Alzheimer’s researchers are pursuing treatments beyond amyloid and tau, and at the 13th International Conference on Alzheimer’s and Parkinson’s Diseases, held March 29 to April 2 in Vienna, they shared news on drugs in Phase 1 and 2 trials that target inflammation, epigenetics, and regeneration. The candidates include two small molecules that soothe microglia and suppress inflammatory signaling, an attempt to tweak epigenetic regulation by way of lysine-specific demethylase, and the neurosteroid allopregnanolone.

Two Strategies to Calm Inflammation
Growing genetic and animal evidence pegs the immune system as a central influencer in both hastening and ameliorating Alzheimer’s progression. Because of this, many groups are looking at ways to tamp down harmful inflammatory signaling. Researchers led by D. Martin Watterson of Northwestern University Feinberg School of Medicine, Chicago, designed and synthesized compounds that specifically suppress pro-inflammatory cytokines, and turned up two related small molecules, MW151 and MW189. Both act on microglia to selectively turn down pro-inflammatory cytokines without affecting anti-inflammatory signaling, said Linda Van Eldik of the University of Kentucky, Lexington, who collaborates with Watterson to develop these candidates as drugs. 

Targeting Inflammation.

The small molecule MW151 turns down harmful inflammatory signaling and is being developed in pill form for Alzheimer’s disease. [Courtesy of Linda Van Eldik.]

The researchers believe the compounds have potential to treat both acute brain injuries, which spark neuroinflammation, and Alzheimer’s and other neurodegenerative diseases. They developed MW189 as an intravenous formulation, and saw that it helped allay the destructive inflammatory effects of both amyloid pathology and brain injury in animal studies, Van Eldik reported in Vienna. To trigger amyloid aggregation, the researchers infused Aβ42 oligomers into mouse ventricles for four weeks. After the third week, half the mice received MW189 once daily for two weeks. MW189 treatment largely prevented the spike in brain astrogliosis and IL-1β seen in untreated control animals. Treated animals maintained normal synaptic density and performed like wild-type in the Y maze, in contrast to deficits on these measures in untreated mice.

Likewise, wild-type mice that received a brain injury followed by four days of twice-daily MW189 were able to cling to a rotarod longer than injured animals that did not receive treatment. They performed better in the Morris water maze than untreated animals, although not as well as wild-type. MW189 helped the animals when given as late as six hours after the injury, and at doses down to 0.1 mg/kg, Van Eldik said (see James et al., 2012). 

In Phase 1a testing, 32 healthy volunteers were given a single IV dose of either placebo or 0.025, 0.05, 0.1, or 0.25 mg/kg MW189. Of eight people in each dose cohort, two received placebo. The researchers recorded no adverse events related to drug. In a separate Phase 1a study, an additional 18 healthy volunteers received 0.25 mg/kg MW189 or placebo, followed by 2 ng/kg lipopolysaccharide to induce an inflammatory reaction. Those who had MW189 in their systems made less pro-inflammatory TNFα and more anti-inflammatory IL-10 over the next 12 hours, supporting the compound’s ability to control inflammation. This protocol also appeared safe, van Eldik said.

MW189 entered a Phase 1b trial, in which 24 healthy participants receive either placebo or 0.075, 0.15, or 0.3 mg/kg by IV twice daily for five days, in the same proportions as the earlier study. If the compound is well-tolerated, the researchers will enroll people who have suffered a blunt-force traumatic brain injury in a Phase 2a trial. MW189 will be administered by IV within six hours of injury and given twice daily for five days, or until patients are discharged. The trial will enroll people whose brain injuries are severe enough to require external ventricular drains, Van Eldik noted. This will allow the researchers to collect CSF and measure changes in pro-inflammatory cytokines during treatment to determine target engagement. Follow-up visits with participants will assess whether the treatment made a difference in long-term outcomes.

The researchers are starting with brain injuries because these trials are shorter and have better-defined endpoints than AD trials, Van Eldik wrote to Alzforum. Ultimately, the researchers would like to apply the insights gained from these trials to the treatment of AD. “Future success with MW189 and TBI endpoints will inform us of the utility of targeting the pro-inflammatory cytokine aspects of neuroinflammation in more chronic CNS disorders, such as AD,” Van Eldik wrote to Alzforum. To that end, they have developed MW151 as an oral formulation. It suppressed inflammation and protected synapses in an APPPS1 knock-in mouse, and prevented a rise in harmful IL-1β and maintained learning and memory after brain injury in rodents (see Bachstetter et al., 2012; May 2012 conference news; Bachstetter et al., 2015). 

Across the Atlantic, Ludwig Aigner of Paracelsus Medical University in Salzburg, Austria, is taking a different approach to modulating the immune system. He evaluates the approved asthma medication montelukast. This small molecule blocks leukotriene receptors that cause bronchial tubes to constrict. Leukotrienes are inflammatory molecules that white blood cells release during an asthma attack. Because neurons and microglia also express leukotriene receptors, Aigner decided to check if this type of signaling plays a role in brain inflammation. In aged, forgetful rats, six weeks of montelukast treatment shrank microglia, restored the blood-brain barrier, and boosted learning in the Morris water maze to that of young rats. Intriguingly, montelukast also boosted neurogenesis, suggesting some regenerative effects (see Oct 2015 news). Notably, leukotrienes rise during aging, and after stroke or brain damage.

The idea of repurposing montelukast for Alzheimer’s disease is buoyed by its excellent safety record; alas, the tablet formulation poorly enters the bloodstream, with only 63 percent of it becoming bioavailable. Because of this, Aigner and colleagues reformulated the drug in collaboration with IntelGenx Corp., Saint-Laurent, Canada. IntelGenx packaged the drug in a thin film that is placed in the mouth, either against the cheek or under the tongue. The film dissolves, delivering the drug to the bloodstream.

The researchers tested this delivery method in eight healthy volunteers, who took 10 mg montelukast, a common asthma dosage, both in the traditional tablet form and by film. The film resulted in higher absorption, reaching 95 percent in the bloodstream, an increase of 50 percent over the tablet’s bioavailability. This formulation will allow lower dosing, Aigner said in Vienna.​

The drug crossed the blood-brain barrier, an essential feature for treating AD. After three hours, participants had 3.5 ng/ml in their CSF, rising to 4.25 ng/ml by seven hours. This is the pharmacologically active range, Aigner noted. The researchers next plan to do a Phase 2 study in AD patients in Canada, in collaboration with the research organization Consortium of Canadian Centres for Clinical Cognitive Research (C5R). Aigner noted that the oral film represents a new product that can be patented for commercial development.

Can the Epigenome Be Safely Targeted?
Many researchers have noted epigenetic changes in AD, but targeting these with a drug has proven difficult (see Nov 2012 conference newsAug 2014 news; Nov 2014 news). Partly this is because histone deacetylase (HDAC) inhibitors, which derepress genes involved in learning and memory, have broad effects that can lead to toxicity with chronic dosing (see Dec 2008 conference news; Aug 2013 conference news). 

Researchers at Oryzon Genomics in Barcelona, Spain, developed a different approach. They identified a small molecule, ORY-2001. It inhibits lysine-specific demethylase 1 (LSD1), an enzyme that forms part of a complex with HDAC1/2 to regulate gene expression. In Vienna, César Molinero of Oryzon noted that ORY-2001 has a second mechanism of action as well, inhibiting the mitochondrial membrane protein monoamine oxidase B (MAOB1). MAOB1 helps break down dopamine in the brain, so inhibiting it can help Parkinson’s patients, who suffer from a dearth of the neurotransmitter. The approved Parkinson’s drug rasagiline targets MAOB1.

ORY-2001 can be taken orally, has favorable pharmacokinetics, and crosses the blood-brain barrier, Molinero said. In senescence-accelerated (SAMP8) mice, a model of age-related cognitive decline, treatment for two to four months lowered the expression of pro-inflammatory genes in the hippocampus and restored learning and memory. The compound appeared to have no ill effects. Because LSD1 stimulates blood cell maturation by repressing stem cell genes, researchers looked for a drop in blood production, but treated mice had none (see Kerenyi et al., 2013). 

The researchers are now testing single and multiple doses in Phase 1. Eighty-eight young, healthy volunteers took either 0.2, 0.6, 1, 1.5, 2.5, or 4 mg of ORY-2001. Of the eight people in each cohort, two got placebo, and multiple doses were given over five days. Molinero reported no serious adverse events so far, but with repeated dosing at 2.5 mg, platelet counts dropped by a third after eight days and took a week to rebound. The researchers added the 4 mg dose to explore these effects, and that testing is still ongoing, Molinero said.

After a single dose, the compound’s half-life in blood was 22 hours, but with repeated dosing, it accumulated, its clearance slowed, and its half-life stretched to four days. To check target engagement, the researchers measured the amount of LSD1 bound to ORY-2001 in peripheral blood mononuclear cells using chemiluminescent ELISA. They found a dose dependence, with the lowest single dose of ORY-2001 binding 20 percent of LSD1 while the highest dose bound 80 percent. After repeated dosing, the 0.2 mg dose resulted in 30 percent bound LSD1, while doses of 1 mg and higher topped out at 80 percent. The effects lasted for about one week, Molinero said in Vienna.

In a separate substudy, the researchers are measuring ORY-2001 in cerebrospinal fluid to find out if it crosses into the brain. They will also test the compound in healthy elderly participants. ORY-2001 will start Phase 2 later this year with support from the Alzheimer’s Drug Discovery Foundation (see news release). 

Regeneration: Could it be Possible?
Neurons wither in the AD brain, and yet the age-old idea of sparking the birth of new neurons to replace them has been considered beyond the reach of reality. Enter the neurosteroid allopregnanolone. It revs up neurogenesis in AD mouse models, restoring learning and memory. All the while, it dials down inflammation and Aβ production. Based on these preclinical data, Roberta Brinton and Lon Schneider of the University of Southern California, Los Angeles, started a Phase 1 trial of the neurosteroid in 24 people diagnosed with mild cognitive impairment due to AD or early AD (see Aug 2013 conference newsDec 2014 conference news). 

Participants sit for a weekly infusion of intravenous allopregnanolone for three months. Each dose cohort consists of eight people, two of whom receive placebo. In Vienna, Brinton presented findings from the first two dose cohorts, who received 2 or 4 mg allopregnanolone. The third cohort, which tests doses from 6 to 18 mg, is still in progress. Administration of the first two doses appeared safe, with no adverse effects, Brinton reported. Toxicology studies in dogs and rats also found no ill effects after six and nine months of chronic dosing with allopregnanolone, she added.

Intriguingly, this short-term treatment revealed hints of efficacy, Brinton told the audience. Cognitive and physical abilities stabilized in people on the drug, whereas those on placebo continued to decline, she said. The treatment group reported being in better moods than the placebo group, and anecdotal reports from caretakers supported better function.

There were indications of possible structural benefits, Brinton added. In the 4 mg cohort, hippocampal volumes of treated participants shrank less than in the placebo group. In three treated participants, the researchers measured improved connectivity between the posterior cingulate cortex and the parietal and prefrontal cortices. These findings are preliminary, as the trial was not powered to detect efficacy. Allopregnanolone is slated to enter a 72-week Phase 2 study of 260 people with early AD, Brinton said.—Madolyn Bowman Roger

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References

News Citations

  1. Keystone: TBI—Learning From Markers, Models, and Diseases
  2. Asthma Drug Revitalizes Memory in Old Rodent Brains
  3. SfN: Epigenetic Changes in Alzheimer’s and Cognitive Decline
  4. Alzheimer’s Brains Mottled with Epigenetic Changes
  5. Epigenetic Alterations Mark Alzheimer’s Disease Genes
  6. DC: Developing But Debatable—Deacetylase Inhibitors for CNS Disease?
  7. Clinical Trials Roundup: Broadening the Lines of Attack
  8. Just for Her? Study of Women’s Biology Offers New Therapeutic Angle

Paper Citations

  1. . TT-301 inhibits microglial activation and improves outcome after central nervous system injury in adult mice. Anesthesiology. 2012 Jun;116(6):1299-311. PubMed.
  2. . Early stage drug treatment that normalizes proinflammatory cytokine production attenuates synaptic dysfunction in a mouse model that exhibits age-dependent progression of Alzheimer's disease-related pathology. J Neurosci. 2012 Jul 25;32(30):10201-10. PubMed.
  3. . Attenuation of traumatic brain injury-induced cognitive impairment in mice by targeting increased cytokine levels with a small molecule experimental therapeutic. J Neuroinflammation. 2015 Apr 10;12:69. PubMed.
  4. . Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation. Elife. 2013 Jun 18;2:e00633. PubMed.

External Citations

  1. Phase 1b
  2. C5R
  3. news release
  4. Phase 1

Further Reading