Reductions in β-amyloid concentrations in vivo by the γ-secretase inhibitors BMS-289948 and BMS-299897

Abstract

A primary pathological feature of Alzheimer's disease is β-amyloid (Aβ)-containing plaques in brain and cerebral vasculature. Reductions in the formation of Aβ peptides by γ-secretase inhibitors may be a viable therapy for reducing Aβ in Alzheimer's disease. Here we report on the effects of two orally active γ-secretase inhibitors. BMS-289948 (4-chloro-N-(2,5-difluorophenyl)-N-((1R)-{4-fluoro-2-[3-(1H-imidazol-1-yl)propyl]phenyl}ethyl)benzenesulfonamide hydrochloride) and BMS-299897 (4-[2-((1R)-1-{[(4-chlorophenyl)sulfonyl]-2,5-difluoroanilino}ethyl)-5-fluorophenyl]butanoic acid) markedly reduced both brain and plasma Aβ_1–40 in APP-YAC mice with ED₅₀ values of 86 and 22 mg/kg per os (po), respectively, for BMS-289948, and 30 and 16 mg/kg po, respectively, for BMS-299897. Both compounds also dose-dependently increased brain concentrations of APP carboxy-terminal fragments, consistent with inhibition of γ-secretase. BMS-289948 and BMS-299897 (100 mg/kg po) reduced brain and plasma Aβ_1–40 rapidly (within 20 min) and maximally within 3 h. BMS-299897 also dose-dependently reduced cortical, cerebrospinal fluid (CSF), and plasma Aβ in guinea pigs with ED₅₀ values of 30 mg/kg intraperitoneally, without affecting CSF levels of α-sAPP. The reductions in cortical Aβ correlated significantly with the reductions in both plasma (r² = 0.77) and CSF (r² = 0.61) Aβ. The decreases in Aβ were apparent at 3 and 6 h post-administration of BMS-299897, but not at 12 h. These results demonstrate that BMS-289948 and BMS-299897 are orally bioavailable, functional γ-secretase inhibitors with the ability to markedly reduce Aβ peptide concentrations in APP-YAC transgenic mice and in guinea pigs. These compounds may be useful pharmacologically for examining the effects of reductions in β-amyloid peptides in both animal models and in Alzheimer's disease.

Introduction

The presence of extracellular neuritic plaques and intracellular neurofibrillary tangles are characteristic features of Alzheimer's disease brain. Although the precise molecular mechanisms by which these lesions develop are unclear, the principal consequences are neuronal death and cognitive impairment [1]. Mutations in the amyloid precursor protein (APP) or in presenilins are associated with early onset, familial forms of Alzheimer's disease [2]. Genetic defects in APP typically result in increased production of β-amyloid peptides including Aβ_1–40 and Aβ_1–42[3], while mutations in presenilin 1 and presenilin 2 result in selective increases in Aβ_1–42 formation [4], [5]. Increased production of β-amyloid peptides, in particular the highly fibrillogenic form, Aβ_1–42, may facilitate amyloidogenesis and the formation of neuritic plaques in Alzheimer's disease [6], [7]. Mounting evidence also suggests that even less-aggregated protofibrils or oligomers of Aβ may produce the synaptic toxicity observed in Alzheimer's disease [8]. Decreasing the formation of Aβ peptides may limit the deposition of Aβ into insoluble plaques and may also diminish the ability of Aβ to form oligomers.

The β-amyloid peptides are formed through proteolytic cleavage of APP by several distinct secretase enzymes [9], [10]. β-Secretase cleaves APP to form the amino-terminus of Aβ (contained within the 99 amino acid-residue carboxy-terminal APP fragment or β-carboxy-terminal fragment) and a soluble APP derivative (β-sAPP) [11]. α-Secretase cleaves APP within the Aβ peptide domain, thereby precluding the generation of Aβ, to produce α-sAPP and an 83-residue carboxy-terminal fragment (α-carboxy-terminal fragment) [12], [13]. Both the β-secretase-cleaved and α-secretase-cleaved carboxy-terminal fragments are substrates for further proteolysis by γ-secretase, which cleaves the former fragment to produce the carboxy-terminus of Aβ and the latter fragment to form p3. Compounds that alter the proteolytic cleavage of APP, including those that inhibit β- or γ-secretase activity or facilitate α-secretase activity, can reduce the production of Aβ peptides and may have potential in treating Alzheimer;s disease [14].

The γ-secretase inhibitor N-[N-(3,5-difluor-phenacetyl)-l-alanyl]-S-phenylglycine (DAPT) was shown to reduce brain concentrations of Aβ following systemic dosing in mice transgenic for human APP_V717F (PDAPP mice) [15]. More recently, DAPT was shown to diminish brain, plasma, and CSF Aβ in mice transgenic for human APP with the “Swedish” mutation (APP_K670N/M671L; Tg2576 mice) [16]. The purpose of the present investigation was to test the effects of two novel, γ-secretase inhibitors on both peripheral and central Aβ peptide levels in two rodent species which express normal (not mutant) human APP. This is important since sporadic forms of Alzheimer's disease, which comprise greater than 90% of all cases of the disease, are generally not associated with mutant forms of APP or over-expression of Aβ[17]. APP-YAC mice were employed since these animals express normal human APP transcripts in a tissue-specific manner and at levels comparable to endogenous mouse APP [18], [19]. Guinea pigs were also utilized since their Aβ peptide sequence is identical to the human peptide [20] and expressed at comparable levels. Both APP-YAC mice and guinea pigs may therefore serve as physiological, rather than pathological, models for testing the effects of γ-secretase inhibitors on Aβ peptide formation. The present study demonstrates that the γ-secretase inhibitors BMS-289948 (4-chloro-N-(2,5-difluorophenyl)-N-((1R)-{4-fluoro-2-[3-(1H-imidazol-1-yl)propyl]phenyl}ethyl)benzenesulfonamide hydrochloride) and BMS-299897 (4-[2-((1R)-1-{[(4-chlorophenyl)sulfonyl]-2,5-difluoroanilino}ethyl)-5-fluorophenyl]butanoic acid) [21] decrease Aβ peptide levels in plasma, CSF, and brain in these two normal APP-expressing animal models.