The 14th International Athens/Springfield Symposium on Advances in Alzheimer Therapy was held March 9 to 12 in Athens, Greece. Researchers met to discuss developments in basic and clinical research, including new approaches to study Aβ oligomers and results of a clinical trial to evaluate a dietary approach to treat early stage Alzheimer’s disease.
Souvenaid Trial Missed Primary, Partially Met Secondary Endpoints
At the 14th International Athens/Springfield Symposium on Advances in Alzheimer Therapy, held March 9 to 12 in Athens, Greece, scientists presented their latest results on Souvenaid, a medical food formulated as a breakfast drink that is being marketed by the Netherlands-based food company Nutricia. A clinical trial within the LipiDiDiet project, funded by the European Union, missed its primary outcome, finding no significant benefit on a composite measure of cognition. Even so, researchers associated with the Souvenaid program highlighted positive results on secondary measures of memory, cognition, and daily function in a subset of trial participants who complied with the daily regimen of Souvenaid and were at the earliest stages of prodromal AD. Hippocampal atrophy also slowed.
“We missed the primary endpoint, but it looks like something is happening cognitively in these people,” said Tobias Hartmann, Saarland University, Homburg, Germany, who coordinates the LipiDiDiet project. A second study, funded by Nutricia, reported in Athens that Souvenaid stimulates brain phospholipid metabolism in mild AD.
Hilkka Soininen, University of Eastern Finland, Kuopio, headed the LipiDiDiet Souvenaid trial, which tested Souvenaid early in disease. Scientists at 11 sites in four countries enrolled people aged 55 to 85 with prodromal Alzheimer’s (Dubois et al., 2007). All had deficits in episodic memory and a biomarker of underlying AD pathology, but their symptoms were mild, with MMSE scores of 24 or higher. In this randomized, double-blind, placebo-controlled study, 153 people drank Souvenaid, and 158 drank placebo every day for two years. Each group had a 20 percent dropout. Volunteers were assessed at baseline, six, 12, and 24 months.
The researchers took as primary outcome a composite score on five subtests from a modified Neuropsychological Test Battery (NTB). Secondary measures included the CDR-SB, other NTB composite scores including one of episodic memory, and MRI measures of brain volume. The Pentara Corporation of Salt Lake City, Utah, led by Suzanne Hendrix, independently analyzed the data. The scientists assessed a subgroup of 119 people taking Souvenaid, and 133 on placebo, who regularly followed the protocol. The scientists also analyzed benefits in people with the highest baseline MMSE scores, meaning those who started at the earliest phases of prodromal AD.
Overall, Souvenaid made no difference on the NTB composite. The treatment group did not perform differently from the placebo group. This is the primary result of the trial. Hartmann said the placebo group declined less than expected, rendering the study underpowered.
For the CDR-SB, the researchers saw a trend toward a benefit for the treatment group, which became a statistically significant 0.55 points only in the subgroup who regularly followed the treatment protocol. Within that compliant subgroup, 68 people who started the trial with an MMSE of 26 or higher scored an average of 0.69 points above placebo; the 37 who began with an MMSE of 28 or above had a 1.3-point advantage. Soininen said these effects were statistically significant. She saw similar benefits on the episodic memory composite score of the NTB. “The gains were most pronounced with high baseline MMSE, and with regular intake. Starting earlier seems to translate to more benefit,” said Hartmann.
The drink affected hippocampal volume. After two years, this brain structure had atrophied 39 percent more in the placebo than the treatment group. Hartmann interpreted this to be a good thing, saying that AD patients have more shrinkage than age-matched controls early in disease, and this may parallel disease progression (Oct 2009 conference news; Hennemen et al., 2009; Barnes et al., 2009). He commented, however, that whether atrophy changes translate into cognitive and clinical benefit remains to be seen. Other studies are less clear about how hippocampal volume loss relates to age, disease, and treatment (Jun 2013 news; Jul 2004 conference news).
Several cases of serious adverse events occurred. They included infections and nervous system disorders, but Hartmann said that as in previous trials, they were considered unrelated to Souvenaid. Patrick Kamphuis heads the neuroscience division of Nutricia research; he told Alzforum that for the time being, the present results would not change the company’s labeling or marketing of Souvenaid, though the company is leaving open that possibility. This medical food is sold over the counter in 15 countries, though not the United States, for about €4 per bottle.
The study will remain double-blind and continue for another four years. During this time, the researchers will analyze blood and cerebrospinal fluid, and progression to AD dementia.
“The significant secondary endpoints seem to indicate an effect on hippocampal functioning and degeneration,” wrote Philip Scheltens, VU University Medical Center, Amsterdam, to Alzforum. Scheltens was an investigator in the current study, but was not involved in the analyses. Co-author Pieter Jelle Visser, also at VU University Medical Centre, agreed. “There is a nice convergence on memory that suggests Souvenaid could be of some benefit fairly early in the disease,” he told Alzforum. The drink may help synapses when damage is still limited, he said. However, Visser said more data and replication are needed before recommending routine treatment. Scheltens pointed out that some participants may not meet the more recent definition of prodromal AD, which requires evidence of amyloid (Dubois et al., 2014).
Another presentation in Athens detailed Souvenaid’s effect on phospholipids in the brain. In a four-week, randomized, placebo-controlled trial funded by Nutricia, Anne Rijpma, a Ph.D. student in the lab of Marcel Olde Rikkert, Radboud University Medical Center, Nijmegen, Netherlands, tested whether the drink stimulated production of phospholipids, the principal component of neural membranes. Thirty-three patients with mild Alzheimer’s, on average 74 years old, took either Souvenaid or a placebo daily. At baseline and at the end of the trial, patients underwent magnetic resonance spectroscopy to measure phospholipid metabolites in the brain.
After a month, the ratio of phospholipid precursors to breakdown products had risen in the hippocampus, anterior cingulate, and retrosplenial cortex of treated patients. This suggests a shift toward more phospholipid formation than breakdown, Rijpma wrote to Alzforum in an email. “The exact effect on neuronal membrane formation requires further research,” she wrote.
“Her results strengthen the existing mechanistic evidence base for Souvenaid,” Hartmann said, adding that further studies will be needed to establish a direct link with results from the LipiDiDiet trial.
Scientists not involved with the Souvenaid trial program were unavailable for independent comment to this report.—Gwyneth Dickey Zakaib
New Tack on Aβ Oligomer Role in Disease and Treatment
As the case builds that soluble oligomers are the most toxic forms of Aβ in Alzheimer’s disease, researchers are working out how best to characterize and target these species. As evidenced by presentations at the 14th International Athens/Springfield Symposium on Advances in Alzheimer Therapy, held March 9 to 12 in Athens, Greece, progress has been slow and breakthroughs remain elusive. Scientists presented snippets of data that suggest Aβ oligomers are physically separate from neurofibrillary tangles, hinting that each of the two spreads through the brain on its own, not together. Others pinpointed a signaling pathway by which Aβ oligomers can lead to the hyperphosphorylation and missorting of tau, and antibodies that might target them therapeutically. “Certainly oligomers are a treatment target,” wrote John Morris, Washington University School of Medicine, St. Louis, to Alzforum. “However, the precise relationship of oligomers to amyloid plaques and neurofibrillary tangles has yet to be determined,” he emphasized. Morris was not involved in the research.
Scientists have puzzled for years about how amyloid plaques and neurofibrillary tangles might intersect, since they form in the brain at different stages of disease and in different places (see Thal et al., 2014). Could there be a tighter relation between soluble oligomers and tangles? So far, much of the data on Aβ oligomers and their location in the brain has come from animal studies. However, Ezio Giacobini, Geneva University Hospitals, who co-organized the conference, wanted to find out where oligomers appear in people’s brains and how they relate to other protein pathologies. He examined postmortem brain tissue from 30 older people with pathologically confirmed mild to severe Alzheimer’s disease and from 43 cognitively healthy controls. Using immunohistochemistry, he detected tau tangles with AT8, amyloid with 4G8, and Aβ oligomers with NU1, an antibody that specifically recognizes synthetic Aβ oligomers. He found that while oligomers and amyloid plaques turned up together in the entorhinal cortices of AD patients, tangles appeared in the hippocampi.
The results suggest that certain oligomers are unlikely to cause tau pathology, said Karen Ashe, University of Minnesota, Minneapolis. However, some forms of oligomer might, she noted. Ashe recently reported that oligomers fall into two main classes. Type 1 are formed from monomers and distributed throughout the brain, whereas type 2 appear stuck close to amyloid plaques that catalyze their formation (June 2015 news). Based on the pattern of staining around plaques, NU1 appears to label type 2 oligomers, Ashe said. Giacobini said that is not certain. Ashe said the results leave open the possibility that type 1 oligomers could co-localize with tangles. A type 1-specific antibody would be required to find out, but none is available, she said. Sylvain Lesné, University of Minnesota, Minneapolis, agreed that multiple antibodies will be required to label different oligomer species and get a fuller picture of their distribution throughout the brain.
Giacobini went on to examine human brain tissue by western blot. In extracts from controls with and without plaques, and from AD patients, he found an NU1-positive band running at 55kDa. AD patients had 40 percent more of this protein in the temporal cortex than did controls. Giacobini told Alzforum that since this oligomer was found in healthy people, he believes it is a normal occurrence in the brain and has some function. Though he detected it in extracts from the cerebellum, which normally are devoid of plaques, he speculated that it reaches toxic levels in disease. While based on its size, this 55kDa protein could be a 14-mer, Giacobini said it may be a dodecamer, like Aβ*56 that was isolated by Ashe’s group. Aβ*56 tracks with cognitive decline in the Tg2576 mouse model of AD, and correlates with neurodegenerative markers in the cerebrospinal fluid of humans (Lesné et al., 2006; Lesné et al., 2008; Zahs and Ashe, 2013).
Lesné, who was not involved in the work, cautioned that the 55kDa band that Giacobini saw may not be Aβ*56. It could be another oligomer with a similar molecular weight to Aβ*56, or it could be a cleavage fragment of the amyloid precursor protein (APP), he suggested. When Lesné measured Aβ*56 in postmortem brain tissue, he found markedly less in those with AD compared to controls, unlike the 55kDa oligomer (Lesné et al., 2013).
For his part, Lesné is looking into the effects of Aβ oligomers on neurons. Scientists have previously reported that oligomers of synthetic Aβ cause hyperphosphorylation and missorting of tau into dendrites (Sep 2010 news on Zempel et al., 2010; De Felice et al., 2008). In Athens, Lesné presented data suggesting Aβ*56 plays a role in this process as well. In the synapses of cultured primary cortical neurons, he found that the oligomer bound NMDA receptors.
This opened the receptors’ ion channels, allowing calcium ions to flood in and activate the Ca2+-dependent calmodulin kinase (CaMKIIα). CaMKIIα then doubled phosphorylation of tau at serine 416. Tau also missorted into the dendritic compartment of these neurons. Blocking CaMKIIα with its endogenous inhibitor, CN21, prevented tau from being hyperphosphorylated and missorted. Lesné saw a similar uptick in activated CaMKIIα in Tg2576 and J20 mice. He also detected elevated CaMKIIα in the inferior temporal gyrus of postmortem brains of cognitively normal elderly.
“We think these results could explain the tau pathology detected in the inferior temporal gyrus of aged subjects at risk for AD,” Lesné wrote to Alzforum. This is one plausible link between Aβ and tau pathology, said Giacobini, though he pointed out that the bulk of these results come from transgenic mice.
Lesné noted that there are likely multiple oligomeric species of Aβ that interact with other receptors and alter tau in different ways. He previously reported that Aβ dimers bind the cellular form of the prion protein, which binds to and activates the Src kinase Fyn, which then hyperphosphorylates tau (Larson et al., 2012). Different oligomer-specific effects on neurons could predominate at various stages of disease, he added, since he had previously found that the relative abundance of Aβ dimers, trimers, and Aβ*56 oligomers changed with age and disease (Lesné et al., 2013).
Some researchers are exploring antibodies that could target various Aβ oligomeric species therapeutically. William Klein, Northwestern University, Evanston, Illinois, who co-founded Acumen Pharmaceuticals, has worked with scientists there to develop an oligomer-specific antibody they called ACU-193 (formerly 19.3). This is the humanized version of a mouse antibody raised to ADDLs, soluble oligomers of synthetic Aβ generated by a procedure developed in Klein’s lab. Their data suggest that ACU-193 binds both type 1 and 2 oligomers, but not Aβ monomers or fibrils, wrote William Goure of Acumen to Alzforum.
In Athens, Klein presented preclinical data on this antibody and introduced a set of single-chain variable fragment antibodies that selectively target oligomer subspecies. In cultured hippocampal neurons, ACU-193 bound no thioflavin-positive plaques. It did bind oligomers, preventing their interaction with the membrane, and this rescued long-term potentiation as well as the oligomer-induced calcium overload. In Tg2576 mice at any age, ACU-193, given intravenously, also bound oligomers. It caused no microhemorrhages, which have been a side effect of some therapeutic antibodies for Aβ. In behavioral assays, the murine precursor of ACU-193, ACU-3B3, reduced hyperactivity in the open field and Y-arm tests in J20 mice, as well as learning deficits seen in the visible, but not hidden version of the Morris water maze. Enough antibody crossed into the brain in mice, rats, dogs, and rhesus monkeys, to be detected by immunofluorescence, and it bound Aβ oligomers with nanomolar affinity, Klein said.
The company has not directly examined binding in postmortem human AD tissues, Goure wrote, but Brian Bacskai and colleagues at Massachusetts General Hospital, Charlestown, found that ACU-3B3 bound soluble oligomers from postmortem AD brain tissue, said Klein. Klein and colleagues previously reported that ACU-193 and ACU-3B3 bind oligomers in human cerebrospinal fluid, and detect more oligomers in people with AD (Savage et al., 2014; Yang et al., 2015). Klein said that given these positive preclinical results and good safety data, he envisions clinical trials for ACU-193 and is trying to raise funds.
Given lingering uncertainty about which forms of oligomer are relevant to Alzheimer’s, Giacobini said it was hard to know whether this antibody would work in humans, and at which stage of disease it would be most effective. Ashe agreed. "Not all oligomers are alike, and people are beginning to accept the idea that there may be more than one type,” she told Alzforum. “It’s important to understand more about the different types of oligomer, and characterize antibodies in terms of whether they target type 1 and/or type 2." David Brody, Washington University, St. Louis, Missouri, agreed. “There is a lot of controversy about which oligomers exist in the human brain; many of the synthetic, cell culture, and transgenic animal-derived forms seem quite different from what has been observed in humans,” he said.—Gwyneth Dickey Zakaib
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