Systemic immune-checkpoint blockade with anti-PD1 antibodies does not alter cerebral amyloid-ß burden in several amyloid transgenic mouse models.

Chronic inflammation represents a central component in the pathogenesis of Alzheimer's disease (AD). Recent work suggests that breaking immune tolerance by Programmed cell Death-1 (PD1) checkpoint inhibition produces an IFN-γ-dependent systemic immune response, with infiltration of the brain by peripheral myeloid cells and neuropathological as well as functional improvements even in mice with advanced amyloid pathology (Baruch et al., (): Nature Medicine, 22:135-137). Immune checkpoint inhibition was therefore suggested as potential treatment for neurodegenerative disorders when activation of the immune system is appropriate. Because a xenogeneic rat antibody (mAb) was used in the study, whether the effect was specific to PD1 target engagement was uncertain. In the present study we examined whether PD1 immunotherapy can lower amyloid-ß pathology in a range of different amyloid transgenic models performed at three pharmaceutical companies with the exact same anti-PD1 isotype and two mouse chimeric variants. Although PD1 immunotherapy stimulated systemic activation of the peripheral immune system, monocyte-derived macrophage infiltration into the brain was not detected, and progression of brain amyloid pathology was not altered. Similar negative results of the effect of PD1 immunotherapy on amyloid brain pathology were obtained in two additional models in two separate institutions. These results show that inhibition of PD1 checkpoint signaling by itself is not sufficient to reduce amyloid pathology and that additional factors might have contributed to previously published results (Baruch et al., (): Nature Medicine, 22:135-137). Until such factors are elucidated, animal model data do not support further evaluation of PD1 checkpoint inhibition as a therapeutic modality for Alzheimer's disease.

Cerebrospinal fluid ß-Amyloid turnover in the mouse, dog, monkey and human evaluated by systematic quantitative analyses.

BACKGROUND: Reducing brain ß-amyloid (Aß) via inhibition of ß-secretase, or inhibition/modulation of γ-secretase, has been widely pursued as a potential disease-modifying treatment for Alzheimer's disease. Compounds that act through these mechanisms have been screened and characterized with Aß lowering in the brain and/or cerebrospinal fluid (CSF) as the primary pharmacological end point. Interpretation and translation of the pharmacokinetic (PK)/pharmacodynamic (PD) relationship for these compounds is complicated by the relatively slow Aß turnover process in these compartments. OBJECTIVE: To understand Aß turnover kinetics in preclinical species and humans. METHODS: We collected CSF Aß dynamic data after ß- or γ-secretase inhibitor treatment from in-house experiments and the public domain, and analyzed the data using PK/PD modeling to obtain CSF Aß turnover rates (kout) in the mouse, dog, monkey and human. RESULTS: The kout for CSF Aß40 follows allometry (kout = 0.395 × body weight(-0.351)). The kout for CSF Aß40 is approximately 2-fold higher than the turnover of CSF in rodents, but in higher species, the two are comparable. CONCLUSION: The turnover of CSF Aß40 was systematically examined, for the first time, in multiple species through quantitative modeling of multiple data sets. Our result suggests that the clearance mechanisms for CSF Aß in rodents may be different from those in the higher species. The understanding of Aß turnover has considerable implications for the discovery and development of Aß-lowering therapeutics, as illustrated from the perspectives of preclinical PK/PD characterization and preclinical-to-clinical translation.

Cerebrospinal fluid amyloid-ß (Aß) as an effect biomarker for brain Aß lowering verified by quantitative preclinical analyses.

Reducing the generation of amyloid-ß (Aß) in the brain via inhibition of ß-secretase or inhibition/modulation of γ-secretase has been pursued as a potential disease-modifying treatment for Alzheimer's disease. For the discovery and development of ß-secretase inhibitors (BACEi), γ-secretase inhibitors (GSI), and γ-secretase modulators (GSM), Aß in cerebrospinal fluid (CSF) has been presumed to be an effect biomarker for Aß lowering in the brain. However, this presumption is challenged by the lack of quantitative understanding of the relationship between brain and CSF Aß lowering. In this study, we strived to elucidate how the intrinsic pharmacokinetic (PK)/pharmacodynamic (PD) relationship for CSF Aß lowering is related to that for brain Aß through quantitative modeling of preclinical data for numerous BACEi, GSI, and GSM across multiple species. Our results indicate that the intrinsic PK/PD relationship in CSF is predictive of that in brain, at least in the postulated pharmacologically relevant range, with excellent consistency across mechanisms and species. As such, the validity of CSF Aß as an effect biomarker for brain Aß lowering is confirmed preclinically. Meanwhile, we have been able to reproduce the dose-dependent separation between brain and CSF effect profiles using simulations. We further discuss the implications of our findings to drug discovery and development with regard to preclinical PK/PD characterization and clinical prediction of Aß lowering in the brain.

Quantitative pharmacokinetic/pharmacodynamic analyses suggest that the 129/SVE mouse is a suitable preclinical pharmacology model for identifying small-molecule γ-secretase inhibitors.

Alzheimer's disease (AD) poses a serious public health threat to the United States. Disease-modifying drugs slowing AD progression are in urgent need, but they are still unavailable. According to the amyloid cascade hypothesis, inhibition of ß- or γ-secretase, key enzymes for the production of amyloid ß (Aß), may be viable mechanisms for the treatment of AD. For the discovery of γ-secretase inhibitors (GSIs), the APP-overexpressing Tg2576 mouse has been the preclinical model of choice, in part because of the ease of detection of Aß species in its brain, plasma, and cerebrospinal fluid (CSF). Some biological observations and practical considerations, however, argue against the use of the Tg2576 mouse. We reasoned that an animal model would be suitable for GSI discovery if the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of a compound for Aß lowering in this model is predictive of that in human. In this study, we assessed whether the background 129/SVE strain is a suitable preclinical pharmacology model for identifying new GSIs by evaluating the translatability of the intrinsic PK/PD relationships for brain and CSF Aß across the Tg2576 and 129/SVE mouse and human. Using semimechanistically based PK/PD modeling, our analyses indicated that the intrinsic PK/PD relationship for brain Aßx-42 and CSF Aßx-40 in the 129/SVE mouse is indicative of that for human CSF Aß. This result, in conjunction with practical considerations, strongly suggests that the 129/SVE mouse is a suitable model for GSI discovery. Concurrently, the necessity and utilities of PK/PD modeling for rational interpretation of Aß data are established.

Enhanced clearance of Abeta in brain by sustaining the plasmin proteolysis cascade.

The amyloid hypothesis states that a variety of neurotoxic beta-amyloid (Abeta) species contribute to the pathogenesis of Alzheimer's disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Abeta production and clearance. Enzymes responsible for the degradation of Abeta are not well understood, and, thus far, it has not been possible to enhance Abeta catabolism by pharmacological manipulation. We provide evidence that Abeta catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Abeta levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Abeta oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Abeta. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Abeta levels, restore long-term potentiation deficits in hippocampal slices from transgenic Abeta-producing mice, and reverse cognitive deficits in these mice.

Design and synthesis of aminohydantoins as potent and selective human ß-secretase (BACE1) inhibitors with enhanced brain permeability.

The identification of small molecule aminohydantoins as potent and selective human ß-secretase inhibitors is reported. These analogs exhibit good brain permeability (40-70%), low nanomolar potency for BACE1, and demonstrate >100-fold selectivity for the structurally related aspartyl proteases cathepsin D, renin and pepsin. Alkyl and alkoxy groups at the meta-position of the P1 phenyl, which extend toward the S3 region of the enzyme, have contributed to the ligand's reduced affinity for the efflux transporter protein P-gp, and decreased topological polar surface area, thus resulting in enhanced brain permeability. A fluorine substitution at the para-position of the P1 phenyl has contributed to 100-fold decrease of CYP3A4 inhibition and enhancement of compound metabolic stability. The plasma and brain protein binding properties of these new analogs are affected by substitutions at the P1 phenyl moiety. Higher compound protein binding was observed in the brain than in the plasma. Two structurally diverse potent BACE1 inhibitors (84 and 89) reduced 30% plasma Aß40 in the Tg2576 mice in vivo model at 30 mg/kg p.o..

High density DNA array analysis reveals distinct genomic profiles in a subset of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) generally harbor activating mutations in KIT or platelet-derived growth facter receptor (PDGFRA). Mutations in these receptor tyrosine kinases lead to dysregulation of downstream signaling pathways that contribute to GIST pathogenesis. GISTs with KIT or PDGFRA mutations also undergo secondary cytogenetic alterations that may indicate the involvement of additional genes important in tumor progression. Approximately 10-15% of adult and 85% of pediatric GISTs do not have mutations in KIT or in PDGFRA. Most mutant adult GISTs display large-scale genomic alterations, but little is known about the mutation-negative tumors. Using genome-wide DNA arrays, we investigated genomic imbalances in a set of 31 GISTs, including 10 KIT/PDGFRA mutation-negative tumors from nine adults and one pediatric case and 21 mutant tumors. Although all 21 mutant GISTs exhibited multiple copy number aberrations, notably losses, eight of the 10 KIT/PDGFRA mutation-negative GISTs exhibited few or no genomic alterations. One KIT/PDGFRA mutation-negative tumor exhibiting numerous genomic changes was found to harbor an alternate activating mutation, in the serine-threonine kinase BRAF. The only other mutation-negative GIST with significant chromosomal imbalances was a recurrent metastatic tumor found to harbor a homozygous deletion in chromosome arm 9p. Similar findings in several KIT-mutant GISTs identified a minimal overlapping region of deletion of approximately 0.28 Mbp in 9p21.3 that includes only the CDKN2A/2B genes, which encode inhibitors of cell-cycle kinases. These results suggest that GISTs without activating kinase mutations, whether pediatric or adult, generally exhibit a much lower level of cytogenetic progression than that observed in mutant GISTs.

Impact of apolipoprotein E (ApoE) polymorphism on brain ApoE levels.

Inheritance of the apoE4 allele (epsilon4) increases the risk of developing Alzheimer's disease; however, the mechanisms underlying this association remain elusive. Recent data suggest that inheritance of epsilon4 may lead to reduced apoE protein levels in the CNS. We therefore examined apoE protein levels in the brains, CSF and plasma of epsilon2/2, epsilon3/3, and epsilon4/4 targeted replacement mice. These apoE mice showed a genotype-dependent decrease in apoE levels; epsilon2/2 >epsilon3/3 >epsilon4/4. Next, we sought to examine the relative contributions of apoE4 and apoE3 in the epsilon3/4 mouse brains. ApoE4 represented 30-40% of the total apoE. Moreover, the absolute amount of apoE3 per allele was similar between epsilon3/3 and epsilon3/4 mice, implying that the reduced levels of total apoE in epsilon3/4 mice can be explained by the reduction in apoE4 levels. In culture medium from epsilon3/4 human astrocytoma or epsilon3/3, epsilon4/4 and epsilon3/4 primary astrocytes, apoE4 levels were consistently lower than apoE3. Secreted cholesterol levels were also lower from epsilon4/4 astrocytes. Pulse-chase experiments showed an enhanced degradation and reduced half-life of newly synthesized apoE4 compared with apoE3. Together, these data suggest that astrocytes preferentially degrade apoE4, leading to reduced apoE4 secretion and ultimately to reduced brain apoE levels. Moreover, the genotype-dependent decrease in CNS apoE levels, mirror the relative risk of developing AD, and suggest that low levels of total apoE exhibited by epsilon4 carriers may directly contribute to the disease progression, perhaps by reducing the capacity of apoE to promote synaptic repair and/or Abeta clearance.

Begacestat (GSI-953): a novel, selective thiophene sulfonamide inhibitor of amyloid precursor protein gamma-secretase for the treatment of Alzheimer's disease.

The presenilin containing gamma-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. gamma-Secretase catalyzes the final step in the generation of Abeta(40) and Abeta(42) peptides from APP. Amyloid beta-peptides (Abeta peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Abeta peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide gamma-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Abeta production with low nanomolar potency in cellular and cell-free assays of gamma-secretase function, and displaces a tritiated analog of GSI-953 from enriched gamma-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Abeta levels, and a reversal of contextual fear-conditioning deficits that are correlated with Abeta load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Abeta levels, confirming pharmacodynamic activity of GSI-953 in humans.

(S)-N-(5-Chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol a Notch-1-sparing gamma-secretase inhibitor.

Accumulation of beta-amyloid (Abeta), produced by the proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase, is widely believed to be associated with Alzheimer's disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) gamma-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol 7.b.2 (Abeta(40/42) EC(50)=28 nM), which is efficacious in reduction of Abeta production in vivo.

BACE-1 inhibitors part 2: identification of hydroxy ethylamines (HEAs) with reduced peptidic character.

This paper describes the discovery of non-peptidic, potent, and selective hydroxy ethylamine (HEA) inhibitors of BACE-1 by replacement of the prime side of a lead di-amide 2. Inhibitors with nanosmolar potency and high selectivity were identified. Depending on the nature of the P(1)(') and P(2)(') substituents, two different binding modes were observed in X-ray co-crystal structures.

BACE-1 inhibitors part 1: identification of novel hydroxy ethylamines (HEAs).

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. Herein, is described the lead generation effort which resulted, with the support of X-ray crystallography, in the discovery of potent inhibitors based on a hydroxy ethylamine (HEA) transition-state mimetic. These inhibitors were capable of lowering amyloid production in a cell-based assay.

BACE-1 inhibitors part 3: identification of hydroxy ethylamines (HEAs) with nanomolar potency in cells.

This article is focusing on further optimization of previously described hydroxy ethylamine (HEA) BACE-1 inhibitors obtained from a focused library with the support of X-ray crystallography. Optimization of the non-prime side of our inhibitors and introduction of a 6-membered sultam substituent binding to Asn-294 as well as a fluorine in the C-2 position led to derivatives with nanomolar potency in cell-based assays.

Design and Synthesis of Clinical Candidate PF-06751979: A Potent, Brain Penetrant, ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Lacking Hypopigmentation.

A major challenge in the development of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors for the treatment of Alzheimer's disease is the alignment of potency, drug-like properties, and selectivity over related aspartyl proteases such as Cathepsin D (CatD) and BACE2. The potential liabilities of inhibiting BACE2 chronically have only recently begun to emerge as BACE2 impacts the processing of the premelanosome protein (PMEL17) and disrupts melanosome morphology resulting in a depigmentation phenotype. Herein, we describe the identification of clinical candidate PF-06751979 (64), which displays excellent brain penetration, potent in vivo efficacy, and broad selectivity over related aspartyl proteases including BACE2. Chronic dosing of 64 for up to 9 months in dog did not reveal any observation of hair coat color (pigmentation) changes and suggests a key differentiator over current BACE1 inhibitors that are nonselective against BACE2 in later stage clinical development.

Aminomethyl-Derived Beta Secretase (BACE1) Inhibitors: Engaging Gly230 without an Anilide Functionality.

A growing subset of ß-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogues with an excellent balance of ADME properties and potency; however, potential drug-drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound 16, which exhibits robust in vivo efficacy as a BACE1 inhibitor.

Chemoproteomic profiling reveals that cathepsin D off-target activity drives ocular toxicity of ß-secretase inhibitors.

Inhibition of ß-secretase BACE1 is considered one of the most promising approaches for treating Alzheimer's disease. Several structurally distinct BACE1 inhibitors have been withdrawn from development after inducing ocular toxicity in animal models, but the target mediating this toxicity has not been identified. Here we use a clickable photoaffinity probe to identify cathepsin D (CatD) as a principal off-target of BACE1 inhibitors in human cells. We find that several BACE1 inhibitors blocked CatD activity in cells with much greater potency than that displayed in cell-free assays with purified protein. Through a series of exploratory toxicology studies, we show that quantifying CatD target engagement in cells with the probe is predictive of ocular toxicity in vivo. Taken together, our findings designate off-target inhibition of CatD as a principal driver of ocular toxicity for BACE1 inhibitors and more generally underscore the power of chemical proteomics for discerning mechanisms of drug action.

Sublethal effects of cadmium on prey choice and capture efficiency in juvenile brook trout (Salvelinus fontinalis).

Prey choice in juvenile brook trout (Salvelinus fontinalis) was investigated under sublethal Cd stress in the presence of alternative prey types. Following 30-d exposures to either 0, 0.5, or 5.0 microg/L Cd, individual fish held in artificial stream channels were presented with both motile (Baetis tricaudatus) and nonmotile (Chironomus tentans) prey items and their foraging behavior and capture success was observed. The Cd-exposed trout were found to express a significant preference for C. tentans larvae as alternative prey to B. tricaudatus nymphs. Nevertheless, these preliminary findings indicate that, irrespective of prey choice, the capture efficiency of Cd-stressed trout declined by 20 to 55% with increasing Cd concentration when compared with control fish, and the activity of individual trout increased by 25% over controls, although these responses were not statistically significant. Cadmium significantly affected fish condition factors: The condition factor of control fish increased by 34% over the 30-d period, and Cd-exposed fish declined in condition by 12 to 18%. It is hypothesized that such a switch by fish to feeding on benthic prey under natural exposure conditions and their proximity to contaminated sediment may further exacerbate the sublethal effects of Cd on these individuals by intensifying or prolonging Cd exposure through a combination of trophic transfer and altered foraging behavior.

Behavioral responses to sublethal cadmium exposure within an experimental aquatic food web.

The foraging and predator-avoidance behaviors of mayfly (Baetis tricaudatus) nymphs and predator-prey interactions were investigated under differing combinations of cadmium concentration and predation risk. Predators consisted of stonefly (Kogotus nonus) nymphs, juvenile brook trout (Salvelinus fontinalis), and longnose dace (Rhinichthys cataractae), either alone or in combination. Organisms were exposed to 0, 0.5, or 5.0 microg/L Cd for 7 d (invertebrates) or 30 d (vertebrates) prior to observation in artificial stream channels. Behavioral observations (20-min duration) were made both day and night over a 24-h period. Mayfly feeding rate was assessed by image analysis of grazed diatom mats cultured on ceramic tiles. Both predators and cadmium produced significant effects on the behavior of B. tricaudatus and K. nonus nymphs, with mayflies showing a hierarchical response in the presence of multiple predators. A significant increase in activity was observed in S. fontinalis exposed to 5.0 microg/L Cd, with significant decreases in capture efficiency of prey items apparent at 0.5 microg/L Cd. Conversely, the foraging behavior of R. cataractae was unaffected by cadmium, possibly as a result of species-specific cadmium tolerance. Such sublethal effects of a contaminant on the energy budgets of individuals and the interactions between species have implications for community structure in aquatic food webs.

Tool compounds robustly increase turnover of an artificial substrate by glucocerebrosidase in human brain lysates.

Mutations in glucocerebrosidase (GBA1) cause Gaucher disease and also represent a common risk factor for Parkinson's disease and Dementia with Lewy bodies. Recently, new tool molecules were described which can increase turnover of an artificial substrate 4MUG when incubated with mutant N370S GBA1 from human spleen. Here we show that these compounds exert a similar effect on the wild-type enzyme in a cell-free system. In addition, these tool compounds robustly increase turnover of 4MUG by GBA1 derived from human cortex, despite substantially lower glycosylation of GBA1 in human brain, suggesting that the degree of glycosylation is not important for compound binding. Surprisingly, these tool compounds failed to robustly alter GBA1 turnover of 4MUG in the mouse brain homogenate. Our data raise the possibility that in vivo models with humanized glucocerebrosidase may be needed for efficacy assessments of such small molecules.

Discovery of a series of efficient, centrally efficacious BACE1 inhibitors through structure-based drug design.

The identification of centrally efficacious ß-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease (AD) has historically been thwarted by an inability to maintain alignment of potency, brain availability, and desired absorption, distribution, metabolism, and excretion (ADME) properties. In this paper, we describe a series of truncated, fused thioamidines that are efficiently selective in garnering BACE1 activity without simultaneously inhibiting the closely related cathepsin D or negatively impacting brain penetration and ADME alignment, as exemplified by 36. Upon oral administration, these inhibitors exhibit robust brain availability and are efficacious in lowering central Amyloid ß (Aß) levels in mouse and dog. In addition, chronic treatment in aged PS1/APP mice effects a decrease in the number and size of Aß-derived plaques. Most importantly, evaluation of 36 in a 2-week exploratory toxicology study revealed no accumulation of autofluorescent material in retinal pigment epithelium or histology findings in the eye, issues observed with earlier BACE1 inhibitors.