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To evaluate the safety and tolerability and pharmacokinetic properties of R-flurbiprofen (Tarenflurbil) in normal elderly individuals and to determine the effect of the drug on amyloid beta 42 (Abeta42) levels, we conducted a double-blind, placebo-controlled study of 48 healthy subjects aged 55 to 80. Three successive cohorts were randomized to doses of 400, 800, or 1600 mg/d, or placebo, given as 2 divided doses for 21 days. Blood and cerebrospinal fluid were collected for pharmacokinetic studies and measurement of Abeta levels at baseline and on day 21. R-flurbiprofen was well-tolerated at all 3 doses. The compound penetrated the blood-brain barrier in a dose-dependent manner. From baseline to 21 days, comparisons between study groups revealed no significant differences in changes of cerebrospinal fluid Abeta42 levels and no significant differences in changes of plasma Abeta42 levels at the time of trough drug level at 21 days of treatment. Further analysis of drug concentration-response for plasma samples showed that at the time of peak plasma concentration, higher plasma drug concentration was related to lower Abeta42 plasma levels (P=0.016). R-flurbiprofen had an excellent safety profile and showed dose-dependent central nervous system penetration. Exploratory analyses of plasma Abeta and peak drug levels suggested a short-term effect in plasma that warrants independent verification. The safety, tolerability, and pharmacokinetic profile of R-flurbiprofen in these older individuals support the ongoing studies of this compound in patients with Alzheimer disease.

Certain non-steroidal anti-inflammatory drugs (NSAIDs) preferentially inhibit production of the amyloidogenic Abeta42 peptide, presumably by direct modulation of gamma-secretase activity. A recent report indicated that NSAIDs could reduce Abeta42 by inhibition of the small GTPase Rho, and a single inhibitor of Rho kinase (ROCK) mimicked the effects of Abeta42-lowering NSAIDs. To investigate whether Abeta42 reduction is a common property of ROCK inhibitors, we tested commercially available compounds in cell lines that were previously used to demonstrate the Abeta42-lowering activity of NSAIDs. Surprisingly, we found that two ROCK inhibitors reduced total Abeta secretion in a dose-dependent manner but showed no selectivity for Abeta42. In addition, ROCK inhibitors did not increase Abeta38 secretion in cell-based assays or reduce Abeta production in gamma-secretase in vitro assays, which are critical characteristics of Abeta42-lowering NSAIDs. The reduction in total Abeta levels by ROCK inhibitors was not accompanied by overall-changes in amyloid precursor protein processing. Targeting ROCK by expression of dominant-negative or constitutively active ROCK mutants failed to modulate Abeta secretion, indicating that ROCK inhibition may either be redundant or insufficient for Abeta reduction by ROCK inhibitors. Taken together, these results seem to exclude a mechanistic involvement of ROCK in the Abeta42-lowering activity of NSAIDs.

Epidemiological evidence suggests that chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of Alzheimer's disease. Recently, NSAIDs have been shown to decrease amyloid pathology in a transgenic mouse model of Alzheimer's disease. This benefit may be partially attributable to the ability of NSAIDs to selectively reduce production of the amyloidogenic A beta 42 peptide in both cultured cells and transgenic mice. Although this activity does not appear to require the action of cyclooxygenases in cultured cells, it is not known whether other NSAID-sensitive targets contribute to this A beta 42 effect. In this study, we have used both pharmacological and genetic means to determine if other known cellular targets of NSAIDs could mediate the reduction in A beta 42 secretion from cultured cells. We find that altered arachidonic acid metabolism via NSAID action on cyclooxygenases and lipoxygenases does not alter A beta 42 production. Furthermore, we demonstrate that alterations in activity of peroxisome proliferator-activated receptors, I kappa B kinase beta or nuclear factor kappa B do not affect A beta 42 production. Thus, NSAIDs do not appear to alter A beta 42 production indirectly through previously identified cellular targets and may interact directly with the gamma-secretase complex itself to affect amyloid production.

Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a lower risk of developing Alzheimer's disease. Recent evidence indicates that some NSAIDs specifically inhibit secretion of the amyloidogenic A beta 42 peptide in cultured cells and mouse models of Alzheimer's disease. The reduction of A beta 42 peptides is not mediated by inhibition of cyclooxygenases (COX) but the molecular mechanism underlying this novel activity of NSAIDs has not been further defined. We now demonstrate that NSAIDs efficiently reduce the intracellular pool of A beta 42 in cell-based studies and selectively decrease A beta 42 production in a cell-free assay of gamma-secretase activity. Moreover, we find that presenilin-1 (PS1) mutations, which affect gamma-secretase activity, differentially modulate the cellular A beta 42 response to NSAID treatment. Overexpression of the PS1-M146L mutation enhances the cellular drug response to A beta 42 lowering NSAIDs as compared with cells expressing wild-type PS1. In contrast, expression of the PS1-Delta Exon9 mutation strongly diminishes the A beta 42 response, showing that PS1 mutations can modulate the cellular drug response to NSAID treatment both positively and negatively. Enhancement of the NSAID drug response was also observed with overexpression of the APP V717F mutation but not with Swedish mutant APP, which affects beta-secretase cleavage. In sum, these results strongly suggest that NSAIDs represent a founding group of compounds that lower A beta 42 production by direct modulation of gamma-secretase activity or its substrate.

Epidemiological studies indicate that long term use of nonsteroidal anti-inflammatory drugs (NSAIDs) confers protection from Alzheimer's disease, and some NSAIDs were shown to specifically decrease production of the amyloidogenic Abeta42 peptide, most likely by direct modulation of gamma-secretase activity. In contrast to gamma-secretase inhibitors, Abeta42-lowering NSAIDs do not impair S3 cleavage in the NOTCH receptor and release of the NOTCH intracellular domain, a finding with conceptual implications for the development of safer drugs targeting Abeta production through gamma-secretase modulation. Intramembrane cleavage and release of an intracellular signaling domain has recently been demonstrated in a number of additional gamma-secretase substrates. We now show in cell-based assays that intramembrane cleavage of APP and ErbB-4 receptor is not impaired by the Abeta42-lowering NSAIDs, sulindac sulfide and ibuprofen. Generation of the APP intracellular domain (AICD) was further not inhibited in a cell-free assay at concentrations far exceeding those effective in reducing Abeta42 production. Closer inspection of AICD signaling showed that stabilization of the AICD peptide by FE65 and AICD-mediated transcription were also retained at Abeta42-lowering concentrations. These results demonstrate that S3-like/intramembrane cleavage is preserved by Abeta42-lowering NSAIDs in at least three substrates of gamma-secretase APP, ErbB-4, and NOTCH and underline the striking specificity by which these drugs target Abeta42 production.

Epidemiologic studies demonstrate that long-term use of NSAIDs is associated with a reduced risk for the development of Alzheimer disease (AD). In this study, 20 commonly used NSAIDs, dapsone, and enantiomers of flurbiprofen were analyzed for their ability to lower the level of the 42-amino-acid form of amyloid beta protein (Abeta42) in a human H4 cell line. Thirteen of the NSAIDs and the enantiomers of flurbiprofen were then tested in acute dosing studies in amyloid beta protein precursor (APP) transgenic mice, and plasma and brain levels of Abeta and the drug were evaluated. These studies show that (a). eight FDA-approved NSAIDs lower Abeta42 in vivo, (b). the ability of an NSAID to lower Abeta42 levels in cell culture is highly predicative of its in vivo activity, (c). in vivo Abeta42 lowering in mice occurs at drug levels achievable in humans, and (d). there is a significant correlation between Abeta42 lowering and levels of ibuprofen. Importantly, flurbiprofen and its enantiomers selectively lower Abeta42 levels in broken cell gamma-secretase assays, indicating that these compounds directly target the gamma-secretase complex that generates Abeta from APP. Of the compounds tested, meclofenamic acid, racemic flurbiprofen, and the purified R and S enantiomers of flurbiprofen lowered Abeta42 levels to the greatest extent. Because R-flurbiprofen reduces Abeta42 levels by targeting gamma-secretase and has reduced side effects related to inhibition of cyclooxygenase (COX), it is an excellent candidate for clinical testing as an Abeta42 lowering agent.