. AMPH. (c) Dopamine overflow; post hoc Sidak’s multiple comparison test, *p<0.05; vehicle (n=3), 6c (n=3). (d) Locomotor activity; post hoc Sidak’s multiple comparison test, *p<0.01; vehicle (n=4), 6c (n=4). (e, f) In all, 6 mg/kg 6c or vehicle was given 18 h prior to the administration of 2 mg/kg i.p. amphetamine (AMPH). (e) In post hoc Sidak’s multiple comparison test, *p<0.05 for dopamine overflow. (f) Post hoc Sidak’s multiple comparison test, *p<0.05 for locomotion. For dopamine and locomotor activity, vehicle (n=7) and 6c (n=6). (g) In all, 6 mg/kg 6c or vehicle (n=3) was administered s.c., and dialysate collected from time 0 to 70 min. Post hoc Sidak’s multiple comparison test, *p<0.05. Levels of 6c were quantified using LC-MS and corrected for recovery.

Figure 5

The effect of 6 mg/kg s.c. 6c on AMPH and food self-administration. (a) Training data for rats used in AMPH self-administration experiments. Rats were escalated from a FR1 to a FR5 schedule with 0.1 mg/kg/infusion of AMPH. Finally rats were trained to stably administer on the FR5 schedule with 0.032 mg/kg/infusion of AMPH. (b) Training data for rats used in food self-administration. Rats escalated from a FR1 to a FR5 schedule for food pellets. (c) On test day, 6 mg/kg 6c or vehicle was given 18 h prior to AMPH self-administration session. Post hoc Sidak’s multiple comparison test, *p<0.0001, a significant difference between vehicle- and 6c-treated rats. (d) On test day, 6 mg/kg 6c or vehicle was given 18 h prior to food self-administration session. (e, f) Inactive nose poke responses during AMPH and food self-administration sessions, respectively. All data sets are represented as mean±SEM.