Despite considerable advances in the understanding of AD, no therapeutic interventions that halt or reverse the underlying disease process are available (33
). On the basis of the amyloid cascade hypothesis, a number of therapeutic strategies targeting various steps in the production, deposition, or clearance of Aβ are being evaluated in preclinical or clinical studies. These treatments include (a) β- and γ-secretase inhibitors that target the proteases that produce Aβ, (b) anti-Aβ immunotherapies to promote Aβ clearance, and (c) agents that target Aβ aggregates. Like any novel therapeutic approach, the development of these treatments may be impeded by potential nontarget- and target-based toxicity. Given that such agents are likely to be administered over long-periods to elderly individuals, these agents must demonstrate excellent safety profiles. Because of these concerns, a great deal of recent attention has focused on potential treatment with agents, such as NSAIDs and statins, implicated as protective factors in epidemiologic studies, since these agents have well-characterized toxicity profiles (11
Numerous epidemiologic studies have provided evidence that chronic intake of NSAIDs is associated with a decreased risk AD (11
). Although the epidemiologic studies would suggest a protective role for NSAIDs in AD, several therapeutic trials of various nonaspirin NSAIDs have been conducted. Two small placebo-controlled pilot studies, one with indomethacin and another with diclofenac in combination with misoprostol, showed some trends toward reducing the cognitive decline in patients with AD (36
). However, the results in each trial were confounded by their small size and the large withdrawal rates among those receiving the NSAIDs. FDA-approved selective COX2 inhibitors, celecoxib (Celebrex) and rofecoxib (Vioxx), have also been evaluated in therapeutic AD clinical trials. Final results from these trials have not been reported, but interim reports have failed to report any efficacy. Also ongoing are treatment trials using ibuprofen, dapsone, naproxen, and celecoxib. Of note, the AD Anti-Inflammatory Prevention trial (ADAPT) is assessing the efficacy of naproxen and celecoxib in preventing or delaying the onset of AD in a large cohort. Thus, ADAPT is the only ongoing trial that will test whether certain NSAIDs may reduce the risk of developing AD.
Inhibition of the two isoforms of cyclooxygenase (COX1 and COX2) is the primary pharmacological action of NSAIDs that results in their anti-inflammatory properties (38
). Some NSAIDs activate the peroxisome proliferator γ nuclear transcription factor; this activation may have anti-inflammatory consequences relevant to AD (39
). Although it has been proposed that NSAIDs exert their beneficial effects by reducing the inflammatory responses in the AD brain, the mechanism of action underlying the therapeutic effects of NSAIDs in AD remains uncertain. Our recent finding that ibuprofen, indomethacin, and sulindac lowered Aβ42 production independently of their effects on COX suggests that this property could contribute to their apparent protective efficacy in AD (15
). To better evaluate this possibility, we analyzed 20 NSAIDs, which represent nearly all of those commonly used, for their effects on Aβ42 in cell culture. NSAIDs capable of lowering Aβ42 levels in cell culture, as well as several that failed to lower Aβ42 levels, were then tested in acute dosing studies in transgenic mice. These data show that diclofenac, diflunisal, ibuprofen, sulindac, fenoprofen, indomethacin, flurbiprofen, and meclofenamic acid reduce Aβ42 levels in the brains of mice. Other compounds, including naproxen, aspirin, nabumetone, and ketoprofen, did not lower Aβ42 levels in vivo.
Overall, there was a good degree of association between Aβ42 lowering effects in cell culture and in vivo. However, several discrepancies are apparent. These discrepancies were only seen in compounds, with modest effects, decreasing Aβ42 levels by 17–20%. To determine if compounds that minimally perturb Aβ42 levels in acute dosing paradigms can affect Aβ deposition, long-term dosing studies in APP transgenic mice, such as those already carried out on ibuprofen (29
), will be needed.
A concern with our previous study was that the Aβ42 lowering effect of ibuprofen, sulindac sulfide, and indomethacin was only apparent in cultured cells at relatively high concentrations (>25–50 μM), and maximal lowering typically did not occur unless cells were treated with even higher concentrations of these drugs. It was also thought that the 50 mg/kg per day dosing level in animals would represent a dose likely to be nonphysiologic in humans. In this study, we directly assessed both the plasma levels and brain levels of several NSAIDs after 3 days of dosing. These drug levels likely represent steady-state or near steady-state levels of the compounds. These data demonstrate that, despite a high dose, the plasma levels of most NSAIDs measured did not exceed the levels that can be achieved with therapeutic doses in humans (30
). On the basis of these studies, we conclude that recommended dosing regimens of certain NSAIDs in humans are likely to achieve drug levels in humans that could potentially lower Aβ42 levels.
One of the perplexing aspects of these data is that they demonstrate a discrepancy between potency in cell culture systems and potency in APP transgenic mice. This is especially true if one presumes that these agents are working against specific targets in the CNS. If this is the case, then the low micromolar levels of these drugs present in the brain appear capable of lowering Aβ42 levels to the same extent as seen with 100 μM treatment in a human cell line. Several factors could account for this large increase in apparent potency. First, if drugs accumulate in specific compartments in the brain where the target is also localized, this colocalization could account for the discrepancy in potency. Second, the target in the brain could differ in some ways from the target in cell culture. For example, it is possible that the brain form of γ-secretase is subtly distinct from the form in cultured cells. Third, it is possible that a metabolite of the NSAIDs exhibits higher potency for the target than the parent compounds. Alternatively, it is possible that the drugs do not act centrally but instead reduce peripheral Aβ42 levels, which results in enhanced efflux of Aβ42 from the brain. This type of “peripheral sink” mechanism has been postulated to account for the Aβ lowering effect of anti-Aβ immunotherapy (41
). To determine if such a mechanism might contribute to the Aβ42 lowering effect we have observed with certain NSAIDs, we also examined plasma Aβ levels in a subset of treated animals. There was no correlation between Aβ42 plasma levels and Aβ42 levels in the brain after these 3-day dosing studies. However, longer-term studies with ibuprofen indicate that selective lowering of plasma Aβ42 does occur with at least one NSAID and loosely correlates with brain Aβ42 levels. Additional studies will be needed to distinguish among these possibilities.
The finding that multiple commonly used NSAIDs can lower Aβ42 provides a framework in which to evaluate both previous and future epidemiologic studies and AD clinical trials with respect to anti-inflammatory versus Aβ42 lowering mechanisms. Although none of the published epidemiologic studies on NSAIDs and AD has reported effects of individual NSAIDs, the Cache County Study, the Baltimore Longitudinal Study of Aging, and the Rotterdam Study have compared aspirin or oral salicylates with nonaspirin NSAIDs (12
). In each of these studies, nonaspirin NSAIDs, when taken for more than 2 years, showed a greater protective effect than aspirin. In the Rotterdam Study, detailed data on NSAID use was provided; but no analysis based on individual drug use was reported (12
). In our study, two of the six most commonly used NSAIDs (ibuprofen and indomethacin) significantly lowered Aβ42 levels in APP transgenic mice. Two other NSAIDs, diclofenac and piroxicam, slightly reduced Aβ42 levels in vivo. Together, these four drugs accounted for 74% of NSAID use in the Rotterdam Study, whereas naproxen and ketoprofen, which had no effect on Aβ42 levels, accounted for 21% of NSAID use. Given these observations, insights into the possible mechanisms of action of NSAIDs in AD could be generated by either reevaluating existing data or collecting new data that focus on whether or not the NSAID analyzed has the potential to lower Aβ42 levels. In a similar fashion, these data may help to evaluate results from previous and ongoing clinical trials of various NSAIDs. Only one of the current trials is using an NSAID, ibuprofen, which has the potential to lower Aβ42 levels. Neither of the selective COX2 inhibitors, celecoxib and rofecoxib, appears capable of lowering Aβ42 in vitro or in vivo (T. Golde and E. Koo, unpublished data).
A drawback to the potential clinical use of conventional NSAIDs in AD, either as Aβ42 lowering agents or as anti-inflammatory medications, is the gastrointestinal and renal toxicity thought to be mediated by their inhibition of COX. Because the Aβ42 lowering effect is independent of COX, we suggested that a rational drug candidate for the treatment or prevention of AD is an Aβ42 lowering agent either lacking or having greatly reduced COX-inhibiting activity (15
). In contrast to rodents, in humans the COX-inactive R
-flurbiprofen is minimally converted to the S
). This enables administration of higher doses of R
-flurbiprofen with a reduced incidence of COX-mediated side effects. Here we show that R
- and S
-flurbiprofen are nearly equipotent in their ability to lower Aβ42 levels. Similar data in cultured cells have recently been reported by another group (43
). Both enantiomers of flurbiprofen and racemic flurbiprofen also seem to function through a mechanism similar that which we previously reported. Like sulindac sulfide, which lowers Aβ42 and selectively increases Aβ38 levels, flurbiprofen and its enantiomers lower Aβ42 and increase Aβ38 levels. Thus, these findings with R
-flurbiprofen are consistent with our prediction that such Aβ42 lowering compounds exist. In this study, we further show that the γ-secretase complex is the target of flurbiprofen and its enantiomers, since these compounds selectively lower Aβ42 in a broken cell γ-secretase assay. Additional studies will be needed to determine the precise mechanism through which Aβ42 lowering NSAIDs such as flurbiprofen interact with γ-secretase. Although we have not completed long-term studies of flurbiprofen and its enantiomers in APP transgenic mice, a nitrous oxide–releasing flurbiprofen derivative, NCX-2216, has been reported to be highly effective at reducing Aβ accumulation in long-term studies in an AD mouse model (44
). This derivative is converted to flurbiprofen in vivo. R
-flurbiprofen is also reported to inhibit NF-κB. Inhibition of NF-κB has been postulated to reduce the inflammatory response in AD (45
). However, the Aβ42 lowering effect of R
-flurbiprofen and its effects on NF-κB appear to be independent of each other (43
). Taken together, these data suggest that R
-flurbiprofen is a compound that can be used to test our hypothesis that Aβ42 levels can be safely lowered in humans with minimal effects on COX activity or the physiological functions of γ-secretase. Although there is a strong rationale for selective targeting of Aβ42, no Aβ therapeutic has been sufficiently tested in humans to determine its possible efficacy in the treatment or prevention of AD. Such evidence will not be forthcoming for a number of years. Rigorous clinical testing of R
-flurbiprofen and other NSAIDs that lower Aβ42 will be necessary to determine if they have the ability to lower Aβ42 in humans and therapeutic efficacy in AD.