In vivo characterization of Abeta(40) changes in brain and cerebrospinal fluid using the novel gamma-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 Abeta peptides are one of the hallmarks of Alzheimer's disease. These Abeta peptides are produced by the final proteolytic cleavage of the amyloid precursor protein by the intramembraneous aspartyl protease gamma-secretase. Thus, one approach to lowering levels of Abeta has been via the inhibition of the gamma-secretase enzyme. Here, we report a novel, bioavailable gamma-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 Abeta 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 Abeta could be maintained for 24 h, and comparable temporal reductions in rat brain and CSF Abeta(40) further suggested that these two pools of Abeta are related. This relationship between the brain and CSF Abeta 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 Abeta levels. These results demonstrate that MRK-560 is an orally bioavailable gamma-secretase inhibitor with the ability to markedly reduce Abeta peptide in the brain and CSF of the rat and confirm the utility of the rat for assessing the effects of gamma-secretase inhibitors on central nervous system Abeta(40) levels in vivo.

Quantitative measurement of changes in amyloid-beta(40) in the rat brain and cerebrospinal fluid following treatment with the gamma-secretase inhibitor LY-411575 [N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide].

The efficacy of gamma-secretase inhibitors in vivo has, to date, been generally assessed in transgenic mouse models expressing increased levels of amyloid-beta (Abeta) peptide thereby allowing the detection of changes in Abeta production. However, it is not clear whether the in vivo potency of gamma-secretase inhibitors is independent of the level of amyloid precursor protein expression. In other words, does a gamma-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 Abeta(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 the gamma-secretase inhibitor LY-411575 [N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide] were assessed and robust dose-dependent reductions in rat brain and CSF Abeta(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 Abeta(40), further suggesting these two pools of Abeta 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 Abeta(40) levels. These data demonstrate the utility of the rat as a novel approach for assessing the effects of gamma-secretase inhibitors on central nervous system Abeta(40) levels in vivo.

Stress-induced increase of cortical dopamine metabolism: attenuation by a tachykinin NK1 receptor antagonist.

The present study examined the potential role of tachykinin NK1 receptors in modulating immobilisation stress-induced increase of dopamine metabolism in rat medial prefrontal cortex. In agreement with previous studies, 20 min immobilisation stress significantly increased medial prefrontal cortex dopamine metabolism as reflected by the concentration of the dopamine metabolite dihydroxyphenylacetic acid (DOPAC). Pretreatment with the high affinity, selective, tachykinin NK1 receptor antagonist (3(S)-(2-methoxy-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethyl amino)-2(S)-phenylpiperidine) ((S)-GR205171, 10 mg/kg, s.c.), a dose that in ex vivo binding studies extensively occupied rat brain tachykinin NK1 receptors for approximately 60 min, significantly attenuated the stress-induced increase of mesocortical DOPAC concentration without affecting cortical DOPAC levels per se. In contrast, pretreatment of animals with the less active enantiomer (R)-GR205171 (10 mg/kg, s.c.), which demonstrated negligible tachykinin NK1 receptor occupancy ex vivo, failed to affect either basal or stress-induced DOPAC concentration in medial prefrontal cortex. Furthermore, pretreatment of animals with the benzodiazepine/GABAA receptor antagonist, flumazenil (15 mg/kg, i.p.), did not affect the ability of (S)-GR205171 to attenuate the increase of medial prefrontal cortex DOPAC concentration by acute stress. Results demonstrate that the selective tachykinin NK1 receptor antagonist, (S)-GR205171, attenuated the stress-induced activation of mesocortical dopamine neurones by a mechanism independent of the benzodiazepine modulatory site of the GABAA receptor.