Puny efficacy signals of monotherapies and a growing awareness of the complexity of Alzheimer’s disease have made combination therapy a hot topic in the past few years. However, researchers are only beginning to explore this strategy in animal models. At the Alzheimer’s Association International Conference 2014, held July 12-17 in Copenhagen, Denmark, Ron DeMattos of Eli Lilly and Company, Indianapolis, Indiana, presented data on his company’s first attempt. In aged PDAPP mice, investigators found that combining a BACE inhibitor and a pyroglutamate Aβ antibody cleared plaques more thoroughly than either therapy alone over a four-month period. Statistics indicated a synergistic effect, meaning that the combination cleared more plaque than the single effects added together. “This provides a strong rationale in terms of moving toward the clinic with this approach,” said DeMattos.

Other scientists were impressed by his talk. “I thought this was a spectacular piece of science,” said Eric Karran, Alzheimer’s Research UK, Cambridge. “DeMattos revealed in an exquisitely compelling way that when you combine a BACE inhibitor with a specific antibody that will target the plaque, you can have very dramatic effects on plaque loads.”

The mE8 antibody (blue) targets deposits (round, yellow), while a BACE inhibitor reduces production of Aβ peptides (yellow rods). [Image courtesy of Neuron, DeMattos et al., 2012.]

To date, the only other study examining a combination therapy came from scientists at F. Hoffmann-La Roche Ltd. in Basel, Switzerland. They demonstrated the additive effects of the company’s discontinued BACE inhibitor R7129 and its Phase 3 antibody gantenerumab in reducing Aβ levels and plaque from transgenic mice (see April 2013 news story). 

DeMattos and colleagues took a similar approach. They shut off Aβ production with the LY2811376 BACE inhibitor (see May et al., 2011), while clearing existing Aβ aggregates with mE8, an anti-pyroglutamate Aβ antibody that is specific to plaque (see DeMattos et al., 2012). 

Lilly scientists assigned 180 18-month-old PDAPP mice to one of five cohorts. The first was sacrificed to determine the levels of plaque in the brain at baseline. The other four cohorts served as the control group, mE8-treated group, BACE-inhibitor treated group, and combination group. Treatment lasted for four months, with BACE inhibitor given through food and mE8 injected subcutaneously each week. At the end of the study, the researchers extracted Aβ biochemically and used quantitative ELISA to measure the mass of the Aβ deposits in the hippocampus and cortex.

In the control group, plaque load in the hippocampus had doubled from baseline. The mE8- and BACE inhibitor-treated groups had 49 and 66 percent less plaque, respectively, than controls. In the combination group, amyloid levels dropped by 86 percent. Statistical analysis suggested the treatment effects were additive, with trends toward synergy.

Staining brain slices of the hippocampus and cortex with 3D6, an Aβ antibody that visualizes both diffuse and cored plaque, DeMattos and colleagues found that in mE8-treated animals, the area covered by amyloid plaques stayed fairly steady, but their morphology changed, whereby plaques appeared mostly diffuse. In contrast, the BACE inhibitor-treated animals had almost no diffuse plaques, but cored plaques were left behind. The combined therapies attacked both types. To DeMattos, these results suggest that the two different therapies attack different forms of plaque, with the BACE inhibitor cutting off the supply of Aβ that feeds formation of diffuse plaques and the mE8 antibody tackling plaque cores.

In a related poster, Margaret Racke, also from Lilly, detailed the Aβ fragments that predominate in the different types of plaque. Both diffuse and cored plaques were made up mostly of Aβ40 and Aβ42, but the mixture of fragments they also contained differed. Probing western blots with antibodies the scientists developed to recognize different N-termini exposed by various secretases, they found that Aβ4-, 9-, and 11-42 were enriched in the diffuse plaques left over from the mE8 therapy. Aβ3- and 8-42 predominated in cored plaques remaining after treatment with the BACE inhibitor. DeMattos said that these results help further understand the Aβ deposition cascade and give insight into the maturation of a plaque from diffuse to the more toxic cored type.

Because the behavior of PDAPP mice is highly variable at 18 months of age, the scientists did not analyze it, DeMattos said. Instead, they analyzed dystrophic neurites and dendrites as a proxy for neuronal damage. These swollen neuronal extensions surround cored plaques. They were prominent in untreated PDAPP mouse brains but appeared more normal upon amyloid removal with a monotherapy, even more so with the combination. 

To confirm these findings, the scientists repeated the entire study with 19-month-old mice and got similar results. This time, the synergistic effect of the combination therapy reached statistical significance, DeMattos noted.

Researchers did not voice skepticism at the lack of behavioral data, noting that improved mouse behavior has not translated to clinical success in the past anyway. For commentators, the data on dystrophic neurites was sufficient to suggest a benefit on the central nervous system. Gunnar Gouras, Lund University, Sweden commented that the Lilly team might also have looked at synaptophysin, a synaptic protein that correlates with behavior.

“This is strong support for the notion of combination therapy,” said Dennis Selkoe, Brigham and Women’s Hospital, Boston. By attacking more than one form of Aβ, the combination is potentially lowering two sources of oligomers (monomers and mature plaques), which Selkoe and others believe are the most synaptotoxic species. He noted that a technically feasible addition to the study would be to examine what happens to soluble oligomer levels upon combination treatment. “Overall, I think this is the way to go in clinical trials, and we should start planning for them now,” he told Alzforum.

“The animal data [from this research and the Roche study] suggest we can more effectively lower amyloid using two complementary mechanisms,” agreed Reisa Sperling, also of Brigham and Women’s Hospital. A potential benefit of the combination approach is that a patient can take low doses of two medications, whereas a monotherapy might require a higher dose associated with more side effects. “Particularly in patients who likely have a significant amyloid burden, we’re going to need to be more aggressive with anti-amyloid therapy to reduce the buildup that’s already there,” she told Alzforum.—Gwyneth Dickey Zakaib

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References

Therapeutics Citations

  1. RG7129
  2. Gantenerumab

News Citations

  1. First Stab at Combination Therapy Yields Additive Effect

Research Models Citations

  1. PDAPP(line109)

Paper Citations

  1. . Robust central reduction of amyloid-β in humans with an orally available, non-peptidic β-secretase inhibitor. J Neurosci. 2011 Nov 16;31(46):16507-16. PubMed.
  2. . A plaque-specific antibody clears existing β-amyloid plaques in Alzheimer's disease mice. Neuron. 2012 Dec 6;76(5):908-20. PubMed.

Further Reading

Papers

  1. . Robust amyloid clearance in a mouse model of Alzheimer's disease provides novel insights into the mechanism of amyloid-beta immunotherapy. J Neurosci. 2011 Mar 16;31(11):4124-36. PubMed.