The novel adenosine A2A antagonist prodrug MSX-4 is effective in animal models related to motivational and motor functions

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Abstract

Adenosine A2A and dopamine D2 receptors interact to regulate diverse aspects of ventral and dorsal striatal functions related to motivational and motor processes, and it has been suggested that adenosine A2A antagonists could be useful for the treatment of depression, parkinsonism and other disorders. The present experiments were performed to characterize the effects of MSX-4, which is an amino acid ester prodrug of the potent and selective adenosine A2A receptor antagonist MSX-2, by assessing its ability to reverse pharmacologically induced motivational and motor impairments. In the first group of studies, MSX-4 reversed the effects of the D2 antagonist eticlopride on a concurrent lever pressing/chow feeding task that is used as a measure of effort-related choice behavior. MSX-4 was less potent after intraperitoneal administration than the comparison compound, MSX-3, though both were equally efficacious. With this task, MSX-4 was orally active in the same dose range as MSX-3. MSX-4 also reversed the locomotor suppression induced by eticlopride in the open field, but did not induce anxiogenic effects as measured by the relative amount of interior activity. Behaviorally active doses of MSX-4 also attenuated the increase in c-Fos and pDARPP-32(Thr34) expression in nucleus accumbens core that was induced by injections of eticlopride. In addition, MSX-4 suppressed the oral tremor induced by the anticholinesterase galantamine, which is consistent with an antiparkinsonian profile. These actions of MSX-4 indicate that this compound could have potential utility as a treatment for parkinsonism, as well as some of the motivational symptoms of depression and other disorders.

Highlights

► MSX-4 is a novel adenosine A2A antagonist prodrug, converted into MSX-2 in vivo. ► MSX-4 reversed the effects of eticlopride on effort-related choice behavior. ► MSX-4 increased locomotion in eticlopride-treated rats. ► MSX-4 suppressed galantamine-induced tremulous jaw movements in rats. ► MSX-4 may be useful for treating parkinsonism and depression.

Introduction

A growing body of evidence indicates that the neuromodulator adenosine plays an important role in regulating the functional circuitry of the basal ganglia (Ferré et al., 1997, Ferré et al., 2001, Ferré et al., 2004, Stromberg et al., 2000, Hauber et al., 2001, Morelli and Pinna, 2002, Bara-Jimenez et al., 2003, Simola et al., 2006, Salamone et al., 2008). A1 and A2A adenosine receptor subtypes are involved in striatal function, and anatomical studies have shown that adenosine A2A receptors have a very high level of expression within both the neostriatum and the nucleus accumbens (Wang et al., 2000, Hettinger et al., 2001, Chen et al., 2001). In these striatal areas, adenosine A2A receptors are present largely on enkephalin-positive ventral and dorsal striatopallidal neurons, and are co-localized with DA D2 receptors (Fink et al., 1992, Rosin et al., 1998, Hillion et al., 2002, Chen et al., 2001, Fuxe et al., 2003, Fuxe et al., 2007, Mingote et al., 2008). Adenosine A2A and DA D2 receptors are thought to form heteromeric complexes that influence neurotransmitter binding, and these receptors also converge onto the same cAMP-related signal transduction pathways (Ferré, 1997, Ferré et al., 2008). A number of behavioral pharmacology studies have shown that drugs acting on adenosine A2A receptors can affect both motivational and motor functions. Adenosine A2A agonists such as CGS 21680 produce effects that resemble DA antagonism or depletion on tests of locomotor activity (Barraco et al., 1993), operant lever pressing (Mingote et al., 2008), effort-related choice behavior (Font et al., 2008) and tremor (Collins-Praino et al., 2011). Furthermore, adenosine A2A antagonists have been shown to act as psychomotor stimulants (Randall et al., 2011), and to reverse many of the behavioral effects of DA D2 antagonists (Farrar et al., 2007, Farrar et al., 2010, Salamone et al., 2008, Salamone et al., 2009a, Mott et al., 2009, Worden et al., 2009, Trevitt et al., 2009, Nunes et al., 2010, Collins et al., 2010a, Collins et al., 2012, Pereira et al., 2011) and cholinomimetics (Collins et al., 2010b, Collins et al., 2011). Based upon preclinical research, it has been suggested that adenosine A2A receptor antagonists could be useful as antiparkinsonian drugs (Ferré et al., 1997, Wardas et al., 2001, Morelli and Pinna, 2002, Hodgson et al., 2009, Salamone et al., 2008), and antidepressants (El Yacoubi et al., 2003, Hanff et al., 2010, Hodgson et al., 2009), or as treatments for motivational or effort-related psychiatric symptoms such as psychomotor retardation, anergia, apathy and fatigue (Salamone et al., 2007, Salamone et al., 2010), which are core symptoms of depression and other disorders (Marin, 1996, Demyttenaere et al., 2005, Salamone et al., 2006, Friedman, 2009).

In view of the preclinical research described above, and the growing clinical interest in adenosine A2A antagonists (LeWitt et al., 2008, Pinna, 2009, Hodgson et al., 2009, Salamone, 2010a), the development and testing of novel adenosine A2A antagonists is becoming an important research priority. One of the main considerations in drug discovery is the development of novel ligands with useful in vivo characteristics. For example, many drugs have difficulties in terms of absorption properties, solubility and blood brain barrier penetrability (Müller, 2009). An effective way of alleviating some of these problems is the use of prodrugs, which are bioreversible derivatives of drug molecules that must undergo a chemical or enzymatic biotransformation to the active forms prior to exerting a pharmacological action (Rautio et al., 2008, Müller, 2009). Prodrugs are traditionally developed by attaching an inactive promoiety to a pharmacologically active parent drug. The drug and promoiety are covalently linked via bioreversible groups that are chemically or enzymatically labile. The ideal prodrug yields the parent drug with high recovery ratios, with the promoiety being non-toxic (Rautio et al., 2008). Ultimately, the drug and promoiety are cleaved by chemical and/or enzymatic transformations, thus releasing the pharmacologically active parent drug. By employing this methodology, the clinical relevance of a drug molecule may be enhanced without modifying the pharmacological activity of a parent drug (Rautio et al., 2008). An example of an adenosine A2A antagonist parent compound that has been used in prodrug development is MSX-2. MSX-2, like other adenosine receptor antagonists and xanthine derivatives, suffers from poor water solubility (Müller, 2009). One approach to increase water solubility is to attach a polar moiety to the drug, which can be cleaved off by an enzymatic reaction to release the active drug. MSX-3 was developed several years ago as a pro-drug of MSX-2 (Hockemeyer et al., 2004), and previous studies have shown that MSX-3 can reverse the behavioral effects of DA D2 antagonists (Farrar et al., 2007, Salamone et al., 2008, Worden et al., 2009, Mott et al., 2009, Collins et al., 2010a, Collins et al., 2010b, Pardo et al., 2012). More recently, a novel valine ester amino acid promoiety for MSX-2 was developed (Vollmann et al., 2008). The new compound, MSX-4, was developed by condensing the carboxylate function of valine with the OH group of MSX-2, resulting in a prodrug with a basic amino group that can be protonated, which leads to a large increase in water solubility. MSX-4 was found to be stable in artificial gastric acid, and readily cleaved by pig liver esterase into MSX-2 (Vollmann et al., 2008).

Although MSX-3 has been widely used in vivo, the behavioral effects of MSX-4 have not been reported; thus, the present studies focused upon the characterization of the behavioral actions of MSX-4. The first group of studies compared MSX-4 and MSX-3 in terms of their ability to reverse the effects of the D2 antagonist eticlopride on effort-related choice behavior using a concurrent lever pressing/chow feeding choice task (Salamone et al., 1991, Salamone et al., 2002, Salamone et al., 2009a, Salamone et al., 2009b, Cagniard et al., 2006, Farrar et al., 2007, Nunes et al., 2010). It has been suggested that tests of effort-based choice behavior are useful for assessing motivational functions that are related to some of the activational or effort-based motivational symptoms of depression and other disorders (Salamone et al., 2006, Salamone et al., 2007, Salamone et al., 2009a, Salamone et al., 2009b, Salamone et al., 2010, Treadway and Zald, 2011). For these experiments, both intraperitoneal (IP) and oral administration were used. Additional studies focused upon the ability of MSX-4 to reverse the suppression of open field locomotion induced by eticlopride, and also used relative inner activity as a marker of any potential anxiogenic effects of MSX-4 (Prut and Belzung, 2003). To provide a cellular marker of the interaction between eticlopride and MSX-4, immunoreactivity of two markers of signal transduction activity (c-Fos and DARPP-32 phosphorylated at the threonine 34 residue, i.e., pDARPP-32(Thr34)) was determined in brain sections of nucleus accumbens. Finally, the ability of MSX-4 to suppress the oral tremor induced by the anticholinesterase galantamine (Collins et al., 2011) was assessed using the tremulous jaw movement test, which is a widely used rodent model of drug-induced parkinsonian tremor (Salamone et al., 1998, Salamone et al., 2005, Salamone et al., 2008, Rodriguez Diaz et al., 2001, Correa et al., 2004, Simola et al., 2004, Miwa et al., 2011, Collins-Praino et al., 2011, Collins et al., 2012). It was hypothesized that MSX-4 would show the behavioral and neurochemical characteristics of an adenosine A2A antagonist, by reversing the operant and locomotor effects of eticlopride, attenuating eticlopride-induced increases in c-Fos and pDARPP-32(Thr34) expression, and suppressing galantamine-induced oral tremor.

Section snippets

Animals

Across all experiments, a total of 140 male Sprague–Dawley rats (Harlan–Sprague–Dawley, Indianapolis, IN, USA) were used. Rats were housed in pairs in a colony maintained at 22–24 °C with a 12 h light/12 h dark cycle (lights on at 0700). Water was available ad libitum in the home cages at all times. The rats that were tested in operant boxes were food restricted to 85% of their free-feeding weight for initial operant training and allowed modest weight gain during the studies. Animal protocols have

Experiments 1 and 2: MSX-3 and MSX-4 reversed the effort-related effects of eticlopride

In experiment 1, there was an overall significant effect of drug treatment on lever pressing (Fig. 1A; F (5,30) = 11.466, p < 0.001). Planned comparisons showed that eticlopride produced a significant reduction in lever pressing compared to vehicle control (p < 0.05). In addition, co-administration of MSX-3 with eticlopride produced a significant increase in lever pressing compared to eticlopride plus vehicle, with the 0.5, 1.0 and 2.0 mg/kg doses of MSX-3 producing significant differences relative to

Discussion

The present studies were conducted to characterize the behavioral effects of the novel adenosine A2A antagonist prodrug MSX-4. MSX-4 was compared to a previously characterized prodrug (MSX-3) in the first 5 experiments, which employed a concurrent lever pressing/chow feeding task. Furthermore, we studied the MSX-4-induced reversal of the locomotor suppression induced by eticlopride, and the ability of MSX-4 to attenuate the signal transduction effects of eticlopride using c-Fos and

Acknowledgments

This work was supported by a grant to J.S. from the National Institute of Mental Health (MH078023), and to M.C. from Conselleria de Empresa, Universitat i Ciència. Generalitat Valenciana (BEST/2009/157). Y.B. and C.E.M. were supported by the BMBF, Germany (01EW0911) in the frame of ERA-NET NEURON.

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    Present address: Cognitive Neuroscience Division, Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University College of Physicians and Surgeons, NY, USA.

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