Keystone Drug News: CoMentis BACE Inhibitor Debuts

05 Apr 2008

At the Alzheimer’s disease Keystone Symposium, held 24-29 March in Keystone, Colorado, a spate of news on potential future treatments capped a week of presentations and casual discussion in the thin air of Rocky Mountain ski slopes that reach up to 12,400 feet. Here’s the lowdown:

Gerald Koelsch, who is at the Oklahoma City site of the biotech company CoMentis, Inc., surprised the audience with his presentation of human data on the company’s β-secretase inhibitor CTS-21166. In a first proof-of-concept human study, the compound appeared safe and reduced plasma amyloid-β levels substantially for an extended period of time. Many pharmaceutical companies are known to pursue BACE inhibitors, and some are rumored to have begun initial human testing, including Merck (see ARF related news story), Eli Lilly and Co., and Takeda in Japan, but this is the first time clinical results of a BACE inhibitor have been reported at a large scientific conference in the U.S.

Scientists at CoMentis have pursued BACE as a drug target since two of its scientific founders, Jordan Tang and Arun Ghosh, published a crystal structure with an inhibitor caught inside it (Hong et al., 2000). That inhibitor was based on the APP sequence, and since then, the company has focused on medicinal chemistry to develop a suitable small-molecule drug. CTS-21166 is a transition-state analog that is the company’s lead candidate at present, Koelsch said. He would not reveal the molecule’s structure but recounted its properties on the list of requirements a drug must meet, from pharmacokinetics to pharmacodynamics to oral availability, and more.

Koelsch noted that the compound’s potency and selectivity passed muster when it proved to bind BACE with a potency of 1.2 to 3.6 nanomolar in cellular assays, but not to bind some 60 other enzymes, channels, and receptors. In response to a question about non-specific plasma protein binding, which has posed problems for some other candidate BACE inhibitors in the past, Koelsch noted that this has not been an issue with CTS-21166.

When injected intra-peritoneally for six weeks at a dose of 4 mg/kg into an aggressive transgenic mouse model that expresses both the Swedish and London APP mutations (Rockenstein et al., 2001), CTS-21166 reduced levels of brain Aβ40 and 42 by 38 and 35 percent, respectively. It also reduced plaque load in the hippocampus and cortex by some 40 percent. Koelsch said he assumed this plaque drawdown happened indirectly through an equilibrium shift, whereby a drop in new Aβ production allows existing Aβ to come off plaques and be degraded (see DeMattos story). Pharmacokinetic measurements in rats and mice indicated that CTS-21166 displays good brain penetration at a concentration of 8 ng/ml relative to that in plasma of 18 ng/ml at a single time point (4 mg/kg over six weeks). Previously, it has been difficult to find BACE inhibitors that cross the blood-brain barrier.

In these experiments, the drug did not damage the myelin sheath in the sciatic nerve of this mouse model, indicating that the BACE enzyme might aid myelination primarily during development (Willem et al., 2006). They will continue to monitor for demyelination, Koelsch said.

With that data, the scientists undertook a Phase 1 study in humans. Single intravenous injections of doses ranging from 7.5 to 225 mg were given to six volunteers plus two placebo recipients per group. In this small trial, the drug was well tolerated at all doses, Koelsch said. As in prior studies, a dose-dependent response was seen in plasma pharmacokinetics. The volunteers varied little in their response to the drug. They cleared it slowly, indicating the drug could possibly be given once a day. (Though this human study used intravenous administration, the inhibitor has been shown to be orally available in mice, rats, dogs, and monkeys, Koelsch said.)

Koelsch showed data suggesting that the drug reduced plasma Aβ levels by up to 80 percent in the volunteers at the highest dose, reaching its trough three hours after injection and gradually returning to baseline over the course of several days. Interestingly, Aβ40 levels did not overshoot baseline after the drug had washed out. Such a “rebound” effect has puzzled scientists with LY450139 treatment (Siemers et al., 2007. This γ-secretase inhibitor has entered a Phase 3 clinical trial called IDENTITY).

The CoMentis study did not include spinal taps; hence, no CSF data are available. Another major question that remains open at this point is by how much brain Aβ levels need to come down for a drug to be clinically meaningful to AD patients. This question will find its answer in the clinic.—Gabrielle Strobel.

References

News Citations

Paper Citations

Hong L, Koelsch G, Lin X, Wu S, Terzyan S, Ghosh AK, Zhang XC, Tang J. Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor. Science. 2000 Oct 6;290(5489):150-3. PubMed.
Rockenstein E, Mallory M, Mante M, Sisk A, Masliaha E. Early formation of mature amyloid-beta protein deposits in a mutant APP transgenic model depends on levels of Abeta(1-42). J Neurosci Res. 2001 Nov 15;66(4):573-82. PubMed.
Willem M, Garratt AN, Novak B, Citron M, Kaufmann S, Rittger A, Destrooper B, Saftig P, Birchmeier C, Haass C. Control of peripheral nerve myelination by the beta-secretase BACE1. Science. 2006 Oct 27;314(5799):664-6. PubMed.
Siemers ER, Dean RA, Friedrich S, Ferguson-Sells L, Gonzales C, Farlow MR, May PC. Safety, tolerability, and effects on plasma and cerebrospinal fluid amyloid-beta after inhibition of gamma-secretase. Clin Neuropharmacol. 2007 Nov-Dec;30(6):317-25. PubMed.

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Keystone Drug News: Agonists for M1, Serotonin Receptors Prime Cholinergic Pump

07 Apr 2008

The cholinergic hypothesis of Alzheimer disease tends to elicit a yawn from scientists who reserve their excitement for anti-amyloid treatments or more radically new biologic approaches such as immunotherapy. Still, it’s what has yielded most AD drugs the doctor can prescribe today, and researchers clearly feel there is more to be gained from it. Even as amyloid and tau pathologies are considered central to the pathogenic process, the cholinergic system is indispensable for learning, attention, and information processing. At the Keystone conference, held 24-29 March in Keystone, Colorado, two speakers—one from a small biotech, one from pharma giant Merck—presented their active approaches of propping up a failing cholinergic system in AD. One targets a serotonin, one a muscarinic acetylcholine receptor.

First, the more clinically advanced drug. J. Thomas Megerian of Epix Pharmaceuticals in Lexington, Massachusetts, updated the audience on PRX-03140, a 5-HT4 serotonin receptor agonist his company is developing together with GlaxoSmithKline. Epix’s claim to fame is its in-silico drug design technology, which has generated some potential drugs against G protein-coupled receptors (GPCRs). This large family of transmembrane signaling proteins is thought to contain targets for a range of diseases, but they have been difficult to exploit in part because no crystal structures are available to guide drug discovery. The target of PRX-03140 is one such GPCR, i.e., the 5-HT4 receptor.

According to Megerian, preclinical studies have suggested that this small-molecule agonist boosts cholinergic transmission by increasing levels of the acetylcholine receptor on demand, that it stimulates release of the growth factor BDNF, decreases Aβ levels, and promotes α-secretase cleavage of APP to generate the neurotrophic sAPPα fragment. Epix is attempting to develop PRX-03140 either alone or as a combination therapy with donepezil, which increases acetylcholine levels in the resting state by simply blocking its degradation.

Last December, Epix released top-line results of their recently concluded Phase 2a study but soon after issued a correction when errors in the initial data analysis emerged. At Keystone, Megerian presented these data: the trial recruited 80 people with mild AD at 17 U.S. sites and treated them for two weeks with PRX-03140. Some patients took donepezil for three months and then added one of five different doses (5 to 200 mg/day) of PRX-03140. Others took one of two doses (50 or 150 mg) of study drug but no donepezil. The trial measured safety, exposure to donepezil, and cognitive endpoints.

PRX-03140 caused no serious side effects in monotherapy, but the higher doses of combination therapy showed expected cholinergic side effects, mostly gastrointestinal. The drug did not alter drug exposure of donepezil. Patients on the high dose of monotherapy improved a statistically significant 3.6 points over baseline on the ADAS-cog battery of tests and 4.5 points over placebo. The dose-response effect between placebo, 50 mg, and 150 mg was also statistically significant. These effect sizes are within the range of modest improvement typically seen in cholinesterase inhibitor trials. The difference Megerian pointed to is that their drug achieves this effect after two weeks, whereas the cholinesterase inhibitors can take months to do so. The combination therapy showed no signal on ADAS-cog. On a commercial, computerized cognitive assessment (called Mindstreams, by NeuroTrax), patients on both types of therapy showed improvement on either the spatial or memory index scores, Megerian added. The company also uses EEG, measuring the ratio of alpha:theta waves, and reports seeing a signal on the high-dose monotherapy.

Based on these results, Epix is planning two larger Phase 2b trials. A three-month monotherapy trial will compare PRX-03140 to donepezil and offer a three-month extension to people who get randomized to placebo; a six-month combination trial will test PRX-03140 added onto a stable dose of donepezil.

Presented by William James Ray of Merck and Co. in West Point, Pennsylvania, a different way of tapping the cholinergic system for future therapies drew wide praise from other meeting attendees. It targets the M1 muscarinic receptor, one of five GPCRs that constitute the metabotropic acetylcholine receptors. Abundant in the hippocampus and neocortex, the M1 receptor mediates γ oscillations in hippocampal networks that are thought to underlie memory (Fisahn et al., 2002). Researchers have long known that stimulating M1 can be effective in AD, but previous trials had to be abandoned because the drugs were insufficiently selective for M1 and produced intolerable cholinergic side effects.

Ray said that his group became excited about M1 activation when Frank LaFerla at the University of California, Irvine, reported that AF267B, the M1 agonist developed by Abraham Fisher at the Israel Institute for Biological Research in Ness-Ziona, removed plaque pathology, selectively reduced Aβ42 levels, and shifted APP processing toward α-cleavage in triple transgenic mice (see ARF related news story; Caccamo et al., 2006). Ray said these data made his group wonder whether the cholinergic system in the brain normally functions to repress accumulation of Aβ42. If this were true, a drug activating the M1 receptor would be a “home run,” Ray said, because it would combine the known symptomatic boost of drugs such as donepezil with a new disease-modifying effect on amyloid. (See also McLaurin Keystone story on links between cholinergic system and Aβ.)

A challenge to making this work is to ensure the drug is truly selective for the M1 receptor, Ray said. This is difficult to achieve with an agonist that binds the acetylcholine site of the receptor, because that site is highly conserved among all types of muscarinic receptor. For this reason, the scientists instead tried to find a drug that binds the M1 receptor in some other pocket where this receptor is distinctive from the M2 to M5 receptors. Such allosteric binding could tweak the M1 receptor and stimulate it. A subsequently developed allosteric potentiation assay identified one compound, called benyzl quinolone carboxylic acid (BQCA). It left unaffected not only the other muscarinic receptors, but also 300 human receptors and enzymes that are routinely screened against new compounds of interest, Ray said.

BQCA binds to a pocket in the extracellular domain of M1, far away from its acetylcholine site. The compound lowers the energy required for the receptor to adopt its active conformation. This effectively reduces the concentration of acetylcholine needed for signaling, and in this way could sensitize the hippocampus to the transmitter’s dwindling supply in AD, Ray said. BQCA meets some requirements for a drug: it can be taken orally, crosses the blood-brain barrier, and is not metabolized too rapidly in the liver. BQCA caused no cholinergic side effects at therapeutic doses in rodents, ferrets, dogs, and monkeys, and it has some functional data in mouse behavioral tests under its belt. Even so, more preclinical work yet needs to be done, particularly on defining the relationship between M1 activation and Aβ42 levels.

In addition to these efforts, a number of biotech and pharmaceutical companies are developing agonists for the α7 nicotinic acetylcholine receptor, but none presented at this conference.—Gabrielle Strobel.

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References

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Paper Citations

Fisahn A, Yamada M, Duttaroy A, Gan JW, Deng CX, McBain CJ, Wess J. Muscarinic induction of hippocampal gamma oscillations requires coupling of the M1 receptor to two mixed cation currents. Neuron. 2002 Feb 14;33(4):615-24. PubMed.
Caccamo A, Oddo S, Billings LM, Green KN, Martinez-Coria H, Fisher A, Laferla FM. M1 receptors play a central role in modulating AD-like pathology in transgenic mice. Neuron. 2006 Mar 2;49(5):671-82. PubMed.

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Keystone Drug News: Phase 2 Anti-oligomer Sugar Alcohol—How Might It Work?

08 Apr 2008

Eight years ago, JoAnne McLaurin’s research suggested that a particular stereoisomer of a inositol sugar alcohol might make a respectable AD drug (McLaurin et al., 2000; McLaurin et al., 2006). Soon after, the Canadian biotech company Transition Therapeutics Inc. (in which McLaurin declared a financial interest) took on the approach, and then Elan Pharmaceuticals licensed it. The FDA has fast-tracked the drug candidate, variously called scyllo-inositol, AZT-103, or ELND005, and a Phase 2 trial comparing three different doses in 340 people began last December. While this three-year trial proceeds, much remains to be learned about this molecule. At the Keystone conference held 24-29 March in Keystone, Colorado, McLaurin presented some of her mechanistic studies. In brief, scyllo-inositol appears to bind oligomers of Aβ42, preventing them from damaging synapses. The small molecule readily crosses the blood-brain barrier thanks to active transport; its accumulation in CSF and brain was shown recently (Fenili et al., 2007).

McLaurin first summarized published data in mice. Adding scyllo-inositol to the drinking water of transgenic CRND8 mice (which are an aggressive mouse model of amyloidosis, tau hyperphosphorylation, cognitive deficit, and early death) returned the Morris water maze performance of these otherwise impaired mice to that of non-transgenic controls. The compounds gave normal mice no boost, suggesting it is no cognitive enhancer. This worked well into the advanced stages of disease in this model. McLaurin noted that an intact cholinergic system is a prerequisite for learning and remembering in the water maze. When looking at the indicator enzyme choline acetyltransferase (ChAT), McLaurin and colleagues found its levels in the requisite brain area rescued to that of non-transgenic controls. This hinted, again, at a connection between Aβ and the cholinergic system (see ARF Keystone story) The scyllo-spiked drinking water also increased synaptophysin staining in the CRND8 mice, reduced their plaque burden, soluble and insoluble Aβ40 and 42 levels, as well as their astrogliosis and CAA. Treated CRND8 mice survived longer than their untreated littermates, McLaurin said.

To find out how the compound works, McLaurin first ruled out effects on γ-secretase activity and Aβ clearance. Next, oligomer-specific Aβ antibodies indicated that scyllo-inositol appears to increase the number of monomers and trimers while reducing the amount of larger oligomeric species, such as 40mers. An even more aggressive mouse model of amyloidosis, where young mice show plaques by the time they wean, responded to scyllo-inositol treatment with decreased plaque load but increased soluble oligomers. To McLaurin, this suggested that this mouse makes too much Aβ for the brain to be able to clear oligomers after scyllo-inositol stops aggregation and deposition. Despite the accumulating Aβ oligomers, the synaptic deficits of this model improved. In reply to a question about that, McLaurin noted that collaborative experiments with Jim Cleary at the University of Minnesota, Minneapolis, suggested that scyllo-inositol added to drinking water of rats rescues errors they make in a lever-pressing task when injected with Aβ oligomers; data on mouse LTP exist, as well (Townsend et al., 2006). “We think scyllo-inositol binds to oligomers and prevents them from interacting with the neurons,” McLaurin said.

Recent collaborative mass spectrometry experiments with Austin Yang, now at the University of Maryland School of Medicine, Baltimore, suggest that one Aβ42 matches up with two scyllo-inositol molecules, whereas Aβ40 does not appear to bind the compound with any measurable stoichiometry. Prior in-vitro experiments already had suggested that scyllo-inositol inhibits aggregation of Aβ42 but not 40, McLaurin said. Scyllo-inositol does not bind membrane lipids, she added.

To explore the molecular mechanism of this compound, McLaurin and colleagues tinkered with the side groups sticking out from its six-carbon ring. These experiments showed that scyllo-inositol needs to be shaped “just so”—not a hydrogen may change. Removing a hydrogen atom from a hydroxyl group to create a double-bonded oxygen, or substituting a hydroxyl with a fluoro, chloro, or methyl group all wiped out the desired activity. Moreover, all six of scyllo-inositol’s hydroxyl groups are in an equatorial plane, and turning even one into an axial orientation extinguished its activity, indicating that the molecule’s stereochemistry is critical (Nitz et al., 2008).—Gabrielle Strobel.

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References

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Paper Citations

McLaurin J, Golomb R, Jurewicz A, Antel JP, Fraser PE. Inositol stereoisomers stabilize an oligomeric aggregate of Alzheimer amyloid beta peptide and inhibit abeta -induced toxicity. J Biol Chem. 2000 Jun 16;275(24):18495-502. PubMed.
McLaurin J, Kierstead ME, Brown ME, Hawkes CA, Lambermon MH, Phinney AL, Darabie AA, Cousins JE, French JE, Lan MF, Chen F, Wong SS, Mount HT, Fraser PE, Westaway D, St George-Hyslop P. Cyclohexanehexol inhibitors of Abeta aggregation prevent and reverse Alzheimer phenotype in a mouse model. Nat Med. 2006 Jul;12(7):801-8. PubMed.
Fenili D, Brown M, Rappaport R, McLaurin J. Properties of scyllo-inositol as a therapeutic treatment of AD-like pathology. J Mol Med (Berl). 2007 Jun;85(6):603-11. PubMed.
Townsend M, Cleary JP, Mehta T, Hofmeister J, Lesne S, O'Hare E, Walsh DM, Selkoe DJ. Orally available compound prevents deficits in memory caused by the Alzheimer amyloid-beta oligomers. Ann Neurol. 2006 Dec;60(6):668-76. PubMed.
Nitz M, Fenili D, Darabie AA, Wu L, Cousins JE, McLaurin J. Modulation of amyloid-beta aggregation and toxicity by inosose stereoisomers. FEBS J. 2008 Apr;275(8):1663-74. PubMed.

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Keystone Drug News: Glimpse of Lilly’s Phase 1 Passive Vaccine

09 Apr 2008

Antibody therapies for AD have galvanized researchers, affected families, and observers from academia, industry, and even the investment community. Speakers at the Keystone meeting, held March 24 to 29 in Keystone, Colorado, deferred major news announcement on this topic to the upcoming ICAD conference this July in Chicago, but some morsels did slip out. Eli Lilly and Company’s passive immunotherapy reportedly was safe in an initial Phase 1 trial, though it may not act solely through a peripheral sink effect as previously proposed. Instead, a small proportion of the antibody enters the brain, where it appears to sequester forms of Aβ that are in equilibrium with aggregation and plaque formation. This increases the total CSF Aβ pool; however, a CSF Aβ fraction that is not bound to antibody appears to decrease. Besides this treatment, a range of other immunotherapies have by now entered human trials.

Ron DeMattos from Eli Lilly and Co., Indianapolis, introduced his talk with an overview of immunotherapies that are wending their way through the clinical pipeline. Active vaccines include Elan’s ACC-OO1 in Phase 2, Novartis’s CAD-106 in Phase 1, and Merck’s V950 in Phase 1. All three are based on the N-terminal of the Aβ peptide and are thought to stimulate an antibody-driven immune response that clears Aβ primarily through a mechanism of Fc-mediated endocytosis of antibody-decorated amyloid into microglia. Passive immunotherapies, where patients receive an antibody infusion, include a pharmacogenomic set of two Phase 3 trials for Elan’s bapineuzimap; Lilly’s LY2062430, which concluded a Phase 2 trial last month; Baxter’s small Phase 2 trial of its pooled IVIg preparation (aka Gammaguard ); as well as Phase 1 trials by Pfizer, GlaxoSmithKline, and Hoffman-La Roche/Morphosys.

While the trials play out, a major research question concerns the mechanisms by which these experimental therapies act. The three that have been proposed—phagocytic clearance, inhibition of fibrillogenesis, and peripheral sink—are not mutually exclusive, but it’s still debated which ones are at play in humans or whether there will be a single consensus mechanism. In his talk, DeMattos focused on the peripheral sink hypothesis, an indirect mechanism of amyloid clearance that his and colleagues’ research had suggested might cause a net efflux of Aβ from brain to plasma (DeMattos et al., 2001). In essence, the idea was that a peripheral antibody might be able to draw Aβ from the brain indirectly by shifting transport equilibria between the brain and blood and delivering brain Aβ to clearance in the liver and kidneys. The star in this hypothesis is m266. Called a “capture antibody,” this IgG binds epitopes 16 to 24 in the mid-section of soluble Aβ very tightly, prying it away from chaperones and other endogenous proteins that otherwise stick to Aβ. By contrast, m266 does not bind Aβ deposited in plaques, as do many other candidate immunotherapy antibodies.

In preparing m266 for the clinic, Lilly scientists humanized it. In parallel, they studied in detail how m266 perturbed the Aβ transport equilibrium between the plasma and CSF in PDAPP mice and non-transgenic rats. In the process, they also developed CSF biomarkers tailored to this specific treatment. In describing this research, DeMattos seemed to be gingerly stepping away from the original peripheral sink mechanism, though that appeared not to affect the therapeutic promise of m266 immunotherapy.

DeMattos reported that, as previously seen in PDAPP mice, intravenous m266 in rats caused m266-bound Aβ to shoot up 250-fold in plasma within a day. But the antibody also showed up in the CSF two hours after the injection, and in this compartment it reached an equilibrium of 0.08 to 0.14 percent with plasma antibody within the day. In the CSF, then, Aβ40 and 42 levels also increased. To understand what that meant, the scientists developed an assay that can distinguish between the total CSF Aβ pool (i.e., Aβ bound and unbound to IgG) and the pool of unbound Aβ. A subsequent rat study injecting three different doses of m266 showed a dose-dependent increase in total CSF Aβ but a dose-dependent decrease of the pool that is not bound to m266.

By this mechanism, plasma m266 would not initially draw Aβ out of the CSF but instead enter the CSF and disrupt an equilibrium there. The idea behind it is that soluble and insoluble Aβ are in a pathogenic equilibrium, and that decreasing the former by means of m266 would reduce the supply of Aβ available for aggregation and deposition, gradually shrinking amyloid pathology in that way. DeMattos noted that because both the peripheral and central mechanisms of the antibody occur simultaneously, it was impossible to identify which one was primarily responsible for the decreased unbound CSF Aβ. How local m266 mechanisms in the CSF interact with peripheral mechanisms in plasma is at yet unclear, DeMattos said.

DeMattos then offered a brief summary of the Phase 1 trial, promising full data of the Phase 2 trial for ICAD this July. In brief, study volunteers received placebo or one of three doses of m266. Their plasma Aβ40 increased as expected, though with a slower time course than seen in the animal studies. Their CSF likewise showed an increase in total Aβ, both 40 and 42. This trial did not have an assay for free Aβ, but that critical piece will come with the Phase 2 data, DeMattos said.

It’s unclear at present what happens to the antibody-bound Aβ accumulating in the CSF—whether it gets swiftly degraded or might cause complications. Antibody-Aβ complex that forms in the CNS may traffic to the periphery and get eliminated via normal IgG catabolism. That would be consistent with the original premise of the peripheral sink, but the time course of this traffic and degradation remains unknown. What is known, DeMattos noted, is that m266’s mechanism does not involve inflammatory processes. Nor have the Lilly scientists seen effects on CAA or CAA-related microhemorrhages with this antibody, at least in PDAPP mice.

This conference data comes as the latest word in an ongoing debate about m266. Earlier this year, Peter Seubert and colleagues at Elan Pharmaceuticals reported that, in their hands, m266 failed to shrink amyloidosis in PDAPP mice; it even tended to increase it. These scientists also noted that binding to m266 prolonged the normal degradation of Aβ (Seubert et al., 2008). As is often the case, this scientific discrepancy may find its resolution in the clinic.—Gabrielle Strobel.

References

News Citations

Paper Citations

DeMattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzman DM. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8850-5. Epub 2001 Jul 3 PubMed.
Seubert P, Barbour R, Khan K, Motter R, Tang P, Kholodenko D, Kling K, Schenk D, Johnson-Wood K, Schroeter S, Gill D, Jacobsen JS, Pangalos M, Basi G, Games D. Antibody capture of soluble Abeta does not reduce cortical Abeta amyloidosis in the PDAPP mouse. Neurodegener Dis. 2008;5(2):65-71. PubMed.

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Keystone Drug News: Repurposing the Anti-Inflammatory Drug CNI-1493?

10 Apr 2008

To borrow IT-speak, “repurposing” is an underused art in Alzheimer disease drug research. Drugs that have proven to be reasonably safe and effective in one condition could be tried for AD, as well. Such drugs tend to target aspects of AD that this disease has in common with other conditions, for example, its inflammatory component, potentially making them suitable for future combination therapies. One such drug is CNI-1493 (aka semapimod), a synthetic anti-cytokine that has been tested for conditions including Crohn’s inflammatory bowel disease, rheumatoid arthritis, and psoriasis. CNI-1493 is a tetravalent guanlhydrazone that inhibits signaling through the p38 MAP kinase pathway and suppresses release of inflammatory cytokines such as TNFα or certain interleukins from macrophages. At the Keystone conference held 24-29 March in Keystone, Colorado, Michael Bacher of Philipps University in Marburg, Germany, suggested that the drug binds tightly to Aβ oligomers and could be explored for potential future trials in AD.

The drug has been tested in Phase 1 and 2 trials for Crohn’s, but human testing stumbled over local side effects to the infused drug formulation. Recently, the company developing the drug came up with an oral preparation and is now trying to resume clinical trials.

Bacher reported first testing CNI-1493 in cell lines overexpressing APP. The compound left APP processing unchanged but, in dose-dependent fashion, reduced levels of secreted oligomers as detected by the A11 antibody (Kayed et al., 2003). Then Bacher and colleagues treated CRND8 mice with the drug for eight weeks between four and six months of age, when this model progresses from moderate to advanced disease (McLaurin Keystone story). Bacher reported seeing a drastic reduction of soluble brain Aβ levels, as well as of plaques in cortex and hippocampus. Bacher showed no data on the Morris water maze but reported that the treated mice performed better on a novel object recognition task. Besides binding to Aβ, the compound also pacifies microglia in the brain, according to Bacher; however, no detailed cytokine analysis was performed to characterize the microglial response in this mouse model further. In this initial study, the scientists did not yet look for changes of Aβ in plasma or effects on CAA. According to Bacher, this data is in press at the Journal of Experimental Medicine.—Gabrielle Strobel.

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Keystone Drug News: Pyroglu Aβ—Snowball That Touches Off Avalanche?

14 Apr 2008

At the Keystone conference on Alzheimer disease held last month in Keystone, Colorado, Takaomi Saido of the RIKEN in Wako, Japan, and Hans-Ulrich Demuth from the German biotech company, Probiodrug AG, based in Halle, presented new data to argue that pyroglutamate (pGlu) derivatives of Aβ may be critically important in the underlying pathology of Alzheimer disease. Far from being some marginal byproduct of Aβ degradation, pGlu forms of Aβ may be the very seeds that start oligomerization, according to Demuth. In a telephone-based rerun of his talk with this reporter, Demuth likened pGluAβ to a snowball perched on a pristine slope of normal Aβ, saying that when that ball starts to roll, it gathers the peptides that would otherwise melt in the glare of proteases and metabolic clearance.

Pyroglutamate derivatives of Aβ have been known since the early 1990s, but these highly stable and amyloidogenic peptides have come in for closer scrutiny only in the last few years. The peptides form when the first two amino acids of Aβ1-x are removed to expose a glutamate residue. Aβ3-x can then undergo a cyclization reaction at the N-terminus to form pyroglutamate species (a similar fate can befall Aβ11-x). Though researchers led by Hiroshi Mori, then at the University of Tokyo, were the first to report the existence of these peptides (see Mori et al. 1992), their presence, amount, and importance was largely underestimated, said Saido. At Keystone, Saido showed why. PyroGluAβ peptides bind avidly to reverse phase HPLC columns, which must be heated to 50 degrees Celsius using a basic solvent for complete elution. pGlu peptides are also underestimated in mass spectroscopic analysis of Aβ mixtures, because their altered charge-to-mass ratio resulting from truncation and cyclization makes them harder to ionize (e.g., in MALDI TOF MS). On top of that, it only recently became apparent that these species need not arise by the spontaneous and inexorably slow chemical cyclization of glutamate. Instead, cyclization is catalyzed by glutaminyl cyclase, an enzyme present in the brain. (For an introduction to pGluAβ, see ARF related news story).

These factors conspired to relegate pGluAβ to minor importance, suggested Saido. Over the years this has begun to change, as use of proper purification techniques shows that pGluAβ represents as much as 50 percent of total Aβ in human tissue, Saido said. Saido and colleagues previously showed that pyroglutamate-derived Aβ species are dominant in senile plaques and actually occur prior to plaques containing normal Aβ in Down syndrome patients (see Saido et al., 1995).

This observation jibes with what is now known about pGluAβ stability, namely, that pGlu-modified peptides have a much longer half-life than full-length Aβ. For its part, however, full-length has a longer half-life than non-cyclized, N-terminally truncated Aβ1, 2, 3, 4 or 5-x. So how do the modified peptides end up as a major constituent of plaques? Saido suggested this could happen if the degradation machinery somehow failed to remove the truncated and cyclized peptides. At Keystone, he showed that just this scenario can occur in transgenic mice.

One thing that sets existing transgenic mice apart from humans is that the rodents normally make very little pGluAβ. This changes when the protease neprilysin is taken out of the picture, Saido said. His group crossed neprilysin knockout mice with APP23 transgenic animals, which express human APP with the Swedish mutation. Mass spectroscopy shows that these crosses produce Aβ3-x and also pGluAβ species. Saido suggested that failure to form pGluAβ in neprilysin-competent animals might be the major limitation of mice as models of AD. He also noted that pGluAβ correlates to the PET signals detected using the Aβ ligand PIB. Researchers led by Makoto Higuchi at the National Institute of Radiological Sciences, Chiba, Japan, recently developed a PIB radiotracer that is suitable for use in small rodents and found that in both human and mouse brain, PIB retention correlates with levels of an N-terminal, pyroglutamate derivative of Aβ. These researchers proposed that one difference between mouse models of AD and the human condition is that the accelerated production of Aβ in the mice does not allow sufficient time for pGluAβ—and consequently “AD-like” plaques enriched in the pyroglutamate derivative—to form (see ARF related news story).

In humans, neprilysin levels inversely correlate with Aβ deposition in both demented and non-demented individuals (see, e.g., Russo et al., 2005 and Hellström-Lindahl et al., 2006), though it is not clear if that correlation results from increased production of pGluAβ.

These forms of Aβ are not only more stable than the unmodified peptide, but also more fibrillogenic, making them potentially more dangerous to have lurking in one’s brain. This was a point Demuth emphasized. He showed that while pGluAβ3-40 undergoes fibrillization faster than Aβ40, even further acceleration occurs when pGluAβ3-40 is used to seed solutions of Aβ40. This hints that in the AD brain, even a small amount of pGluAβ3-40, or of the even more amyloidogenic pGluAβ3-42, may be sufficient for such a snowball effect. A similar dynamic was seen pGluAβ3-38 in comparison to Aβ38, raising the question of whether γ-secretase modulation to increase Aβ38 production at the expense of Aβ42 production will by itself have the desired effect on amyloid pathology.

To test the relevance of this idea, Demuth and colleagues, together with Thomas Bayer and colleagues at the University of Göttingen, Germany, have created a transgenic mouse model where the third position of Aβ, the glutamate (E), is changed to a glutamine (Q). Glutamine gets cyclized more than three orders of magnitude faster by endogenous glutaminyl cyclase than is glutamate, so if N-terminal pyroglutamate is important for neurodegeneration, one might expect these animals to have accelerated pathology. This is exactly what Demuth showed at Keystone. These transgenic animals, called TBA2, show extremely rapid onset of symptoms, having behavioral problems and motor activity deficits (they cannot find or reach food) by the age of two months. At the same age, the animals’ hippocampus and cortex reacts with antiserum that recognizes both monomeric and oligomeric Aβ.

The Aβ in these animals is produced as part of a prothyrotropin-releasing hormone (proTRH)-Aβ chimera driven by a brain-specific promoter. The N3QAβ is cleaved from proTRH-Aβ by prohormone convertases in the secretory pathway. A similar construct (release of Aβ by furin from a BRI-Aβ sequence) was used by Eileen McGowan and colleagues at the Mayo Clinic, Jacksonville, Florida, to circumvent the complications of γ-secretase cleavage at both the 40 and 42 positions. McGowan and colleagues found that Aβ40 produced from this construct is actually neuroprotective (see ARF related news story). That N3QAβ driven from a similar system is destructive is indicative of the toxicity of pGluAβs. “This is the first Alzheimer’s-related model that shows a tremendous behavioral phenotype after only two months of existence,” said Demuth. He further demonstrated the toxicity of N3QAβs in hippocampal rat brain slices. Conditioned medium from HEK293 cells transfected with APP-NLQ (a construct having asparagine, leucine, and glutamine in the first three positions of Aβ) more strongly depressed hippocampal LTP than did medium from cells expressing normal APP or APP-NLE, which would yield full-length Aβ or Glu3Aβ with slower N-terminal cyclization rates. To Demuth, the in-vivo and tissue culture experiments together confirm the toxicity of pGluAβ.

Human Glutaminyl Cyclase—A Therapeutic Target? If pGluAβ drives AD pathology, then what is driving cyclization of the N-terminal glutamate in the diseased state that is not at work in normal brain? Possible answers here include enhanced Aβ production, enhanced removal of the N-terminal dipeptide, or more rapid cyclization.

Glutaminyl cyclase (QC), the enzyme that cyclizes N3Aβ glutamine in the above mouse model, is normally found in the brain, but at Keystone, Demuth presented data to suggest that this enzyme is massively overexpressed in AD brain. Enzyme and pGluAβ levels show the same pattern, with QC levels being increased up to 40 times depending on the severity of the investigated stages. The enzyme is expressed in brain areas affected in AD, including the CA3 layer of the hippocampus and the cortex.

Such data hint that targeting QC might be one approach to tackling AD pathology, and that’s just what Probiodrug has in mind. Demuth presented data on one experimental inhibitor, PBD150, suggesting efficacy in animal models. Used as a prophylactic in Tg2576 mice beginning at four or six months, PBD150 reduced both pGluAβ and total Aβ, and it improved memory in a contextual fear paradigm. Its efficacy was similar when used as a treatment in older (10 months) animals that have formed plaques. PBD150 reduced pGluAβ and improved memory. Prophylaxis worked in the TASD41 mouse, which produces very high amounts of human APP carrying both Swedish and London mutations (see Rockenstein et al., 2001). Demuth said that Probiodrug has profiled better compounds for passage of the blood-brain barrier.

One potential bonus of QC inhibition is that it may also reduce reactive gliosis. In characterizing the effect of PBD150, Demuth’s collaborators Steffen Rossner at the Brain Research Institute at University of Leipzig, and Manfred Windisch of the contract research organization JSW Research in Graz, Austria, noticed reduction in the number of reactive glia surrounding plaques in treated Tg2576 and TASD41 mice, respectively. This may simply be due to the reduction of plaque load or the reduction of seed peptides, but it may also be related to a direct inhibition of glial responses. In this regard, Demuth noted that other pyroglutamate peptides, in particular pGlu derivatives of monocyte chemoattractant protein (MCP-1), which has an N-terminal glutamine, trigger glial cell migration. Therefore, blocking QC may also reduce inflammatory responses in the brain and even elsewhere. Probiodrug is screening QC inhibitors for inflammatory diseases such as atherosclerosis, as well.

Could this anti-inflammatory aspect be a downside to the strategy, leading to side effects? “The binding constants for the cyclization of glutamine peptides are much lower than for glutamate peptides, so you don’t have to fully inhibit QC’s negative side function of glutamate cyclization within a certain time frame to get an effect on Aβ but still have sufficient glutamine cyclization for maturation of proteins, for example, peptide hormones,” said Demuth.

There may be other surprises in store from this pyroglutamate story. Demuth briefly described experiments that lead him to believe BACE may not be the only important enzyme that leads to Aβ production. This possibility has been entertained before but currently represents a fringe view (see ARF related news story). Cultured HEK293 cells produce much less pGluAβ when transfected with APP containing the Swedish mutation, which promotes cleavage at the BACE site. This suggests that there may be another protease that cleaves Aβ in such a way that the glutamate is unmasked at the N-terminus and can be cyclized. One possibility is that an alternate protease cleaves APP at the three position of Aβ.

Whether another protease is important in vivo remains to be seen. Robert Vassar of Northwestern University, Chicago, attended the meeting but is not involved in this work. He told ARF that the PDAPP mouse used by Elan is wild-type at the β-secretase cleavage site and, when crossed with BACE knockouts, produces no Aβ or amyloid plaques, suggesting that BACE is the major protease for Aβ even in wild-type APP (McConlogue et al., 2007). “There are other enzymes that cut [at the β site], but they cut rarely and the amounts of their Aβ are very low, so I don’t think they play a significant role in AD. They do start to cut at higher levels when you overexpress APP. It is possible that under condition of overexpression, and when you inhibit BACE so you have more full-length APP, that some of these aberrant cleavages appear to increase,” Vassar suggested. Perhaps any BACE doppelganger will reveal itself at the next Keystone meeting.—Tom Fagan.

References

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Paper Citations

Mori H, Takio K, Ogawara M, Selkoe DJ. Mass spectrometry of purified amyloid beta protein in Alzheimer's disease. J Biol Chem. 1992 Aug 25;267(24):17082-6. PubMed.
Saido TC, Iwatsubo T, Mann DM, Shimada H, Ihara Y, Kawashima S. Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3(pE), in senile plaques. Neuron. 1995 Feb;14(2):457-66. PubMed.
Russo R, Borghi R, Markesbery W, Tabaton M, Piccini A. Neprylisin decreases uniformly in Alzheimer's disease and in normal aging. FEBS Lett. 2005 Nov 7;579(27):6027-30. PubMed.
Hellström-Lindahl E, Ravid R, Nordberg A. Age-dependent decline of neprilysin in Alzheimer's disease and normal brain: inverse correlation with A beta levels. Neurobiol Aging. 2008 Feb;29(2):210-21. PubMed.
Rockenstein E, Mallory M, Mante M, Sisk A, Masliaha E. Early formation of mature amyloid-beta protein deposits in a mutant APP transgenic model depends on levels of Abeta(1-42). J Neurosci Res. 2001 Nov 15;66(4):573-82. PubMed.
McConlogue L, Buttini M, Anderson JP, Brigham EF, Chen KS, Freedman SB, Games D, Johnson-Wood K, Lee M, Zeller M, Liu W, Motter R, Sinha S. Partial reduction of BACE1 has dramatic effects on Alzheimer plaque and synaptic pathology in APP Transgenic Mice. J Biol Chem. 2007 Sep 7;282(36):26326-34. PubMed.

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Keystone: BACE, Age, Stress, Energy—Is Translation to Blame?

06 May 2008

At the Alzheimer’s Disease conference, held 24-29 March at the Keystone resort in the Colorado Rocky Mountains, Robert Vassar of Northwestern University in Chicago briefed the audience on his group’s latest work on the β-secretase BACE1. Vassar, along with Bart de Strooper and Philip Wong, won the MetLife Award earlier this year for his role in cloning and characterizing this important enzyme. More recently, the Vassar lab has hit upon the translational stress controller eIF2α in its ongoing efforts to try to work out how BACE1 figures in late-onset AD. (For their part, the De Strooper and Wong labs keep exploring BACE1, as well. Just this past month, the former reported that a particular group of microRNAs normally keep a lid on BACE1 translation but fail in sporadic AD [see Hebert et al., 2008], while the latter strengthened the notion that BACE1 might indeed play a role in schizophrenia, as well [see Savonenko et al., 2008]).

Unlike APP and presenilin, BACE1 mutations have never been found to cause familial forms of AD; however, the protease has nonetheless assumed a central place in AD research. The field has widely accepted the finding made by different groups over the past six years that BACE1 activity and protein levels (but not its mRNA) are increased in the brains of people with late-onset AD (e.g., Holsinger et al., 2002; Fukumoto et al., 2002; Tyler et al., 2002; Yang et al, 2003). This finding has raised the question of what regulates BACE1 translation. Vassar approached the issue starting from the broader premise that both aging and Aβ42 contribute to AD pathogenesis early on. The lab began a research program to see if age-related stressors in a person’s physiology might integrate with specific AD processes through BACE1. Broadly speaking, BACE1 levels go up in response to various stressors—published work exists on apoptosis, hypoxia, ischemia, and oxidative stress, as well as traumatic brain injury.

At Keystone, Vassar focused on energy production, hypothesizing that age-related energy impairments induce a stress response that drives up BACE1 in the brain. Brain imaging has shown that glucose use is down in AD, MCI, and even asymptomatic middle-aged people whose ApoE4 genotype puts them at risk of AD. And work going back to 1994 showed that blocking energy metabolism boosted the amyloidogenic processing of APP in vitro (Gabuzda et al. 1994). To model where in all this there could be a connection to BACE1, Rod Velliquette in Vassar’s lab blocked energy production in Tg2576 APP-transgenic mice with injections of various energy production inhibitors. Published work indicates that single injections of either insulin (which causes hypoglycemia), 2-deoxyglucose (which mimics hypoglycemia), or 3-nitropropionic acid (which reduces ATP production), all increased BACE1 levels and Aβ levels (Velliquette et al., 2005).

To get at how this might work, Tracy O’Connor in the lab studied energy inhibition in a cell line overexpressing BACE1 and in cultured primary cortical neurons from Tg2576 mice. This work ruled out the possibility that BACE1’s half-life grew longer under conditions of energy starvation. But it did point to a change in eIF2α. This translation factor normally helps initiate global translation. Under conditions of stress it becomes increasingly phosphorylated, arrests global translation, and switches to supporting selective translation of certain stress-response proteins instead. The BACE1 mRNA 5’ untranslated region contains signature features of mRNAs that are translated with increased efficiency following physiological stresses, an effect induced by phosphorylated eIF2α. Experiments driving up eIF2α phosphorylation pharmacologically with an inhibitor of its phosphatase increased BACE1 protein levels, whereas experiments driving down eIF2α phosphorylation genetically prevented the stress-induced BACE1 increase. “We think we have nailed the mechanism. We can modulate eIF2α phosphorylation up and down and get a corresponding change in BACE1,” Vassar told the audience.

With those data, O’Connor went back in vivo and measured whether eIF2α was more phosphorylated in Tg2576 mice that had undergone chronic energy inhibition. Three months of weekly injections of either 2-deoxyglucose or 3-nitropropionic acid likewise increased both eIF2α phosphorylation and BACE1 protein levels but, again, not BACE1 mRNA. Aβ levels and amyloid plaque deposition went up in parallel in these energy-deprived mice. And a series of postmortem samples of AD brain from Rush University Medical Center confirmed not only the previously reported BACE1 increase but also a corresponding increase in eIF2α-P in AD patients.

When the scientists looked in a particularly aggressive transgenic mouse model of autosomal-dominant AD (Oakley et al., 2006), they noted an eIF2α-P increase even without depressing the mice’s energy production, Vassar reported. This suggested to him that amyloid itself can drive up eIF2α phosphorylation and subsequent selective BACE1 translation as part of a positive feedback loop, in line with a localized BACE1 increase near plaques the lab had published last spring (Zhao et al., 2007).

The data available to date comes down to a working hypothesis by which different kinds of age-related stress may all funnel into impaired brain energy metabolism, Vassar said. The stressors could be age, high cholesterol, cardiovascular disease, traumatic brain injury, ApoE4. Each of these could cause physiological stress, which leads to eIF2α phosphorylation and a subsequent BACE1 increase. This is initially a protective response to short-term stress, but if the chain of events becomes chronic, then the increased Aβ production will lead to amyloid accumulation. If amyloid degradation and clearance cannot keep up, amyloid itself can feed back to fuel eIF2α phosphorylation, more amyloid, and then downstream tau pathology and sporadic AD.

This presentation generated numerous questions, including why some people get AD without having had energy deprivation (e.g., a stroke) while others who sustain trauma do not go on to develop AD. In response, Vassar said that sporadic AD is a syndrome and this pathway may only operate in a fraction of patients. On the other hand, age-related chronic physiological stress is likely to be low level, and therefore difficult to detect, but could increase amyloidogenesis over a long period of time. There is at present no epidemiological data on energy inhibition in a subset of AD samples. Another questioner noted that when eIF2α switches from global translation to selective translation, other proteins may get selectively upregulated and other disease states may follow. A third area that needs follow-up research is whether vascular disease, which constricts the brain’s blood supply and often comes with amyloid deposits in blood vessels, might act in part through this change in translational regulation, as well (Cole and Vassar, 2008).—Gabrielle Strobel.

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References

Paper Citations

Hébert SS, Horré K, Nicolaï L, Papadopoulou AS, Mandemakers W, Silahtaroglu AN, Kauppinen S, Delacourte A, De Strooper B. Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/beta-secretase expression. Proc Natl Acad Sci U S A. 2008 Apr 29;105(17):6415-20. Epub 2008 Apr 23 PubMed.
Savonenko AV, Melnikova T, Laird FM, Stewart KA, Price DL, Wong PC. Alteration of BACE1-dependent NRG1/ErbB4 signaling and schizophrenia-like phenotypes in BACE1-null mice. Proc Natl Acad Sci U S A. 2008 Apr 8;105(14):5585-90. PubMed.
Holsinger RM, McLean CA, Beyreuther K, Masters CL, Evin G. Increased expression of the amyloid precursor beta-secretase in Alzheimer's disease. Ann Neurol. 2002 Jun;51(6):783-6. PubMed.
Fukumoto H, Cheung BS, Hyman BT, Irizarry MC. Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease. Arch Neurol. 2002 Sep;59(9):1381-9. PubMed.
Tyler SJ, Dawbarn D, Wilcock GK, Allen SJ. alpha- and beta-secretase: profound changes in Alzheimer's disease. Biochem Biophys Res Commun. 2002 Dec 6;299(3):373-6. PubMed.
Yang LB, Lindholm K, Yan R, Citron M, Xia W, Yang XL, Beach T, Sue L, Wong P, Price D, Li R, Shen Y. Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease. Nat Med. 2003 Jan;9(1):3-4. PubMed.
Gabuzda D, Busciglio J, Chen LB, Matsudaira P, Yankner BA. Inhibition of energy metabolism alters the processing of amyloid precursor protein and induces a potentially amyloidogenic derivative. J Biol Chem. 1994 May 6;269(18):13623-8. PubMed.
Velliquette RA, O'Connor T, Vassar R. Energy inhibition elevates beta-secretase levels and activity and is potentially amyloidogenic in APP transgenic mice: possible early events in Alzheimer's disease pathogenesis. J Neurosci. 2005 Nov 23;25(47):10874-83. PubMed.
Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, Guillozet-Bongaarts A, Ohno M, Disterhoft J, Van Eldik L, Berry R, Vassar R. Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation. J Neurosci. 2006 Oct 4;26(40):10129-40. PubMed.
Zhao J, Fu Y, Yasvoina M, Shao P, Hitt B, O'Connor T, Logan S, Maus E, Citron M, Berry R, Binder L, Vassar R. Beta-site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques: implications for Alzheimer's disease pathogenesis. J Neurosci. 2007 Apr 4;27(14):3639-49. PubMed.
Cole SL, Vassar R. Linking vascular disorders and Alzheimer's disease: potential involvement of BACE1. Neurobiol Aging. 2009 Oct;30(10):1535-44. PubMed.

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