20 Aug 2022

Alzheimer’s trials targeting β-secretase screeched to a halt when it turned out that the drugs slightly worsened cognition. Alarmed, pharmaceutical companies ditched their BACE programs, or put them on ice. Still, since the last such trial ended in 2019, a small cadre of scientists has been urging the dementia field not to forget these inhibitors. At the Alzheimer’s Association International Conference held July 31 to August 4 in San Diego, and at a BACE symposium on July 26 at the University of Connecticut School of Medicine, Farmington, these researchers honed their argument for reinvigorating these programs, claiming that a dose could be found that both is safe and would sufficiently slow or stop the trickle of Aβ that causes AD.

Some scientists outlined trial designs to test this hypothesis. Others suggested biomarker or cognitive tests that might track cognitive side effects. All agreed that these inhibitors are worth another look and that they could provide a simpler and cheaper alternative to long-term immunotherapy.

Though academics had voiced concerns about moving forward too quickly with BACE inhibition, the first clinical trials started more than a decade ago. Despite early setbacks with compounds that damaged the eye or the liver, a slew of candidates with good safety profiles soon emerged that looked promising in early trials. Then, one by one, BACE inhibitor programs were terminated in Phase 3. Merck’s verubecestat, Janssen/Shionogi Pharma’s atabecestat, Novartis/Amgen’s umibecestat, Biogen/Eisai’s elenbecestat, and Lilly's lanabecestat all fell by the wayside (Feb 2018 news; May 2018 news; Jul 2019 news; Sep 2019 news). The most obvious problem was that these drugs worsened performance on cognitive tests, though some people also lost weight or had brain atrophy as well.

What happened? Scientists at these two conferences believe that the cognitive loss directly resulted from BACE inhibition, not from some off-target effect. This is because in addition to APP, BACE cleaves dozens of other substrates, including seizure protein 6, aka Sez6-like (Sez6L), neuregulin, and NCAML1, any of which might be indispensable for synaptic activity. That these other substrates might be affected had always been a red flag for these programs, but because BACE1 knockout mice seemed mostly normal, drug sponsors had calculated that if they didn’t completely block the enzyme, pharmacological inhibition would be a net positive.

Some BACE afficionados believe it still can be. “I think that industry was too quick to abandon these inhibitors,” said Stefan Lichtenthaler, DZNE, Munich. “We know that clinical development of BACE inhibitors is on hold, but this is not automatically the end of the development. Let’s keep in mind that setbacks in clinical trials are completely normal,” he said. This is also the view of Riqiang Yan, University of Connecticut School of Medicine, who organized the mini symposium there, and Robert Vassar, Northwestern University, Chicago. At AAIC, Vassar co-chaired a focused topic session on BACE inhibition for the prevention of AD with Maria Carrillo of the Alzheimer’s Association. Vassar noted that statins were almost abandoned for cardiovascular disease because of side effects, yet went on to become widely used. “That’s the paradigm to keep in mind,” he said.

So, how to continue from here? Vassar recommended beginning with small, three-month, dose-finding trials in healthy controls, aiming to block 12, 25, or at most 50 percent of BACE activity. Most of the prior clinical trials inhibited the protease by around half, and in some cases by more than 70 percent. Scientists think this was too high and likely spelled doom for these trials.

Once a suitable dose is found, Vassar thinks the next step would be a Phase 2 trial in older, cognitively unimpaired people who are at risk for AD, testing about 100-200 people per dose. These volunteers might be homozygous for ApoE4, or, in a primary prevention trial, carry a familial AD mutation. If such trials can pinpoint a dose that show signs of efficacy without worsening cognition or causing other serious side effects, then a large Phase 3 secondary prevention trial akin to the AHEAD trials would be on order. This might take four to five years, or up to 10 years in a primary prevention trial in FAD carrier. “Despite the challenges, BACE inhibition offers exquisite sensitivity and specificity for Aβ lowering, and represents a powerful, practical, simple approach to AD prevention,” said Vassar. Others envision this playing out similarly. “Whatever BACE inhibitor we use, we have to determine if it is going to have that negative [cognitive] signal,” noted Bruce Albala, who is now at University of California, Irvine, at the AAIC. Albala had worked at Eisai during the elenbecestat trials.”

Reisa Sperling, Brigham and Women’s Hospital, Boston, agreed with this reasoning. She noted that in BACE trials, the greatest cognitive decline seemed to occur in people who were least impaired to begin with, suggesting this might be the best population in which to rule out detrimental effects on cognition. Though cognitive decline in BACE trials was apparent by three months, it might start earlier. “Three months was the earliest we looked, but we might actually see it at three to four weeks,” she suggested.

Alas, the same scientists agree that more work needs to be done on understanding BACE function before the pharmaceutical industry is likely to get on board with this type of plan. “What is the mechanism of the cognitive loss? That’s the key question for moving into low-dose clinical trials,” said Vassar.

There is some progress on that front. Working with Matthew Kennedy from Merck, Lichtenthaler has been analyzing samples from verubecestat trials to identify substrates that might have been improperly processed under BACE inhibition. Previously, he had reported that soluble ectodomains of Sez6 and gp130 plummeted in CSF shortly after people began taking the drug (Apr 2021 conference news). Sez6 is known for supporting synaptic spines, and expressed by neurons, gp130 forms a complex with interleukin 6 receptors that’s essential for IL-6 signaling. This cytokine can suppress appetite, and Lichtenthaler suggested that this might help explain the weight loss seen in clinical trials and in preclinical mouse studies of BACE inhibitors. He believes that the soluble ectodomains of Sez6 and gp130 in the CSF could serve as pharmacodynamic activity markers to track side effects in clinical trials.

Tracking BACE2. Levels of the soluble ectodomain of VEGFR3 fell in plasma of mice treated with verubecestat (left). Plasma sVEGFR3 also fell in four people who were treated with atabecestat (right). [Courtesy of Stefan Lichtenthaler.]

What about BACE2? Most inhibitors tested in trials cannot distinguish between the two BACE isoforms. Although BACE2 expression in the central nervous system is low, Lichtenthaler suspects that its inhibition might also affect cognition. Tracking BACE2 activity has been challenging because there are no easy redouts bar fur color in mice, which grays when BACE2 is blocked. This warning sign is not only slow and unquantitative, it also breaks a human trial's blind.

At the UConn symposium, which was attended by a few dozen people from a handful of leading BACE labs, Lichtenthaler reported a new substrate that could be a much better marker, even in furless humans. It turns out levels of the soluble ectodomain of vascular endothelial growth factor receptor plummet in the plasma of BACE2 knockouts compared to normal mouse plasma. Likewise, BACE inhibitors reduced VEGFR3 ectodomain shedding in cell culture and in vivo, verubecestat dose-dependently reduced sVEGFR3 in mouse plasma. In fact, its levels dropped by about 25 percent after only one day on a high dose of the drug; a week later, sVEGFR3 was down by 75 percent. The ectodomain dropped by about 60 percent in mice fed a low dose, even though their coats kept their hue. The data suggest that this ectodomain could be a sensitive and fast-responding marker of BACE2 inhibition.

What about in people? Preliminary data suggests there here, too, sVEGFR3 might be useful. Lichtenthaler's lab found that, in the plasma of four people who had been on atabecestat for a month, the protein level had dropped, in one person by up to 50 percent.

Do any of these substrate changes track with cognitive changes? That’s a question several labs are going after. In ongoing work, Lichtenthaler and Kennedy are correlating potential markers with cognitive outcomes from verubecestat trials. In Vassar’s lab, Elyse Watkins is hunting substrates responsible for cognitive deficits by genetically or pharmacologically knocking them down one by one and checking if that mimics the effect of BACE inhibition. “We hope to be able to correlate behavior with specific substrates to see if we can find a sweet spot that will significantly lower Aβ without affecting cognitive decline,” said Vassar.

Are these substrate cleavages responsible for the biological effect, or do they merely reflect a signaling change on the surface of the cells bearing the receptors? To address this question, Watkins plans to treat PDAPP mice with a BACE inhibitor, then inject adeno-associated virus expressing either the soluble shed fragments of various substrates or their plasma membrane C-terminal domains to see if any will rescue phenotypes that have been linked to BACE inhibition, such as loss of dendritic spines or mossy fiber disorganization (Dec 2013 conference news; Sep 2018 news). Watkins told Alzforum that ideally she would like to have a cognitive test for mice that better reflects the subtle changes seen in people on BACE inhibitors. Many scientists believe that the standard memory tests used for mice poorly reflect cognition in people.

For his part, Kennedy wants to skirt this problem by studying nonhuman primates. At Merck, Kennedy uses a colony of rhesus monkeys to monitor BACE substrate changes in the CSF, as well as cognition in a visuospatial paired-associate learning task. He wants to see if he can reproduce the cognitive worsening seen in people on BACE inhibitors, relate that to CSF markers of BACE substrate cleavage, and look for a dosing window where Aβ production can be reduced without worsening cognition. This work was interrupted by the COVID pandemic; Kennedy said it has now resumed, and the monkeys are relearning the cognitive task.

Even if a dose window can be found for these drugs, questions remain. Whom to treat, and when? At AAIC, Mathias Jucker, University of Tübingen, Germany, argued that BACE inhibition would have to start very early in the disease process, before plaques had become established, if it is to head off neurodegeneration. Scientists in his lab fed a BACE inhibitor to APPPS1 mice at various ages, then measured the effect on various markers of AD pathology, including Aβ, sTREM2, soluble tau species, and neurofilament light (NfL), a marker of neurodegeneration. They found that both chronic dosing and three-month stints on the inhibitor reduced Aβ, sTREM2, and tau, regardless of when the treatment was started. NfL, however, was another matter. If treatment began when plaques were already established, then NfL just kept rising, albeit at a slightly slower pace (see image below).

“This is reminiscent of what we have seen in clinical trials, that NfL just doesn’t really follow Aβ once the brain was full of Aβ,” said Jucker. He believes that once amyloid seeding activity reaches a plateau, which happens at about 50 percent maximal plaque load, then the brain reaches a tipping point and neurodegeneration proceeds independently of Aβ, in keeping with the proposed cellular phase of AD (De Strooper and Karran 2016). Eric McDade, Washington University, St. Louis, saw this as a problem for the field. “It is very concerning that in treating with just a BACE inhibitor after NfL has started to change, it is really difficult to have a dramatic effect in a positive direction,” McDade said.

In his talk, McDade made a case for using BACE inhibitors as an adjunct to immunotherapy. “We have a number of immunotherapies that will potentially be approved that significantly reduce amyloid pathology, but we have to know how we will use these therapies long-term,” he said. If those therapies are stopped, then plaque load would rebound by about 20 percent over 18 months. “Do we continue to dose at a low level, or do we combine with a therapy that will prevent amyloid production?” McDade asked.

Here is where the BACE inhibitors come in. By preventing Aβ production, they can keep amyloid from creeping back up. McDade sees primary prevention trials as an essential first step to establishing whether BACE inhibitors can be used safely. He thinks frequent cognitive and psychiatric testing, combined with monitoring by MRI, plasma markers of AD pathology, and other BACE substrate cleavages, now offer the necessary tools to make this happen.

Sperling agreed. She thinks some of these tests can be run now on existing samples. “We have really good tools now, but we should look hard at the plasma data we have from prevention trials to try to get hints at the pharmacodynamic levels of BACE inhibitor exposure and when we can we start to see biomarker changes,” she said. “We should even look to see if there is any evidence of a beneficial change on p-tau measures as well,” Sperling said.

BACE and DAMs. In microglia, BACE1 attenuates signaling through toll-like receptors. Blocking the protease activates PI3K and p38 MAPK pathways, facilitating the transition to the DAM state and enhancing phagocytosis of amyloid. [Courtesy of Singh et al., 2022, Science Advances.]

One surprise benefit of BACE inhibition came from Yan’s lab. At the UConn meeting, Neeraj Singh reported that microglia in 5xFAD mice increase expression of BACE1 as plaques accumulate, and that this restricts plaque clearance. When Singh conditionally knocked out the gene in microglia, the cells increased phagocytosis and degradation of amyloid. As a result, synaptic function improved, as did learning and memory. It turns that that BACE1 activity helps microglia transition from a homeostatic state to a more phagocytic disease-associated microglia. Similarly, Singh found that the transition from homeostatic to DAM cells correlated with reduced microglial expression of BACE1. Digging into the mechanism, Singh found evidence that BACE inhibition reduced signaling through interleukin 1R2 and toll-like receptors 2 and 4. Reduced cleavage of these receptors associated with activation of p38 MAPK and phosphatidyl inositol 3-kinase pathways and the expression of DAM genes. The findings were recently published in two papers in Science Advances (Singh et al., 2022; Singh et al., 2022). 

Yan suggested that a microglial-specific BACE1 inhibitor would be better than one that systemically blocks the enzyme—a tough ask for medicinal chemists.—Tom Fagan

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References

Therapeutics Citations

1. Verubecestat
2. Atabecestat
3. Umibecestat
4. Elenbecestat
5. Lanabecestat

News Citations

1. Merck Axes Verubecestat for Prodromal AD, Researchers Say ‘Go Earlier’ 14 Feb 2018
2. Liver Tox Ends Janssen BACE Program 18 May 2018
3. Cognitive Decline Trips Up API Trials of BACE Inhibitor 12 Jul 2019
4. End of the BACE Inhibitors? Elenbecestat Trials Halted Amid Safety Concerns 13 Sep 2019
5. What BACE Hits: New Substrates Create New Headaches 9 Apr 2021
6. Blocking BACE—Do Adult Mouse Phenotypes Predict Side Effects? 13 Dec 2013
7. In Conditional BACE1 Knockouts, Hippocampal Axons Compromised 21 Sep 2018

Paper Citations

1. De Strooper B, Karran E. The Cellular Phase of Alzheimer's Disease. Cell. 2016 Feb 11;164(4):603-15. PubMed.
2. Singh N, Benoit MR, Zhou J, Das B, Davila-Velderrain J, Kellis M, Tsai LH, Hu X, Yan R. BACE-1 inhibition facilitates the transition from homeostatic microglia to DAM-1. Sci Adv. 2022 Jun 17;8(24):eabo1286. PubMed.
3. Singh N, Das B, Zhou J, Hu X, Yan R. Targeted BACE-1 inhibition in microglia enhances amyloid clearance and improved cognitive performance. Sci Adv. 2022 Jul 22;8(29):eabo3610. Epub 2022 Jul 20 PubMed.

Further Reading

News

1. In Conditional BACE1 Knockouts, Hippocampal Axons Compromised 21 Sep 2018