Jonathan D. Best

The Novel γ Secretase Inhibitor N-[cis-4-[(4-Chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) Reduces Amyloid Plaque Deposition without Evidence of Notch-Related Pathology in the Tg2576 Mouse

There is a substantial body of evidence indicating that β-amyloid peptides (Aβ) are critical factors in the onset and development of Alzheimers disease (AD). One strategy for combating AD is to reduce or eliminate the production of Aβ through inhibition of the γ-secretase enzyme, which cleaves Aβ from the amyloid precursor protein (APP). We demonstrate here that chronic treatment for 3 months with 3 mg/kg of the potent, orally bioavailable and brain-penetrant γ-secretase inhibitor N-[cis-4-[(4-chlorophenyl)-sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) attenuates the appearance of amyloid plaques in the Tg2576 mouse. These reductions in plaques were also accompanied by a decrease in the level of reactive gliosis. The morphometric and histological measures agreed with biochemical analysis of Aβ(40) and Aβ(42) in the cortex. Interestingly, the volume of the plaques across treatment groups did not change, indicating that reducing Aβ levels does not significantly alter deposit growth once initiated. Furthermore, we demonstrate that these beneficial effects can be achieved without causing histopathological changes in the ileum, spleen, or thymus as a consequence of blockade of the processing of alternative substrates, such as the Notch family of receptors. This indicates that in vivo a therapeutic window between these substrates seems possible—a key concern in the development of this approach to AD. An understanding of the mechanisms whereby MRK-560 shows differentiation between the APP and Notch proteolytic pathway of γ-secretase should provide the basis for the next generation of γ-secretase inhibitors.

In Vivo Characterization of Aβ(40) Changes in Brain and Cerebrospinal Fluid Using the Novel γ-Secretase Inhibitor N-[cis-4-[(4-Chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) in the Rat

Plaques in the parenchyma of the brain containing Aβ peptides are one of the hallmarks of Alzheimers disease. These Aβ peptides are produced by the final proteolytic cleavage of the amyloid precursor protein by the intramembraneous aspartyl protease γ-secretase. Thus, one approach to lowering levels of Aβ has been via the inhibition of the γ-secretase enzyme. Here, we report a novel, bioavailable γ-secretase inhibitor, N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) that displayed oral pharmacokinetics suitable for once-a-day dosing. It was able to markedly reduce Aβ in the brain and cerebrospinal fluid (CSF) in the rat, with ED50 values of 6 and 10 mg/kg, respectively. Time-course experiments using MRK-560 demonstrated these reductions in Aβ could be maintained for 24 h, and comparable temporal reductions in rat brain and CSF Aβ(40) further suggested that these two pools of Aβ are related. This relationship between the brain and CSF Aβ was maintained when MRK-560 was dosed once a day for 2 weeks, and accordingly, when all the data for the dose-response curve and time courses were correlated, a strong association was observed between the brain and CSF Aβ levels. These results demonstrate that MRK-560 is an orally bioavailable γ-secretase inhibitor with the ability to markedly reduce Aβ peptide in the brain and CSF of the rat and confirm the utility of the rat for assessing the effects of γ-secretase inhibitors on central nervous system Aβ(40) levels in vivo.

In vivo characterisation of Aβ(40) changes in brain and CSF using the novel γ-secretase inhibitor MRK-560 (N-[cis-4-[(4-chlorophenyl)sulfonyl]-4- (2,5-difluorophenyl)cyclohexyl]-1,1,1- trifluoromethanesulfonamide) in the rat

Plaques in the parenchyma of the brain containing Aβ peptides are one of the hallmarks of Alzheimers disease. These Aβ peptides are produced by the final proteolytic cleavage of the amyloid precursor protein by the intramembraneous aspartyl protease γ-secretase. Thus, one approach to lowering levels of Aβ has been via the inhibition of the γ-secretase enzyme. Here, we report a novel, bioavailable γ-secretase inhibitor, N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) that displayed oral pharmacokinetics suitable for once-a-day dosing. It was able to markedly reduce Aβ in the brain and cerebrospinal fluid (CSF) in the rat, with ED50 values of 6 and 10 mg/kg, respectively. Time-course experiments using MRK-560 demonstrated these reductions in Aβ could be maintained for 24 h, and comparable temporal reductions in rat brain and CSF Aβ(40) further suggested that these two pools of Aβ are related. This relationship between the brain and CSF Aβ was maintained when MRK-560 was dosed once a day for 2 weeks, and accordingly, when all the data for the dose-response curve and time courses were correlated, a strong association was observed between the brain and CSF Aβ levels. These results demonstrate that MRK-560 is an orally bioavailable γ-secretase inhibitor with the ability to markedly reduce Aβ peptide in the brain and CSF of the rat and confirm the utility of the rat for assessing the effects of γ-secretase inhibitors on central nervous system Aβ(40) levels in vivo.

A comparative assessment of γ-secretase activity in transgenic and non-transgenic rodent brain

Abstract Amyloid-beta (Aβ) deposits are one of the hallmarks of the neuropathological degeneration observed in Alzheimers disease (AD) and Aβ concentrations have been reported to vary in different brain regions of AD patients. Aβ is produced by the sequential cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase, respectively. Previous studies have shown that over-expression of the γ-secretase complex leads to increased γ-secretase proteolytic activity increasing Aβ production. However, it is not known whether brain regions with highest Aβ concentration also express relatively higher levels of γ-secretase activity. Accordingly, the relationship between Aβ levels and γ-secretase activity across brain regions was investigated and correlated in the brains of transgenic and non-transgenic rodents commonly used in AD research. The data demonstrated that Aβ levels do vary in different brain regions in both transgenic and non-transgenic mice but are not correlated with regional γ-secretase activity. Furthermore, this study demonstrated that while mutations in the APP and PS1 sequences affect the absolute Aβ levels this is not reflected in an increase in γ-secretase proteolytic activity. The data in the current paper indicate that this assay is able to measure the level of γ-secretase activity in rodent species. Using this methodology will aid our understanding of physiological γ-secretase function.