Plaques in the parenchyma of the brain containing A peptides are one of the hallmarks of Alzheimer's disease. These A peptides are produced by the final proteolytic cleavage of the amyloid precursor protein by the intramembraneous aspartyl protease ␥-secretase. Thus, one approach to lowering levels of A has been via the inhibition of the ␥-secretase enzyme. Here, we report a novel, bioavailable ␥-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 A in the brain and cerebrospinal fluid (CSF) in the rat, with ED 50 values of 6 and 10 mg/kg, respectively. Time-course experiments using MRK-560 demonstrated these reductions in A could be maintained for 24 h, and comparable temporal reductions in rat brain and CSF A (40) further suggested that these two pools of A are related. This relationship between the brain and CSF A 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 A levels. These results demonstrate that MRK-560 is an orally bioavailable ␥-secretase inhibitor with the ability to markedly reduce A peptide in the brain and CSF of the rat and confirm the utility of the rat for assessing the effects of ␥-secretase inhibitors on central nervous system A(40) levels in vivo.The neuropathology of AD is characterized by extracellular protein deposits in the brain parenchyma known as plaques, along with intracellular neurofibrillary tangles, which are comprised of hyperphosphorylated tau protein. The plaques are mainly made up of the A proteins A(1-40) and (1-42), cleavage products of the APP of which the majority is the more fibrillogenic (1-42) form (Selkoe, 2001). These observations, along with a number of mutations in the APP gene that lead to early onset familial AD, give evidence of the direct involvement of aberrant APP processing in AD (Hardy, 1997).Consequently, much effort has focused on ways to inhibit the production of A. The enzymes responsible for processing APP into A are the aspartyl proteases, -site APP cleaving enzyme (-secretase) and ␥-secretase (Churcher and Beher, 2005). Although an attractive target for drug discovery, inhibition of -secretase has proved challenging in terms of identification of small molecules for therapeutic use (Selkoe and Schenk, 2003;Middendorp et al., 2004). An alternative strategy has been to inhibit ␥-secretase, an enzyme complex composed of at least four different protein subunits: presenilin (an aspartyl protease, mutations of which are associated with familial AD), nicastrin, APH-1, and PEN-2. ␥-Secretase is responsible for the intramembraneous proteolytic cleavage of the C-terminal fragment of APP, resulting in mainly Article, publication date, and citation information can be found at
The efficacy of ␥-secretase inhibitors in vivo has, to date, been generally assessed in transgenic mouse models expressing increased levels of amyloid- (A) peptide thereby allowing the detection of changes in A production. However, it is not clear whether the in vivo potency of ␥-secretase inhibitors is independent of the level of amyloid precursor protein expression. In other words, does a ␥-secretase inhibitor have the same effect in nontransgenic physiological animals versus transgenic overexpressing animals? In the present study, an immunoassay has been developed which can detect A(40) in the rat brain, where concentrations are much lower than those seen in transgenic mice such as Tg2576 (c. 0.7 and 25 nM, respectively) and in cerebrospinal fluid (CSF, c. 0.3 nM). Using this immunoassay, the effects of thewere assessed and robust dose-dependent reductions in rat brain and CSF A(40) levels were observed with ID 50 values of 1.3 mg/kg for both brain and CSF. These values were comparable with those calculated for LY-411575 in transgenic mice.Time course experiments using LY-411575 demonstrated comparable temporal reductions in rat brain and CSF A(40), further suggesting these two pools of A are related. Accordingly, when all the data for the dose-response curve and time course were correlated, a strong association was observed between the brain and CSF A (40) levels. These data demonstrate the utility of the rat as a novel approach for assessing the effects of ␥-secretase inhibitors on central nervous system A(40) levels in vivo.Alzheimer's disease (AD) is one of the major neurological diseases of the elderly, characterized histopathologically by protein deposits in the brain parenchyma (plaques) or blood vessels and intracellular neurofibrillary tangles of abnormally phosphorylated protein. The plaques mainly consist of the A peptides originating from cleavage of the amyloid precursor protein (APP), with the majority being the more hydrophobic A(42) which is particularly prone to aggregation (Selkoe 2001). Furthermore, the identification of APP gene mutations in familial Alzheimer's disease (FAD) cases gives evidence of the involvement of APP processing in AD (Goate et al., 1991;Hardy, 1997).The enzymes responsible for processing APP into A are the aspartyl proteases, -amyloid cleaving enzyme (BACE or 1 These authors contributed equally to this publication. Article, publication date, and citation information can be found at
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