Conclusion of our 11-part Eibsee series. See also Parts 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and complete PDF.
Therapy development efforts in AD have diversified to include statins, PPARγ agonists, antioxidants, and certain NSAIDs as existing drug candidates, as well as the tau kinase GSK3β as a target for preclinical drug development. But most of the effort is still centered around the amyloid hypothesis. At the conference, two speakers detailed their efforts to find a better NSAID.
Sascha Weggen told the audience what his lab has done since he set up shop at the University of Mainz. By studying NSAIDs, Weggen can test whether Aβ42 truly is a good target in AD. This stands to reason because many FAD mutations increase the levels of this peptide only modestly (see ARF Discussion). Conceivably, Weggen said, a modest increase in the Aβ42/Aβ40 ratio over many years might lead to sporadic AD, and an equally modest reduction might therefore stave off disease. Initial presenilin inhibitors have proven toxic, but NSAIDS offer a new chance to tackle the question.
NSAIDs became a hot topic in AD research when epidemiological studies detected protection against AD in chronic users, but figuring out how they might do so has been all but straightforward. These mechanisms have been proposed:
- Cox inhibition, (few AD researchers believe it plays the major role)
- PPARγ activation to affect expression of inflammatory mediators (e.g., IL-1b, IL-6)
- PPARγ activation to reduce BACE expression
- γ-secretase modulation to lower Aβ42
- the Rho-Rock pathway (several groups have largely ruled this out)
- inhibition of Aβ aggregation
- reduced expression of α1-antichymotrypsin, an acute phase protein that accelerates amyloid pathology in APP-transgenic mice (this has only been shown for ibuprofen)
- sAPPα stimulation
Some of these ideas rest exclusively on in-vitro data, and Weggen offered some negative data of the kind that rarely see formal publication. When his group used pulse-chase experiments to test a claim for stimulation of sAPPα production and a subsequent signaling pathway (Avramovich et al., 2002), the scientists were unable to reproduce these findings in the same cells as originally reported. Seeing no effect with ibuprofen and indomethacin on sAPPα secretion, Weggen concluded that sAPPα stimulation is not an alternative mechanism of NSAID action.
Weggen focuses on the fourth category, that is, NSAIDS that lower Aβ42 production while leaving Aβ40 unchanged and increasing Aβ38 production. They include sulindac sulfide, ibuprofen, indomethacin, and flurbiprofen. New experiments into their mechanism of action show that they are unlikely to cause the mechanism-based toxicity that bedeviled previous γ-secretase inhibitors (Weggen et al., 2001; Lanz et al., 2005). Even so, their COX activity has gastrointestinal side effects that preclude their chronic, much less preventive, use at high doses. The way forward, then, would lie with new compounds that modulate γ-secretase in this desired way but don’t block COX.
Aβ-lowering NSAIDs do not modulate γ-secretase through the active site. They probably tweak a third, allosteric binding site proposed for γ-secretase, and several labs are in hot pursuit of exactly where they interact with members of the complex. For his part, Weggen noticed that certain FAD mutations change the secretase’s response to the NSAIDs. For example, wild-type cells produce less Aβ42 when treated with sulindac sulfide, but cells carrying certain FAD mutations that aggressively increase Aβ42 production do not. This suggests that the residues changed by these FAD mutations, or conformational effects of these mutations, affect the NSAID binding site.
Clinical studies of existing NSAIDs have largely failed or been halted, though an ongoing phase 3 trial of a purified stereoisomer may yet be successful (see R-flurbiprofen story). While encouraging, even this compound does not fill the need for new modulators that are more potent, penetrate the brain better, and stay active longer. To this end, Weggen has teamed up with Herbert Waldman at the Max-Planck Institute in Dortmund, Germany, whose collection of 500 sulindac sulfide and indomethacin analogs Weggen is screening for Aβ42-lowering and against COX activity. The scientists will test the hits in long-term animal models. Incidentally, Weggen noted, this will allow him to test whether anti-inflammatory effects have contributed to the AD protection reported in the epidemiological NSAID studies, after all. This possibility has gotten lost amid the current focus on Aβ42 lowering, but inflammation has never been formally ruled out.
Like Weggen, Boris Schmidt, who is at the Technical University of Darmstadt, Germany, searches for Aβ42-lowering compounds that are more potent than the four drugs known to do so. These four have IC50s that are in the micromolar range, and that is too high for wide use. Schmidt, a medicinal chemist, tested all commercially available NSAIDs in Germany for their ability to change the Aβ42/40/38 ratio and made about 150 analogs to selected ones, but none had the desired properties. The most potent one proved to be carprofen, a prescription drug approved for use in dogs only. (The only legal way to obtain this drug from pharmacies, even for scientific study in cells, is to obtain an arthritic dog, Schmidt said, adding, tongue in cheek, that his grant application for such an animal is pending.)
Schmidt also picked up research by Greg Cole and Sally Frautschy on Aβ lowering by the curry spice curcumin, and found that curcumin is an all-out inhibitor of γ-secretase but would be toxic at the concentrations needed. His lab made milder analogs that fit the desired γ-secretase modulation profile in the low micromolar range. Schmidt’s group also analyzed the mechanism of action of many γ-secretase modulators reported in the literature.
As take-home lessons of these studies, Schmidt noted that modulation is fundamentally different from inhibition, as the former need not change total Aβ load much at all. Secondly, Schmidt found that all modulators that achieve a favorable ratio of Aβ42/40/38 have a carboxylic residue in common and that certain lipophilic substitutions can improve the compounds’ potency.
Schmidt’s other effort has been to use NSAIDs as “bait” to “fish” for individual γ-secretase complex components in order to define the NSAID binding site. This fishing expedition has not caught anything whatsoever, Schmidt said, and the lesson to be learned is that the true binding site of the NSAID γ-secretase modulators most likely comprises a protein-protein interaction that gets disturbed by the bait and its attached label.
Overall, Schmidt said, the field of AD drug discovery is advancing but as of yet, no brain-penetrating β- or γ-secretase inhibitors have made it into a phase 3 trial. In Schmidt’s estimation, too, allosteric modulation may be a better ticket to success.
No matter which position they take on the amyloid hypothesis, scientists agree that its definitive test will occur in the clinic. Christoph Hock of the University of Zurich, offered 3-year follow-up data on the Zurich cohort of the aborted AN1792 active immunotherapy trials. (To read up on this topic, see Fox et al., 2005; Gilman et al., 2005; Orgogozo et al., 2003; Hock et al., 2003; Alzforum discussion; workshop coverage on AN1972 biomarker use.)
Hock showed a movie sequence of the first Zurich patient receiving a shot of the AN1792 vaccine into his upper left arm. A second film sequence showed a different, 68-year-old trial participant taking a line-marking test that is part of the trial’s cognitive assessment package. This man had developed the meningoencephalitis (ME) with an MRI lesion, a deficit in this particular test, but few other symptoms. He recovered from the encephalitis, and his mild-to-moderate AD still remains stable, Hock said. By contrast, another trial patient who developed ME had a normal MRI throughout, even though he was severely ill. “This just shows how difficult these trials are,” Hock said. He recalled a challenging time in 2002, as the investigators found themselves under assault from newspapers and colleagues who accused them of rushing into human tests prematurely. Since then, Hock and colleagues have observed the patients in hopes of learning if the stalled trial can still yield information about the underlying questions of whether immunotherapy can remove amyloid and whether that changes the disease.
In short, the trial appears to have worked inasmuch as it induced antibodies that crossed the blood-brain barrier and removed patches of parenchymal amyloid. People who developed high levels of antibody tended to show an improvement on certain memory tests, Hock said. In the overall AN1792 cohort, the control group declined less steeply than the control group of the Zurich cohort did, so the total measured effect came out small. Among the Zurich patients, the responders still decline more slowly on MMSE than the non-responders after 3 years, Hock noted. An additional MRI scan at 36 months showed that hippocampal volume in the responders is still at the level measured at 24 months. At 24 months, it had begun to recover after an unexpected decline at 12 months. “This is an indication that there might be a restoration of hippocampal volume after the immunization,” Hock said. Three years after the immunization, antibody titers, hippocampal volume, and cognitive performance correlated, indicating that the immune response is long-lasting. At 30, the number of patients in this cohort was small to begin with. Now, close to 4 years after the trial began, about 20 percent are dropping out of the follow-up study every year, though the six highest responders are still doing well, Hock said.
Despite its setbacks, the AN1792 trial has stimulated interest in immunotherapy to where these companies are now said to pursue their own programs: Elan and Wyeth; Novartis; Hoffman LaRoche; Eli Lilly and Co.; Rinat Neuroscience; Neurochem; Eisei Co./BioArctic Neuroscience; Abmaxis Inc./BioArctic; AC Immune; AC-Immune (Lausanne); Boehringer Ingelheim; Merck/Acumen; and Abbot, with all except Elan/Wyeth at the preclinical or phase 1 stage.—Gabrielle Strobel.