Discovery of (R)-4-cyclopropyl-7,8-difluoro-5-(4-(trifluoromethyl)phenylsulfonyl)-4,5-dihydro-1H-pyrazolo[4,3-c]quinoline (ELND006) and (R)-4-cyclopropyl-8-fluoro-5-(6-(trifluoromethyl)pyridin-3-ylsulfonyl)-4,5-dihydro-2H-pyrazolo[4,3-c]quinoline (ELND007): metabolically stable γ-secretase Inhibitors that selectively inhibit the production of amyloid-ß over Notch.

Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aß generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds 30 (ELND006) and 34 (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aß generation in vivo between 30, 34, Semagacestat 41, Begacestat 42, and Avagacestat 43 in mice is made. 30 lowered Aß in the CSF of healthy human volunteers.

Amyloid precursor protein selective gamma-secretase inhibitors for treatment of Alzheimer's disease.

INTRODUCTION: Inhibition of gamma-secretase presents a direct target for lowering Aß production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. METHODS: In vitro assays monitoring inhibitor potencies at APP γ-site cleavage (equivalent to Aß40), and Notch ε-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of Aß production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain Aß was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain Aß reduction vs. Notch signaling endpoints in periphery. RESULTS: The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting Aß production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain Aß in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain Aß was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested. CONCLUSIONS: The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.

Design of an orally efficacious hydroxyethylamine (HEA) BACE-1 inhibitor in a preclinical animal model.

In this Letter, we describe our efforts to design HEA BACE-1 inhibitors that are highly permeable coupled with negligible levels of permeability-glycoprotein activity. These efforts culminate in producing 16 which lowers Αß by 28% and 32% in the cortex and CSF, respectively, in the preclinical wild type Hartley guinea pig animal model when dosed orally at 30mpk BID for 2.5days.

Antibody capture of soluble Abeta does not reduce cortical Abeta amyloidosis in the PDAPP mouse.

BACKGROUND: In vivo administration of antibodies against the amyloid-beta (Abeta) peptide has been shown to reduce and reverse the progressive amyloidosis that develops in a variety of mouse models of Alzheimer's disease (AD). This work has been extended to clinical trials where subsequent autopsy cases of AD subjects immunized against Abeta showed similar reductions in parenchymal amyloid plaques, suggesting this approach to reduce neuropathology in man is feasible. OBJECTIVE: Multiple hypotheses have been advanced to explain how anti-Abeta antibodies may lower amyloid burden. In this report, we compare approaches utilizing either plaque-binding or peptide-capturing anti-Abeta antibodies for effectiveness in reducing amyloidosis in a mouse model of AD. METHODS: A plaque-binding monoclonal antibody (3D6) and an Abeta peptide-capturing monoclonal antibody (266) were compared in chronic treatment and prevention paradigms using a transgenic mouse model of AD. The effects of antibody therapy on plaque burden and plasma clearance of Abeta were investigated by quantitative imaging and clearance studies of intravenously injected (125)I-Abeta. RESULTS: The plaque-binding antibody 3D6 was highly effective in either treatment or prevention of amyloidosis. In these studies, the peptide-capture antibody 266 showed no reduction in amyloidosis in either paradigm and showed trends towards increasing amyloidosis. Antibody 266 was also found to greatly prolong (>180-fold) the normally rapid peripheral clearance of Abeta, in contrast to that found with 3D6 (>24-fold). CONCLUSION: Reversing and preventing Alzheimer's type amyloidosis is most effectively accomplished with anti-amyloid antibodies that avidly bind plaque.

Epitope and isotype specificities of antibodies to beta -amyloid peptide for protection against Alzheimer's disease-like neuropathology.

Transgenic PDAPP mice, which express a disease-linked isoform of the human amyloid precursor protein, exhibit CNS pathology that is similar to Alzheimer's disease. In an age-dependent fashion, the mice develop plaques containing beta-amyloid peptide (Abeta) and exhibit neuronal dystrophy and synaptic loss. It has been shown in previous studies that pathology can be prevented and even reversed by immunization of the mice with the Abeta peptide. Similar protection could be achieved by passive administration of some but not all monoclonal antibodies against Abeta. In the current studies we sought to define the optimal antibody response for reducing neuropathology. Immune sera with reactivity against different Abeta epitopes and monoclonal antibodies with different isotypes were examined for efficacy both ex vivo and in vivo. The studies showed that: (i) of the purified or elicited antibodies tested, only antibodies against the N-terminal regions of Abeta were able to invoke plaque clearance; (ii) plaque binding correlated with a clearance response and neuronal protection, whereas the ability of antibodies to capture soluble Abeta was not necessarily correlated with efficacy; (iii) the isotype of the antibody dramatically influenced the degree of plaque clearance and neuronal protection; (iv) high affinity of the antibody for Fc receptors on microglial cells seemed more important than high affinity for Abeta itself; and (v) complement activation was not required for plaque clearance. These results indicate that antibody Fc-mediated plaque clearance is a highly efficient and effective process for protection against neuropathology in an animal model of Alzheimer's disease.