Nrf2 is a transcription factor regulating expression of the Phase II Antioxidant Response and plays an important role in neuroprotection and detoxification. Nrf2 activation is inhibited by interaction with Keap1. Covalent Keap1 inhibitors such as dimethyl fumarate (DMF) and RTA-408 are either on the market or in late stage clinical trials which implies potential benefit of Nrf2 activation. Activation of Nrf2 by disrupting Nrf2-Keap1 interaction through a non-covalent small molecule is an attractive approach with the promise of greater selectivity. However, there are no known non-covalent Nrf2 activators with acceptable pharmacokinetic properties to test the hypothesis in vivo. Based on our early reported work, using structural-based design, followed by extensive SAR exploration, we have identified a novel series of non-covalent Nrf2 activators, with sub-nanomolar binding affinity on Keap1 and single digit nanomolar activity in an astrocyte assay. A representative analog shows excellent oral PK and good Nrf2-dependent gene inductions in kidney. These results provide a peripheral in vivo tool compound to validate the biology of non-covalent activation of Nrf2.

Dimethyl fumarate (DMF) is indicated for the treatment of relapsing multiple sclerosis and may exert therapeutic effects via activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway. Following oral DMF administration, central nervous system (CNS) tissue is predominantly exposed to monomethyl fumarate (MMF), the bioactive metabolite of DMF, which can stabilize NRF2 and induce antioxidant gene expression; however, the detailed NRF2-dependent mechanisms modulated by MMF that lead to cytoprotection are unknown. Our data identify a mechanism for MMF-mediated cytoprotection in human astrocytes that functions in an OSGIN1-dependent manner, specifically via upregulation of the OSGIN1-61 kDa isoform. NRF2-dependent OSGIN1 expression induced P53 nuclear translocation following MMF administration, leading to cell-cycle inhibition and cell protection against oxidative challenge. This study provides mechanistic insight into MMF-mediated cytoprotection via NRF2, OSGIN1, and P53 in human CNS-derived cells and contributes to our understanding of how DMF may act clinically to ameliorate pathological processes in neurodegenerative disease.

Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology, and neurology. N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A (11) C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding.

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aβ in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aβ EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a β-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aβ in guinea pigs. The therapeutic index between Aβ reductions and changes in B-cell populations were studied for compound 10 h.

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel gamma-secretase inhibitor that reduces amyloid-beta (Abeta) production with an in vitro IC(50) of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC(50) of 2.1 microM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Abeta in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Abeta. To further characterize Abeta dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Abeta, and the magnitude and duration of Abeta lowering exceeded those of the reductions in B-cell endpoints. Other gamma-secretase inhibitors have shown high potency at elevating Abeta in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Abeta11-40 and Abeta1-43 at doses that potently inhibited Abeta1-40 and Abeta1-42. PF-3084014, like previously described gamma-secretase inhibitors, preferentially reduced Abeta1-40 relative to Abeta1-42. Potency at Abeta relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

Tau accumulation and hyperphosphorylation occurs in brain regions associated with neurodegeneration and synaptic loss in Alzheimer's Disease (AD) and correlates with cognitive decline. Multiple sites on tau are hyperphosphorylated in human AD brain. Mutagenesis studies in Drosophila suggest the KXGS phosphorylation sites within the microtubule binding domain, Ser262 and Ser356, are required for both downstream phosphorylation of Tau within PDPK sites and Tau-induced neurodegeneration. Here we sought to determine if the KXGS sites played a similar role in cultured mouse hippocampal neurons. Neuronal cultures were prepared from either Tg4510 (inducible Tau) transgenic or control mouse embryos at E17. For AAV transductions, hippocampal neurons were transfected with an Adeno-associated virus serotype 8(AAV8) expressing wild type Tau 441 and destabilized YFP (ds YFP) at 4 DIV and cultured for 14 days. To assess the effects of Ser262 and Ser356 phosphorylation, a virus was constructed expressing a mutant form of Tau where S262 and S356 were mutated to alanine residues by site directed mutagenesis (AAV8 S262AS356A). Transfections with virus expressing only the ds YFP (AAV8 dsYFP) were used as a negative control. Immunocytochemistry was used to evaluate effects of Tau expression on Tau localization, phosphorylation and synaptic marker expression. Changes in VGLUT and Drebrin were quantified using the Cellomics Arrayscan. Neurons cultured from the Tg4510 P301L Tau transgenic mice showed abnormal axonal morphology and a loss of pre-synaptic markers, synapsin and VGLUT on the soma and proximal dendrites of human Tau expressing cells. Quantification of the VGLUT and Drebrin spots showed a significant reduction of VGLUT and drebrin spot intensity, area and number in the absence of changes in neuronal number. Overexpression of wild type Tau showed a similar effect. In contrast, the AAV8 Tau S262AS356A virus showed significantly less VGLUT or Drebrin loss relative to the AAV8 Tau virus. These data suggests that Tau overexpression can disrupt formation and/or maintenance of synapses through both autonomous and non-autonomous mechanisms and that phosphorylation at the KXGS sites plays a role in Tau-induced synaptic loss.

Utilizing structure-based drug design, a 4-aminoimidazole heterocyclic core was synthesized as a replacement for a 2-aminothiazole due to potential metabolically mediated toxicity. The synthetic route utilized allowed for ready synthesis of 1-substituted-4-aminoimidazoles. SAR exploration resulted in the identification of a novel cis-substituted cyclobutyl group that gave improved enzyme and cellular potency against cdk5/p25 with up to 30-fold selectivity over cdk2/cyclin E.

Reducing levels of Aβ, the principal component of amyloid plaques, has been proposed as a disease-modifying approach for the treatment of Alzheimer's disease. Here we report the efficacy and exposure over time of PF-3084014, a novel potent γ-secretase inhibitor. PF-3084014 was dosed at 0.03–10 mg/kg subcutaneously in guinea pigs at multiple time points ranging from 0.75 to 30 hours. Aβ1-X was measured in brain, CSF, and plasma at each dose and time point, and PF-3084014 levels were measured in brain and plasma at selected time points. Dose-response relationships were observed in all compartments. At 10 mg/kg, Aβ levels were reduced by 70% in brain and plasma, and 50% in CSF, which was maintained at 30 hours post-dose. At all time points assessed, a linear relationship was observed between efficacy and exposure. PF-3084014 exhibited a brain-to-plasma ratio of approximately 1, and brain and plasma Aβ levels were reduced to a similar degree. Other γ-secretase inhibitors have been shown to strongly elevate plasma Aβ at low doses or in a rebound following a period of inhibition in guinea pigs; however, this pattern was not observed with PF-3084014. To further determine changes in specific Aβ isoforms, PF-3084014 was administered to young (plaque-free) Tg2576 mice. Brain, CSF, and plasma were harvested following PF-3084014 dosing at 1–18 mg/kg. In mice, a lower brain-to-plasma ratio correlated with greater Aβ reductions in plasma versus brain. Aβ1-X, Aβ1–40, and Aβ1–42 were reduced in a dose-responsive manner; at 18 mg/kg Aβ levels were reduced by 78% in brain, 72% in CSF, and 92% in plasma. As in guinea pigs, a linear efficacy-exposure relationship across doses was observed in mice. Aβ1–40 was most potently inhibited in all compartments, followed closely by Aβ1-X. Aβ1–42 showed approximately 20% less reduction than Aβ1–40 in all compartments, a feature shared by other published “nonselective” γ-secretase inhibitors. PF-3084014 effectively reduces Aβ levels in brain, plasma, and CSF, and exhibits a linear efficacy-exposure relationship across doses.

Postoperative cognitive dysfunction, confusion, and delirium are common after general anesthesia in the elderly, with symptoms persisting for months or years in some patients. Even middle-aged patients are likely to have postoperative cognitive dysfunction for months after surgery, and Alzheimer's disease (AD) patients appear to be particularly at risk of deterioration after anesthesia. Several investigators have thus examined whether general anesthesia is associated with AD, with some studies suggesting that exposure to anesthetics may increase the risk of AD. However, little is known on the biochemical consequences of anesthesia on pathogenic pathways in vivo. Here, we investigated the effect of anesthesia on tau phosphorylation and amyloid precursor protein (APP) metabolism in mouse brain. We found that, regardless of the anesthetic used, anesthesia induced rapid and massive hyperphosphorylation of tau, rapid and prolonged hypothermia, inhibition of Ser/Thr PP2A (protein phosphatase 2A), but no changes in APP metabolism or Abeta (beta-amyloid peptide) accumulation. Reestablishing normothermia during anesthesia completely rescued tau phosphorylation to normal levels. Our results indicate that changes in tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity and subsequent hyperphosphorylation of tau. These findings call for careful monitoring of core temperature during anesthesia in laboratory animals to avoid artifactual elevation of protein phosphorylation. Furthermore, a thorough examination of the effect of anesthesia-induced hypothermia on the risk and progression of AD is warranted.

Increasing beta-amyloid (Aβ) clearance may alter the course of Alzheimer's disease progression and attenuate amyloid plaque pathology. Insulin-like growth factor I (IGF-1) augmentation has been suggested to increase Aβ clearance by facilitating transport of Aβ out of the brain. The availability of safe agents that increase IGF-1 levels therefore makes IGF-1 elevation an attractive target for disease modifying therapy in AD. The present series of studies sought to replicate published paradigms in which peripheral IGF-1 administration lowered brain Aβ acutely, with reduction in plaque pathology after chronic treatment. Thus Aβ levels were measured in several animal models following treatments that elevated IGF-1. Administration of IGF-1 to young or old rats for up to 3 days had no effect on Aβ levels in brain, CSF, or plasma. In adult beagles, 4 days of dosing with the growth hormone secretagogue, CP-424391, doubled baseline plasma IGF-1 levels, yet failed to alter CSF or plasma Aβ. 5-day treatment of young Tg2576 mice with IGF-1 produced robust elevations of IGF-1 levels in plasma, but no effects on Aβ were detected in brain, CSF, or plasma. Finally, 11-month-old Tg2576 mice were implanted with subcutaneous minipumps delivering IGF-1 for 1 month. No significant changes in Aβ (by ELISA or Western blot), plaque pathology, or phospho-tau epitopes were detected. These results do not demonstrate acute or chronic actions of peripherally administered IGF-1 on Aβ levels or the phosphorylation state of tau and therefore do not suggest any disease-modifying benefits of IGF-1 restorative therapy for AD through these mechanisms.

1Among many documented changes in Alzheimer's Disease brain, increased tau kinase activities have become a focus of therapeutic strategies due to their proposed role in contributing to the neurofibrillary pathology of the disease. Several distinct kinases have been suggested to regulate the phosphorylation state of tau at a variety of sites. While no consensus has been reached regarding the relative roles of specific kinases in initiation or progression of neuropathology, the pharmaceutical industry and some academic institutions are actively engaged in the development of selective kinase inhibitors as an approach to prevent AD progression. Many factors figure into the process of selecting which kinase to target for development of a therapeutic agent. This presentation will summarize some of the approaches used and obstacles encountered during efforts to develop inhibitors for two tau kinases, cdk5 and GSK3β. To evaluate the relative roles of cdk5 and GSK3β in regulating the phosphorylation of tau at various sites in cellular and neuronal systems. Semi- quantitative solution immunoassays were used to measure tau phosphoepitopes in CHO cells engineered with inducible expression of human tau and either cdk5/p25 or GSK3β. Tau phosphorylation sites were also evaluated in metabolically active mouse brain miniprisms or neurons incubated with kinase inhibitors. Cellular expression of either kinase increased tau phosphorylation at the AT-8 epitope. However, the two kinases displayed distinct profiles of tau phosphorylation at other epitopes. The AT-8 immunoreactivity in each cell line was selectively reversed by the addition of the respective tau kinase inhibitor, demonstrating specificity of this common response for each kinase. Similarly to observations in the GSK3β cell line, evaluations in mouse brain miniprisms and neurons revealed inhibition of tau phosphorylation at AT-8 and other epitopes by GSK3 inhibitors. In contrast, inhibitors of cdk5 demonstrated little or no effect on AT-8 or other measured tau phosphorylation sites in these tissues. While many kinases are likely involved in the normal regulation of tau phosphorylation in neural tissue, these data support a more dominant role of GSK3, as compared to cdk5, in the regulation of normal tau phosphorylation.

LY-450139 is a gamma-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-beta (Abeta) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Abeta responses to LY-450139 in the guinea pig, a nontransgenic model that has an Abeta sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma Abeta levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Abeta levels at early time points, with return to baseline within hours. Higher doses inhibited Abeta levels in all compartments at early time points, but elevated plasma Abeta levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma Abeta was significantly inhibited at 10-30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Abeta elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted Abeta were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the gamma-secretase complex, eliciting Abeta lowering at high concentrations but Abeta elevation at low concentrations.

LY-450139 is a (cid:2) -secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust ele- vations of plasma amyloid- (cid:1) (A (cid:1) ) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate A (cid:1) responses to LY-450139 in the guinea pig, a nontransgenic model that has an A (cid:1) sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2–60 mg/kg), and brain, cerebrospinal fluid, and plasma A (cid:1) levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma A (cid:1) levels at early time points, with return to baseline within hours. Higher doses inhibited A (cid:1) levels in all compartments at early time points, but elevated plasma A (cid:1) levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcu- taneous minipumps delivering LY-450139 (0.3–30 mg/kg/day) for 5 days. Plasma A (cid:1) was significantly inhibited at 10–30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of A (cid:1) elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted A (cid:1) were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the (cid:2) -secretase complex, eliciting A (cid:1) lowering at high concentrations but A (cid:1) elevation at low concentrations.

The present study evaluated the effects of two novel N-methyl-D-aspartate (NMDA) receptor blockers and ifenprodil derivatives, CP-101,606 and CP-101,581, and their racemic mixture CP-98,113, on spatial memory and regional cerebral edema following experimental fluid-percussion (FP) brain injury in the rat (n = 66). Fifteen minutes after brain injury (2.5 atm), animals received either (1) CP-98,113 (5 mg/kg, i.p., n = 11), (2) CP-101,581 (5 mg/kg, i.p., n = 13), (3) CP-101,606 (6.5 mg/kg, i.p., n = 12), or (4) DMSO vehicle (equal volume, n = 12); followed by a continuous 24-h subcutaneous infusion of drug at a rate of 1.5 mg/kg/h by means of miniature osmotic (Alzet) pumps implanted subcutaneously. Control (uninjured) animals were subjected to identical anesthesia and surgery without injury and received DMSO vehicle (n = 8); CP-98,113 (5 mg/kg, i.p., n = 3); CP-101,581 (5 mg/kg, i.p., n = 3); or CP-101,606 (6.5 mg/kg, i.p., n = 3). FP brain injury produced a significant cognitive impairment assessed at 2 days postinjury using a well-characterized testing paradigm of visuospatial memory in the Morris Water Maze (MWM) (p < 0.001). Administration of either CP-98,113, CP-101,581, or CP-101,606 had no effect on sham (uninjured) animals, but significant attenuated spatial memory impairment assessed at 2 days postinjury (p = 0.004, p = 0.02, or p = 0.02, respectively). Administration of CP-89,113 but not CP-101,581 or CP-101,606 significantly reduced the extent of regional cerebral edema in the cortex adjacent to the site of injury (p < 0.05) and in the ipsilateral hippocampus (p < 0.05) and thalamus (p < 0.05). These results suggest that excitatory neurotransmission may play a pivotal role in the pathogenesis of memory dysfunction following traumatic brain injury (TBI) and that blockade of the NMDA receptor may significantly attenuate cognitive deficits associated with TBI.

The synthesis of GYKI 52466 has been achieved in a short six step sequence which proceeds in an overall yield of 11.5%.

We examined the ability of the neurotoxin,l-ß-methylaminoalanine (l-BMAA), to inhibit [3H]glutamate binding to rat brain synaptic junctions. In a tris(hydroxymethyl)aminomethane acetate buffer,l-BMAA did not affect [3H]glutamate binding (IC50 > 10 mM). However, in the presence of ammonium bicarbonate (20 mM)l-BMAA blocked [3H]glutamate binding with an IC50 of 1 mM. This inhibition was not caused by ammonium ion since other ammonium salts were inactive. Furthermore, identical inhibition was obtained in the presence of potassium bicarbonate. Bicarbonate ion did not alter the ability ofN-methyl-d-aspartic acid to block glutamate binding. These results indicate that bicarbonate ion is required for the interaction ofl-BMAA with the glutamate receptor and may account for the observation that beta-methylaminoalanine is neurotoxic in vitro only in the presence of bicarbonate.