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Analgesic properties of orthosteric agonists of the muscarinic M4 receptor subtype have been documented in literature reports, with evidence from pharmacological and in vivo receptor knock out (KO) studies. Constitutive M4 receptor KO mice demonstrated an increased response in the formalin pain model, supporting this hypothesis. Two novel positive allosteric modulators (PAM) of the M4 receptor, Compounds 1 and 2, were characterized in rodent models of acute nociception. Results indicated decreased time spent on nociceptive behaviors in the mouse formalin model, and efficacy in the mouse tail flick assay. The analgesic-like effects of Compounds 1 and 2 were shown to be on target, as the compounds lacked any activity in constitutive M4 KO mice, while retaining activity in wild type control littermates. The analgesic-like effects of Compounds 1 and 2 were significantly diminished in KO mice that have selective deletion of the M4 receptor in neurons that co-express the dopaminergic D1 receptor subtype, suggesting a centrally-mediated effect on nociception. The opioid antagonist naloxone did not diminish the effect of Compound 1, indicating the effects of Compound 1 are not secondarily linked to opioid pathways. Compound 1 was evaluated in the rat, where it demonstrated analgesic-like effects in tail flick and a subpopulation of spinal nociceptive sensitive neurons, suggesting some involvement of spinal mechanisms of nociceptive modulation. These studies indicate that M4 PAMs may be a tractable target for pain management assuming an appropriate safety profile, and it appears likely that both spinal and supraspinal pathways may mediate the antinociceptive-like effects.

SummaryMetabolites in the kynurenine pathway, generated by tryptophan degradation, are thought to play an important role in neurodegenerative disorders, including Alzheimer's and Huntington's diseases. In these disorders, glutamate receptor-mediated excitotoxicity and free radical formation have been correlated with decreased levels of the neuroprotective metabolite kynurenic acid. Here, we describe the synthesis and characterization of JM6, a small-molecule prodrug inhibitor of kynurenine 3-monooxygenase (KMO). Chronic oral administration of JM6 inhibits KMO in the blood, increasing kynurenic acid levels and reducing extracellular glutamate in the brain. In a transgenic mouse model of Alzheimer's disease, JM6 prevents spatial memory deficits, anxiety-related behavior, and synaptic loss. JM6 also extends life span, prevents synaptic loss, and decreases microglial activation in a mouse model of Huntington's disease. These findings support a critical link between tryptophan metabolism in the blood and neurodegeneration, and they provide a foundation for treatment of neurodegenerative diseases.

The kynurenine pathway of tryptophan degradation is hypothesized to play an important role in Huntington disease, a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin. Neurotoxic metabolites of the kynurenine pathway, generated in microglia and macrophages, are present at increased levels in the brains of patients and mouse models during early stages of disease, but the mechanism by which kynurenine pathway up-regulation occurs in Huntington disease is unknown. Here we report that expression of a mutant huntingtin fragment was sufficient to induce transcription of the kynurenine pathway in yeast and that this induction was abrogated by impairing the activity of the histone deacetylase Rpd3. Moreover, numerous genetic suppressors of mutant huntingtin toxicity that are functionally unrelated converged unexpectedly on the kynurenine pathway, supporting a critical role for the kynurenine pathway in mediating mutant huntingtin toxicity in yeast. Histone deacetylase-dependent regulation of the kynurenine pathway was also observed in a mouse model of Huntington disease, in which treatment with a neuroprotective histone deacetylase inhibitor blocked activation of the kynurenine pathway in microglia expressing a mutant huntingtin fragment in vitro and in vivo. These findings suggest that a mutant huntingtin fragment can perturb transcriptional programs in microglia, and thus implicate these cells as potential modulators of neurodegeneration in Huntington disease that are worthy of further investigation.