BACKGROUND:Acute neurological insults caused by infection, systemic inflammation, ischemia, or traumatic injury are often associated with breakdown of the blood-brain barrier (BBB) followed by infiltration of peripheral immune cells, cytotoxic proteins, and water. BBB breakdown and extravasation of these peripheral components into the brain parenchyma result in inflammation, oxidative stress, edema, excitotoxicity, and neurodegeneration. These downstream consequences of BBB dysfunction can drive pathophysiological processes and play a substantial role in the morbidity and mortality of acute and chronic neurological insults, and contribute to long-term sequelae. Preserving or rescuing BBB integrity and homeostasis therefore represents a translational research area of high therapeutic potential.METHODS:Induction of general and localized BBB disruption in mice was carried out using systemic administration of LPS and focal photothrombotic ischemic insult, respectively, in the presence and absence of the monoacylglycerol lipase (MAGL) inhibitor, CPD-4645. The effects of CPD-4645 treatment were assessed by gene expression analysis performed on neurovascular-enriched brain fractions, cytokine and inflammatory mediator measurement, and functional assessment of BBB permeability. The mechanism of action of CPD-4645 was studied pharmacologically using inverse agonists/antagonists of the cannabinoid receptors CB1 and CB2.RESULTS:Here, we demonstrate that the neurovasculature exhibits a unique transcriptional signature following inflammatory insults, and pharmacological inhibition of MAGL using a newly characterized inhibitor rescues the transcriptional profile of brain vasculature and restores its functional homeostasis. This pronounced effect of MAGL inhibition on blood-brain barrier permeability is evident following both systemic inflammatory and localized ischemic insults. Mechanistically, the protective effects of the MAGL inhibitor are partially mediated by cannabinoid receptor signaling in the ischemic brain insult.CONCLUSIONS:Our results support considering MAGL inhibitors as potential therapeutics for BBB dysfunction and cerebral edema associated with inflammatory brain insults.

Prominent cerebral amyloid angiopathy is often observed in the brains of elderly individuals and is almost universally found in patients with Alzheimer's disease. Cerebral amyloid angiopathy is characterized by accumulation of the shorter amyloid-β isoform(s) (predominantly amyloid-β40) in the walls of leptomeningeal and cortical arterioles and is likely a contributory factor to vascular dysfunction leading to stroke and dementia in the elderly. We used transgenic mice with prominent cerebral amyloid angiopathy to investigate the ability of ponezumab, an anti-amyloid-β40 selective antibody, to attenuate amyloid-β accrual in cerebral vessels and to acutely restore vascular reactivity. Chronic administration of ponezumab to transgenic mice led to a significant reduction in amyloid and amyloid-β accumulation both in leptomeningeal and brain vessels when measured by intravital multiphoton imaging and immunohistochemistry. By enriching for cerebral vascular elements, we also measured a significant reduction in the levels of soluble amyloid-β biochemically. We hypothesized that the reduction in vascular amyloid-β40 after ponezumab administration may reflect the ability of ponezumab to mobilize an interstitial fluid pool of amyloid-β40 in brain. Acutely, ponezumab triggered a significant and transient increase in interstitial fluid amyloid-β40 levels in old plaque-bearing transgenic mice but not in young animals. We also measured a beneficial effect on vascular reactivity following acute administration of ponezumab, even in vessels where there was a severe cerebral amyloid angiopathy burden. Taken together, the beneficial effects ponezumab administration has on reducing the rate of cerebral amyloid angiopathy deposition and restoring cerebral vascular health favours a mechanism that involves rapid removal and/or neutralization of amyloid-β species that may otherwise be detrimental to normal vessel function.

A considerable body of genetic evidence supports the pivotal role of γ-secretase in AD. Through the proteolytic cleavage of APP, the γ-secretase enzyme complex is responsible for producing Aβ peptides containing various carboxy-termini. As the final step in the production of Aβ, the γ-secretase complex represents a compelling target for pharmacological intervention aimed at reducing the levels of Aβ42, the most amyloidogenic Aβ isoform, thereby slowing or halting deposition of amyloid plaques and subsequent neuronal damage. Our goal was to use novel γ-secretase modulator (GSM) compounds to assess Aβ modulation, its impact in a plaque-bearing mouse model and multiple preclinical species. The effect of 2 months of GSM treatment on Aβ modulation and brain plaque-progression was assessed in PS1-APP mice by 2-photon imaging, immunohistochemistry and DELFIA. The effect of acute GSM dosing was assessed in brain and CSF by measuring Aβ modulation in multiple preclinical species by IP/MALDI-MS and DELFIA. Chronic administration of a GSM halts amyloid plaque accumulation in a transgenic mouse model of Alzheimer's disease as evaluated by 2-photon imaging and immunohistochemistry. Biochemical analysis of brain homogenates by DELFIA indicates Aβ42 and Aβ40 lowering in parenchyma and vascular brain fractions and no apparent accumulation of shorter Aβ fragments following chronic GSM treatment. In addition, acute administration of a GSM compound in multiple preclinical species revealed a complex shift in the brain and CSF of up to fifteen Aβ peptides as measured by DELFIA and IP/MALDI-MS. The efficacy data from preclinical models provides evidence for a potentially beneficial effect of GSM treatment in AD, by shifting cleavage away from Aβ42 and Aβ40, toward production of carboxy-terminal truncated forms Aβ37 and Aβ38; these fragments are considered less amyloidogenic and neurotoxic forms of Aβ.

Herein we describe the design and synthesis of a novel series of γ-secretase modulators (GSMs) that incorporates a pyridopiperazine-1,6-dione ring system. To align improved potency with favorable ADME and in vitro safety, we applied prospective physicochemical property-driven design coupled with parallel medicinal chemistry techniques to arrive at a novel series containing a conformationally restricted core. Lead compound 51 exhibited good in vitro potency and ADME, which translated into a favorable in vivo pharmacokinetic profile. Furthermore, robust reduction of brain Aβ42 was observed in guinea pig at 30 mg/kg dosed orally. Through chemical biology efforts involving the design and synthesis of a clickable photoreactive probe, we demonstrated specific labeling of the presenilin N-terminal fragment (PS1-NTF) within the γ-secretase complex, thus gaining insight into the binding site of this series of GSMs.

Cerebrovascular amyloid angiopathy (CAA) is characterized by accumulation of the shorter b -amyloid isoform(s) (predominantly A b 40) in the walls of leptomeningeal and cortical arterioles and is likely a contributory factor to vascular dysfunction leading to stroke and dementia in the elderly. We utilized transgenic mouse models that display prominent CAA to investigate the ability of ponezumab, an anti-A b 40 monoclonal antibody, to attenuate cerebral vessel A b accrual and to restore vascular reactivity. Following chronic administration of ponezumab to PS1APP transgenic mice, we measured amyloid and A b 40 by immunohistochemistry and intravital 2 photon imaging in both leptomeningeal and cortical vessels. We also measured A b levels in brain tissue extracts that were enriched for cerebral vascular elements using a sensitive ELISA. Following acute administration of ponezumab to APP transgenic mice, we measured a brain interstitial fluid (ISF) pool of A b 40 via microdialysis as well as pial arteriole reactivity using cranial window imaging in living animals. Chronic administration of ponezumab to PS1APP transgenic mice, led to a significant attenuation in amyloid accrual in both leptomeningeal and cortical vessels, and a significant reduction in A b levels in brain vasculature. Acute administration of ponezumab effectively mobilized a brain ISF pool of A b 40 in plaque bearing mice that was accompanied by a beneficial effect on cerebrovascular function even in vessels with a significant CAA burden. These results demonstrate that ponezumab administration to transgenic mice can decrease CAA burden, as well as restore, even after a single peripheral administration, vasomotor responses in vessels with a high CAA burden.

gamma-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic beta-amyloid peptides (i.e. A beta 42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule gamma-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of A beta 42 while not blocking the processing of gamma-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active gamma-secretase) but not full-length PS1 (inactive gamma-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the gamma-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of gamma-secretase and the modulatory binding site of certain GSMs.

We investigated the efficacy of ponezumab, an anti-Aβ 1–40 monoclonal antibody, in reducing brain vascular amyloid-beta and parenchymal plaque in the PS1APP transgenic mouse model of amyloidosis. A comprehensive study employing multiple methods to ascertain effect on the two major amyloid-beta deposits in the PS1APP mouse model has not been previously undertaken. PS1APP animals were chronically dosed with vehicle or ponezumab weekly (IP, 10mg/kg) for 6 months. The amount of amyloid in brain parenchyma and brain vasculature was assessed by immunohistochemistry and western blot analysis. DELFIA ELISA was also utilized to assess Abeta x-40, Abeta x-42 and Abeta 1-X levels in brain parenchyma, brain vasculature, and plasma. Unpaired, two-tailed t-tests were used to determine statistical significance across treatment groups. All procedures performed on animals were in accordance with regulations and established guidelines, and approved by Pfizer's Institutional Animal Care and Use Committee. Chronic ponezumab treatment led to a statistically significant reduction in the number of amyloid-beta positive brain vessels in PS1APP animals as assessed by immunohistochemistry and western blot compared to vehicle control. The number of parenchymal plaques in PS1APP mouse brain was not affected by chronic treatment with ponezumab. When administered weekly for 6 months at 10mg/kg, ponezumab was found to be effective at lowering brain vascular amyloid-beta load in the PS1APP mouse model of amyloid-beta deposition. This treatment regimen was not effective in lowering parenchymal amyloid-beta plaque numbers in this model.

The g-secretase complex, which is composed of at least four membrane bound proteins, has an intra-membrane catalytic pore for C-terminal sequence determination of the A b peptide. Our goal was to use a g -secretase modulator (GSM) tool compound, GSM-A, to further refine how to predict efficacy, from in vitro to higher order species. Photoaffinity labeling was used to elucidate the target protein within the g -secretase complex. In vitro A b modulation was measured in multiple systems: CHO and H4 cells overexpressing wild-type human APP, and Tg2576 primary cortical neurons. The effect of GSM-A on other g -secretase substrates was assessed in Tg2576 neurons and compared to that of a g -secretase inhibitor. Brain A b modulation and drug levels were measured in rat and guinea pig. CSF and Plasma A b modulation and drug levels were measured in rat, guinea pig, dog and non-human primate. All procedures performed on animals were in accordance with regulations and established guidelines, and approved by Pfizer's Institutional Animal Care and Use Committee. Photoaffinity cross-linked competition studies revealed that first and second generation imidazole GSMs do not have identical binding sites. PK-PD modeling was utilized to predict an exposure-response relationship. Free and total brain exposures were compared for prediction of efficacy using an in vitro to in vivo correlation among structurally related compounds. A β immunoprecipitation of GSM-A treated guinea pig brain followed by MALDI-MS revealed a complex shift in the A b profile. Finally, inter-compartmental biomarker relationships were established for plasma and CSF A b from rat, guinea pig, dog and non-human primate. Using empirical data and modeling from in vitro cell lines to higher order species, we have a greater understanding of A b efficacy through modulation of the g -secretase complex.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease with no known cure. Apolipoprotein E, (apoE), a major lipid transport protein in brain, is strongly linked to late onset forms of AD. Of the three human isoforms, apoE4 is associated with the highest risk and apoE2 is associated with the lowest risk for developing AD. ApoE has been found to play an important role in the clearance of toxic Aß fragments from brain, therefore increasing brain apoE levels could be an important therapeutic target for treating AD. Since apoE also plays an important role as a plasma lipid transport protein, we sought to establish an in vitro-in vivo model using T0901317, a potent LXRα agonist. Mice were dosed with T0901317 (0–50 mg/kg) for 5 consecutive days. Brain and plasma apoE levels were evaluated using a sensitive ELISA. We also measured triglyceride levels in plasma and liver and assessed mRNA levels of relevant lipogenic genes by RT-qPCR. De novo lipogenesis in Hepatoblastoma (HepG2) cells was used to correlate the effect of T0901317 on de novo lipogenesis. Brain apoE mRNA and protein levels increased in a dose-dependent fashion following treatment with T0901317. Plasma triglyceride levels and mRNA expression of hepatic lipogenic genes (FASN, ABCA1, ABCG5 and ABCG8) increased in a dose dependent manner following T0901317 treatment. We also observed a dose-dependent increase in de novo lipogenesis and expression of lipogenic genes in HepG2 cells following exposure to T0901317. We observed a significant dose-dependent increase in triglyceride levels (both plasma and liver) following administration of T0901317 that correlated well with de novo lipogenesis in treated HepG2 cells. Using T0901317 as a tool compound, we were able to establish an in vitro- in vivo model system to study the effect of increasing brain apoE levels on cardiovascular safety.

g -Secretase modulators (GSMs) which reduce the production of A b 42 have emerged as a promising class of compounds for the treatment of Alzheimer's disease (AD). The target and mechanism of action of GSMs is a topic of much interest. Some evidence has suggested that GSMs function by binding the substrate, APP, while other evidence has suggested action through direct modulation of the enzyme, γ-secretase. We have developed several GSM and GSIphotoaffinity probes as tools to gain a better understanding of their binding sites and mechanism of modulation or inhibition. These include clickable photoaffinity probes based on acid and imidazole GSMs as well as sulfonamide GSIs that contain a photoreactive group for crosslinking the compound to cellular target proteins and an alkyne handles to attach a reporter tag by click chemistry for visualization and identification of the target(s). All 3 classes of photoprobes were found to specifically label the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling of PS1-NTF by the imidazole GSM photoprobe E2012-BPyne was competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, while labeling by the acid GSM photoprobe was competed in the presence of the parent acid GSM-1, but not with E2012. In both cases, the photolabeling was not competed by the allosteric GSI BMS-708163 or the substrate docking site peptide inhibitor pep11 providing evidence that all of these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active-site directed GSI L458. Taken together our results support the hypothesis that multiple binding sites within the g -secretase complex exist, each of which may contribute to different modes of modulatory or inhibitory action. In addition, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of g -secretase and the modulatory binding site of certain GSMs.

γ‐Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to generate Aβ species, including the neurotoxic Aβ‐42. Aβ‐42 is a component of β‐amyloid plaques, and is believed to play a causative role in Alzheimer's disease (AD). γ‐Secretase modulators (GSMs) have emerged as a potential treatment for AD because they decrease the production of Aβ‐42 without affecting the processing of other critical γ‐secretase substrates. We used clickable GSM photoaffinity probes to identify the protein target of different classes of GSMs and to probe their mechanism‐of‐action. Probes (i.e., E2012‐BPyne and GSM‐5) were synthesized with a benzophenone or aryl azide for photocrosslinking the compound to the target proteins and an alkyne handle for click chemistry. Cell membranes or live cells were treated with the GSM probe with or without competitor compounds from various GSM and γ‐secretase inhibitor (GSI) classes. Samples were UV‐irradiated followed by click chemistry with biotin‐azide, enriched on Streptavidin and detected by Western blotting. These studies revealed distinct allosteric binding sites on the N‐terminal fragment of presenilin 1 (PS1‐NTF). Furthermore, E2012‐BPyne labeling of PS1‐NTF was potentiated by the presence of the active site‐directed GSI, L458, suggesting a degree of cooperativity between the protein's active site and modulatory binding sites of certain GSMs.

We have developed clickable active site-directed photoaffinity probes for γ-secretase which incorporate a photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated conjugation with azide-linked reporter tags for visualization (e.g., TAMRA-azide) or enrichment (e.g., biotin-azide) of labeled proteins. Specifically, we synthesized clickable analogs of L646 (2) and L505 (3) and validated specific labeling to presenilin–1N-terminal fragment (PS1-NTF), the active site aspartyl protease component within the γ-secretase complex. Additionally, we were able to identify signal peptide peptidase (SPP) by Western blot analysis. Furthermore, we analyzed the photo-labeled proteins in an unbiased fashion by click chemistry with TAMRA-azide followed by in-gel fluorescence detection. This approach expands the utility of γ-secretase inhibitor (GSI) photoaffinity probes in that labeled proteins can be tagged with any number of azide-linked reporters groups using a single clickable photoaffinity probe for target pull down and/or fluorescent imaging applications.

γ-Secretase is involved in the final processing of the amyloid precursor protein into a heterogeneous pool of β-amyloid (Aβ) peptides. Current Alzheimer’s disease drug discovery efforts include targeting γ-secretase activity in brain to attenuate production of the neurotoxic Aβ species. The resulting pharmacology may be affected by species-specific differences in the γ-secretase core complex or its associated proteins. Therefore, we utilized partially purified γ-secretase membranes derived from the brains of different species, including human cortex, to quantitatively assess the de novo production of both Aβ42 and Aβ40 following treatment with known γ-secretase inhibitors and modulators. We determined that the inhibitory activity of a Notch-1 sparing γ-secretase inhibitor and the modulatory activity of two classes of γ-secretase modulators were equipotent at affecting the production of Aβ across rodent and human brain membrane preparations. Additionally, the observed modulator-specific Aβ profile in isolated brain membranes across species was similar to that observed in HeLa cell membranes, and the brain and CSF of guinea pigs following oral administration. By utilizing rapidly purified γ-secretase, we were able to probe and compare the complex pharmacology of γ-secretase in the brain across common rodent species and human cortex.

The "Notch-sparing" γ-secretase inhibitor (GSI) BMS-708,163 (Avagacestat) is currently in phase II clinical trials for Alzheimer's disease. Unlike previously failed GSIs, BMS-708,163 is considered to be a promising drug candidate because of its reported Notch-sparing activity for the inhibition of Aβ production over Notch cleavage. We now report that BMS-708,163 binds directly to the presenilin-1 N-terminal fragment and that binding can be challenged by other pan-GSIs, but not by γ-secretase modulators. Furthermore, BMS-708,163 blocks the binding of four different active site-directed GSI photoaffinity probes. We therefore report that this compound acts as a nonselective γ-secretase inhibitor.

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.

Aβ42 is believed to play a causative role in Alzheimer's disease (AD) pathogenesis. γ-Secretase modulators (GSMs) are actively being pursued as potential AD therapeutics because they selectively alter the cleavage site of the amyloid precursor protein (APP) to reduce the formation of Aβ42. However, the binding partner of acid based GSMs was unresolved until now. We have developed clickable photoaffinity probes based on piperidine acetic acid GSM-1 and identified PS1 as the target within the γ-secretase complex. Furthermore, we provide evidence that allosteric interaction of GSMs with PS1 results in a conformational change in the active site of the γ-secretase complex leading to the observed modulation of γ-secretase activity.

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.