Sarah A. Sagi

A subset of NSAIDs lower amyloidogenic Aβ42 independently of cyclooxygenase activity

Epidemiological studies have documented a reduced prevalence of Alzheimers disease among users of nonsteroidal anti-inflammatory drugs (NSAIDs). It has been proposed that NSAIDs exert their beneficial effects in part by reducing neurotoxic inflammatory responses in the brain, although this mechanism has not been proved. Here we report that the NSAIDs ibuprofen, indomethacin and sulindac sulphide preferentially decrease the highly amyloidogenic Aβ42 peptide (the 42-residue isoform of the amyloid-β peptide) produced from a variety of cultured cells by as much as 80%. This effect was not seen in all NSAIDs and seems not to be mediated by inhibition of cyclooxygenase (COX) activity, the principal pharmacological target of NSAIDs. Furthermore, short-term administration of ibuprofen to mice that produce mutant β-amyloid precursor protein (APP) lowered their brain levels of Aβ42. In cultured cells, the decrease in Aβ42 secretion was accompanied by an increase in the Aβ(1–38) isoform, indicating that NSAIDs subtly alter γ-secretase activity without significantly perturbing other APP processing pathways or Notch cleavage. Our findings suggest that NSAIDs directly affect amyloid pathology in the brain by reducing Aβ42 peptide levels independently of COX activity and that this Aβ42-lowering activity could be optimized to selectively target the pathogenic Aβ42 species.

NSAIDs and enantiomers of flurbiprofen target γ-secretase and lower Aβ42 in vivo

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 β protein (Aβ42) in a human H4 cell line. Thirteen of the NSAIDs and the enantiomers of flurbiprofen were then tested in acute dosing studies in amyloid β protein precursor (APP) transgenic mice, and plasma and brain levels of Aβ and the drug were evaluated. These studies show that (a) eight FDA-approved NSAIDs lower Aβ42 in vivo, (b) the ability of an NSAID to lower Aβ42 levels in cell culture is highly predicative of its in vivo activity, (c) in vivo Aβ42 lowering in mice occurs at drug levels achievable in humans, and (d) there is a significant correlation between Aβ42 lowering and levels of ibuprofen. Importantly, flurbiprofen and its enantiomers selectively lower Aβ42 levels in broken cell γ-secretase assays, indicating that these compounds directly target the γ-secretase complex that generates Aβ from APP. Of the compounds tested, meclofenamic acid, racemic flurbiprofen, and the purified R and S enantiomers of flurbiprofen lowered Aβ42 levels to the greatest extent. Because R-flurbiprofen reduces Aβ42 levels by targeting γ-secretase and has reduced side effects related to inhibition of cyclooxygenase (COX), it is an excellent candidate for clinical testing as an Aβ42 lowering agent.

Substrate-targeting γ-secretase modulators

Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer’s disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the γ-secretase complex, but instead labelled the β-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-β peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP γ-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28–36 of amyloid-β, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-β act as GSMs, and some GSMs alter the production of cell-derived amyloid-β oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Aβ42 production and inhibition of amyloid-β aggregation, which may synergistically reduce amyloid-β deposition in Alzheimer’s disease. These data also demonstrate the existence and feasibility of ‘substrate targeting’ by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of ‘druggable’ targets.

Diverse compounds mimic Alzheimer disease–causing mutations by augmenting Aβ42 production

Increased Aβ42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Aβ42. Among the more potent Aβ42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2–selective NSAID. Many COX-2–selective NSAIDs tested raised Aβ42, including multiple COX-2–selective derivatives of two Aβ42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Aβ42. These compounds seem to target the γ-secretase complex, increasing γ-secretase–catalyzed production of Aβ42 in vitro. Short-term in vivo studies show that two Aβ42-raising compounds increase Aβ42 levels in the brains of mice. The elevations in Aβ42 by these compounds are comparable to the increases in Aβ42 induced by Alzheimer disease–causing mutations in the genes encoding amyloid β protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Aβ42 production in humans.

Safety, tolerability, pharmacokinetics, and Aβ levels after short-term administration of R-flurbiprofen in healthy elderly individuals

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 (Aβ42) 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 Aβ 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 Aβ42 levels and no significant differences in changes of plasma Aβ42 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 Aβ42 plasma levels (P=0.016). R-flurbiprofen had an excellent safety profile and showed dose-dependent central nervous system penetration. Exploratory analyses of plasma Aβ 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.

P2-338: Substrate-targeting gamma-secretase modulators

trifluoro-1-(hydroxyl[ H2 ]methyl)-2-methylbutyl]thiophene-2sulfonamide). GSI-953 and benchmark GSIs (DAPT, LY411575, LY450139, DuPont E, L-685458, a BMS sulfonamide and an Amgen sulfonamide) were profiled in this assay for their ability to displace the radiolabeled GSI-953 analog. For comparison to GS binding affinity, the GSIs were also profiled for inhibition of synthesis in a cellular assay (hAPPCHO cells). Results: GSI-953 competitively displaced TGSI from the GS complex in the cell-free binding assay (IC50 8 nM) and its affinity for GS was comparable to its GSI potency in the cellular assay (EC50 42 15 nM). The stereospecific nature of both the binding to and inhibition of GS by GSI-953 was confirmed by the low affinity (IC50 10,000 nM) and GSI activity (EC50 42 30,000 nM) observed for the enantiomer of GSI-953. Benchmark GSIs DAPT (IC50 29 nM), LY411575 (IC50 3 nM), LY450139 (IC50 26 nM), DuPont E (IC50 10 nM), a BMS sulfonamide (IC50 57 nM) and an Amgen sulfonamide (IC50 986 nM) were able to competitively displace TGSI from GS at concentrations comparable to their EC50s for inhibition of synthesis. The transition state inhibitor L-685458 was only able to partially displace TGSI. Conclusions: GSI-953 and benchmark GSIs (except L-685458) can competitively displace TGSI from GS suggesting that these GSIs may bind to the same site. Among the GSIs profiled, a good correlation was observed between GS binding affinity and GSI potency.