Background: An accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimer's disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38 MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (A ) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the bloodbrain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38 MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes.

A corollary of the neuroinflammation hypothesis is that selective suppression of neurotoxic products produced by excessive glial activation will result in neuroprotection. We report here that daily oral administration to mice of the brain-penetrant compound 4,6-diphenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine (MW01-5-188WH), a selective inhibitor of proinflammatory cytokine production by activated glia, suppressed the human amyloid-␤ (A␤) 1-42-induced upregulation of interleukin-1␤, tumor necrosis factor-␣, and S100B in the hippocampus. Suppression of neuroinflammation was accompanied by restoration of hippocampal synaptic dysfunction markers synaptophysin and postsynaptic density-95 back toward control levels. Consistent with the neuropathophysiological improvements, MW01-5-188WH therapy attenuated deficits in Y maze behavior, a hippocampal-linked task. Oral MW01-5-188WH therapy begun 3 weeks after initiation of intracerebroventricular infusion of human A␤ decreased the numbers of activated astrocytes and microglia and the cytokine levels in the hippocampus without modifying amyloid plaque burden or altering peripheral tissue cytokine upregulation in response to an in vivo inflammatory challenge. The results provide a novel integrative chemical biology proof in support of the neuroinflammation hypothesis of disease progression, demonstrate that neurodegeneration can be attenuated independently of plaque modulation by targeting innate brain proinflammatory cytokine responses, and indicate the feasibility of developing efficacious, safe, and selective therapies for neurodegenerative disorders by targeting key glial activation pathways.

We report the development of a novel, aqueous-soluble, safe, small molecule, experimental therapeutic that suppresses injury-induced, proinflammatory cytokine increases in the brain, with resultant attenuation of synaptic protein biomarker loss and improvement in hippocampus-dependent behavioral deficits. A GMP production scheme for the active pharmaceutical ingredient, compound 17, is presented. The development and large-scale availability of this novel compound allow exploration of new, potentially disease-modifying, therapeutic approaches to CNS disorders.Over-production of proinflammatory cytokines contributes to the progression of pathology in a diverse array of diseases, 1-5 and the recent approval of protein therapies 5-8 that seek to control the level or activity of proinflammatory cytokines provides a proof-of-concept for targeting proinflammatory cytokine levels in drug discovery. However, the macromolecular therapeutic approach is not as amenable to clinical use in some diseases, such as central nervous system (CNS) disorders, creating an unmet need for small molecule, brain-penetrant compounds as new classes of disease-modifying drugs. An accumulating body of evidence from animal model studies and clinical observations supports the concept 1,3,4,9,10 that development of such therapies for CNS disorders could have potential impact on both acute and chronic disorders as diverse as Alzheimer's disease (AD), stroke, traumatic brain injury, neuropathic pain, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, agerelated macular degeneration, and non-AD dementias. The prevailing hypothesis in this regard is that targeting the up-regulation of proinflammatory cytokine production by activated glia, the main cellular source of cytokines in the CNS, will alter disease progression through attenuation of the subsequent neuronal synaptic dysfunction, the cellular basis of clinical symptoms and behavioral alterations. However, there are no such current therapies available and there are no consensus molecular targets for such discovery efforts. The current state of the art requires, therefore, the use of the more classical and unbiased functional approach to drug discovery. 11 *To Whom Correspondence Should be Addressed: Phone: 312-503-0656, FAX: 312-503-0007, E-mail: m-watterson@northwestern.edu. SUPPORTING INFORMATION AVAILABLE:The contents of Supporting Information include the following: details of synthetic procedures, analytical chemistry methods, physical property determinations, NMR spectra, and biological procedures, including screening data for oral bioavailability.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affec...

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Following acute brain injury, albumin may gain access to the brain parenchyma. Clinical studies indicate a protective role for albumin in stroke but an increase in mortality associated with albumin administration following traumatic brain injury. We investigated the effects of albumin on astrocyte and microglial activation, and the role of mitogen activated protein kinases (MAPK) in these responses. Albumin activated ERK1/2, p38 MAPK and JNK signaling pathways in astrocytes, and induced the production of interleukin (IL)-1β, inducible nitric oxide (NO) synthase, the NO metabolite nitrite, and the chemokine CX3CL1 while reducing the level of S100B. The release of inflammatory markers by astrocytes was partially dependent on p38 MAPK and ERK1/2 pathways, but not JNK. In microglia, albumin exposure activated all three MAPK pathways and produced an increase in IL-1 β and nitrite. Inhibition of p38 MAPK in microglia lead to an increased level of IL1β, while inhibition of all three MAPKs suppressed the release of nitrite. These results suggest that albumin activates astrocytes and microglia, inducing inflammatory responses involved both in the mechanisms of cellular injury and repair via activation of MAPK pathways, and thereby implicate glial activation in the clinical responses to administration of albumin.