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Caffeine and more specific antagonists of the adenosine A2Areceptor recently have been found to be neuroprotective in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. Here we show that 8-(3-chlorostyryl)caffeine (CSC), a specific A2Aantagonist closely related to caffeine, also attenuates MPTP-induced neurotoxicity. Because the neurotoxicity of MPTP relies on its oxidative metabolism to the mitochondrial toxin MPP+, we investigated the actions of CSC on striatal MPTP metabolism in vivo. CSC elevated striatal levels of MPTP but lowered levels of the oxidative intermediate MPDP+and of MPP+, suggesting that CSC blocks the conversion of MPTP to MPDP+in vivo. In assessing the direct effects of CSC and A2Areceptors on monoamine oxidase (MAO) activity, we found that CSC potently and specifically inhibited mouse brain mitochondrial MAO-B activity in vitrowith aKivalue of 100 nm, whereas caffeine and another relatively specific A2Aantagonist produced little or no inhibition. The A2Areceptor independence of MAO-B inhibition by CSC was further supported by the similarity of brain MAO activities derived from A2Areceptor knockout and wild-type mice and was confirmed by demonstrating potent inhibition of A2Areceptor knockout-derived MAO-B by CSC. Together, these data indicate that CSC possesses dual actions of MAO-B inhibition and A2Areceptor antagonism, a unique combination suggesting a new class of compounds with the potential for enhanced neuroprotective properties.

Dopamine neurons from various animal species differ in sensitivity to the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP+). Compared with striatal vesicles isolated from mice, those from rats have a higher density of the brain vesicular monoamine transporter (VMAT2) and a greater ability to sequester MPP+, suggesting a larger storage capacity for MPP+in rat vesicles. In the present study, we examined whether striatal VMAT2-containing vesicles might provide protection against the neurotoxic effects of MPP+in vivo. Dose-response curves for striatally infused MPP+were determined in animals pretreated with or without a VMAT2 inhibitor. Ro 4-1284 administration (10 mg/kg i.p.; VMAT2 inhibitor) produced a 5-fold leftward shift in the MPP+dose-response curve and a significant lowering of the EC50concentration for MPP+-induced damage. These findings provide evidence for a substantial accumulation of MPP+in VMAT2-containing vesicles in vivo in the rat striatum and support the hypothesis that MPP+sequestration in vesicles can provide protection against its toxic actions. In mice, VMAT2 inhibition did not reliably enhance toxicity produced by a striatal infusion of MPP+or by systemic administration of MPTP. These data suggest that vesicular sequestration of MPP+may be of less importance in mice than in rats as relates to protection from the toxin. The present results also reveal that although VMAT2 inhibition enhanced striatal MPP+toxicity in the rat, the potency of MPP+in the rat striatum was less than that in mouse striatum. This implies that there are other factors that either exacerbate MPP+toxicity in the mouse or attenuate MPP+toxicity in rats.

Significant differences exist in the sensitivity of mice and rats to the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that cannot be explained by differences in exposure to or uptake of 1-methyl-4-phenylpyridinium (MPP+) into dopamine (DA) neurons. MPP+is also a substrate for the brain vesicular monoamine transporter (VMAT2), and sequestration into synaptic vesicles may be one mechanism of protection against MPP+toxicity. A greater sequestration of MPP+into vesicles of DA neurons in rats versus mice could explain the lower vulnerability of DA neurons in the rat to MPP+toxicity. To test this hypothesis, the kinetics of uptake for [3H]MPP+and [3H]DA as well as [3H]dihydrotetrabenazine binding to VMAT2 were compared in vesicles isolated from the striata of rats and mice. The Kmvalue of [3H]MPP+transport was similar in the two species. In contrast, the maximal transport rate (Vmax) was 2-fold greater in vesicles from rats than in those from mice. Likewise, theKmvalue for [3H]DA transport was similar in both preparations, but theVmaxvalue was 2-fold greater in rat than in mouse vesicles. The Bmaxvalue for [3H]dihydrotetrabenazine binding was also 2-fold greater in striatal vesicles from rats than in those from mice. Electron micrographs demonstrated that vesicles isolated from rats and mice were approximately the same size. Based on these observations, we propose that striatal vesicles from rats have more VMAT2 than vesicles from mice and that this species difference in VMAT2 density may help explain the reduced vulnerability of rat DA neurons to MPP+neurotoxicity.

There remains an insufficient number of P2X7 receptor antagonists with adequate rodent potency, CNS permeability, and pharmacokinetic properties from which to evaluate CNS disease hypotheses preclinically. Herein, we describe the molecular pharmacology, safety, pharmacokinetics, and functional CNS target engagement of Lu AF27139, a novel rodent-active and CNS-penetrant P2X7 receptor antagonist. Lu AF27139 is highly selective and potent against rat, mouse, and human forms of the receptors. The rat pharmacokinetic profile is favorable with high oral bioavailability, modest clearance (0.79 L/(h kg)), and good CNS permeability. In vivomouse CNS microdialysis studies of lipopolysaccharide (LPS)-primed and 2′(3′)-O-(benzoylbenzoyl)adenosine-5′-triphosphate (BzATP)-induced IL-1β release demonstrate functional CNS target engagement. Importantly, Lu AF27139 was without effect in standard in vitroand in vivotoxicity studies. Based on these properties, we believe Lu AF27139 will be a valuable tool for probing the role of the P2X7 receptor in rodent models of CNS diseases.