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

Neuropsychopharmacology (2017) 42, 1940–1949 © 2017 American College of Neuropsychopharmacology. All rights reserved 0893-133X/17 www.neuropsychopharmacology.org Direct and Systemic Administration of a CNS-Permeant Tamoxifen Analog Reduces Amphetamine-Induced Dopamine Release and Reinforcing Effects Colleen Carpenter1,5, Alexander G Zestos1,2,5, Rachel Altshuler1, Roderick J Sorenson3,4, Bipasha Guptaroy1, Hollis D Showalter3,4, Robert T Kennedy1,2, Emily Jutkiewicz1 and Margaret E Gnegy*,1 1 Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA; 2Department of Chemistry, University of Michigan, Ann Arbor, MI, USA; Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI, USA; 4Vahlteich Medicinal Chemistry Core, University of Michigan, Ann Arbor, MI, USA 3 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 AMPHstimulated 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 AMPHstimulated 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. Neuropsychopharmacology (2017) 42, 1940–1949; doi:10.1038/npp.2017.95; published online 14 June 2017 INTRODUCTION Amphetamine (AMPH) and its congeners are highly addictive stimulants and their abuse remains a significant health, social, and economic burden (Berman et al, 2008; Carvalho et al, 2012). Yet an effective treatment for AMPH abuse remains elusive. Similar to other drugs of abuse, the reinforcing effects of AMPH are attributed to its ability to significantly increase extracellular dopamine in the nucleus accumbens (Di Chiara and Imperato, 1988; Wise and Bozarth, 1985). AMPH achieves this effect through its action at the dopamine transporter (DAT). The primary role of DAT is to clear extracellular dopamine, thereby terminating presynaptic and postsynaptic dopamine signaling (Zhu and *Correspondence: Dr ME Gnegy, Department of Pharmacology, University of Michigan, 2220E MSRB III, 1150 W. Medical Center Drive, Ann Arbor MI 48109, USA, Tel: +1 734 763 5358, Fax: +1 734 763 4450; E-mail: 5 These authors are co-first authors. Received 8 March 2017; revised 29 April 2017; accepted 3 May 2017; accepted article preview online 11 May 2017 Reith, 2008). AMPH, a substrate of DAT, disrupts this process by competitively blocking dopamine reuptake and also promoting reverse transport of dopamine via DAT (McMillen, 1983). Unlike stimulants such as cocaine, whose actions are more reliant on storage pools of monoamines, the release of newly synthesized dopamine also contributes to AMPH action (Chiueh and Moore, 1975; Parker and Cubeddu, 1986). We found that protein kinase C (PKC) enhances AMPHstimulated dopamine efflux. AMPH increases striatal particulate PKC activity (Giambalvo, 1992, 2004) and PKC stimulates the phosphorylation of N-terminal DAT residues (Foster et al, 2002). Phosphorylation of DAT is permissive for AMPH-stimulated dopamine release (Khoshbouei et al, 2004; Wang et al, 2016). Selective PKC inhibitors and genetic deletion of PKC significantly reduce AMPH-stimulated dopamine release from striatal synaptosomes and slices (Chen et al, 2009; Kantor and Gnegy, 1998). PKC inhibition, however, does not alter the normal uptake functioning of the transporter (Johnson et al, 2005; Kantor and Gnegy, 1998; Utility of tamoxifen analogs in reducing AMPH effects C Carpenter et al 1941 Zestos et al, 2016). Therefore, PKC represents a novel therapeutic target for the treatment of AMPH abuse. The selective estrogen receptor (ER) modulator tamoxifen stands as the only commercially available central nervous system (CNS)-permeant PKC inhibitor (Zarate and Manji, 2009). Tamoxifen is commonly used to reduce ER-positive breast cancer recurrence and to prevent breast cancer in high-risk women (Fisher et al, 1998; Jordan, 2003). Early reports using purified PKC show that tamoxifen inhibits the calcium- and phospholipid-dependent activity of classical PKC isoforms, with IC50s between 25 and 100 μM (Su et al, 1985). In cells, tamoxifen inhibits PKC at more pharmacologically relevant concentrations (1–5 μM) (Gundimeda et al, 1996; Horgan et al, 1986; Lien et al, 1991; O'Brian et al, 1985). There are findings that suggest PKC activity is elevated in patients suffering from bipolar mania, a disorder modeled by repeated AMPH administration in animals (Wang and Friedman, 1996). Interestingly, systemic tamoxifen reduces manic symptoms in patients with bipolar mania, and this effectiveness is believed to stem from the action of tamoxifen at PKC (Kulkarni et al, 2006; Zarate et al, 2007). These data point to the clinical relevance of tamoxifen as a CNS-permeant PKC inhibitor. Although tamoxifen is well tolerated overall, it can cause ER-mediated adverse effects, including increased risk of hot flashes, thromboembolisms, and endometrial cancers (Fisher et al, 1998; Gradishar, 2004). Therefore, a CNS-permeant tamoxifen analog lacking ER activity could be useful in the context of AMPH abuse treatment. Extensive structure–activity relationship (SAR) studies have investigated tamoxifen substructures that contribute to its ability to bind to the ER and inhibit PKC (de Medina et al, 2004). We used this wealth of knowledge to synthesize a new generation of tamoxifen analogs with increased selectivity for PKC over ER (Carpenter et al, 2016). In this paper, we investigate the effect of our most promising novel compound, 6c (Figure 1a), at DAT and also on the neurochemical, behavioral, and reinforcing actions of AMPH. Our key findings show that 6c modula (...truncated)