26 May 2017

Could tyrosine kinase inhibitors, a standard tool of cancer treatment, help people with amyotrophic lateral sclerosis? Converging evidence suggests that this drug class may slow ALS progression, perhaps through multiple mechanisms. In the May 24 Science Translational Medicine, researchers led by Haruhisa Inoue at Kyoto University, Japan, report that numerous different inhibitors of the tyrosine kinases Src and c-Abl improve the survival of motor neurons from ALS patients. The compounds act by stimulating autophagy, which accelerates the removal of toxic proteins. One of the most potent inhibitors, bosutinib, boosted motor neuron survival by 50 percent and modestly lengthened the lives of ALS model mice, the authors report.

In related news, researchers recently reported positive findings from a Phase 3 clinical trial of another tyrosine kinase inhibitor, masitinib, at the European Network for the Cure of ALS (ENCALS) annual meeting, held May 18 to 20 in Ljubljana, Slovenia. This inhibitor, which is approved to treat tumors in animals but not people, reportedly doused neuroinflammation in the spinal cord. Patients on the drug maintained motor abilities four months longer than did those on placebo, a statistically significant improvement. AB Science in Paris, the manufacturer, has applied to the European Medicines Agency for approval to use the drug in people, and is planning to start another Phase 3 trial this year before applying for approval from the U.S. Food and Drug Administration.

The data suggest that tyrosine kinase inhibitors might help in other neurodegenerative diseases such as Alzheimer’s and Parkinson’s, which also accumulate toxic proteins and cause neuroinflammation, said Charbel Moussa at Georgetown University, Washington, D.C. He noted that many of these compounds are already FDA-approved for other conditions, and can be used at much lower doses for neurodegenerative disease than for cancer. “These drugs represent a promising alternative to antibody and vaccination strategies,” he told Alzforum. He was not involved in either of these studies.

ALS in a Dish. Stem cells derived from people with familial ALS differentiate into neurons in culture that express motor neuron markers HB9, ChAt, and SMI-32. Nuclei are stained blue. [Courtesy of Science Translational Medicine/AAAS.]

The need for new drugs for ALS is immense. In this devastating disease, spinal motor neurons wither, robbing people of motor control and killing them typically within three to five years. Approved treatments are limited to riluzole and edaravone, which was just approved in the U.S. this month (see May 2017 news). Both modestly slow functional decline, though efficacy data for edaravone remains sparse. Researchers are still seeking better options.

To cast a wider net, Inoue and colleagues screened 1,416 compounds that are either approved for human use or in clinical trials. First author Keiko Imamura generated induced pluripotent stem cells (iPSCs) from a single ALS patient who carried a SOD1 mutation. The authors differentiated these cells into spinal motor neurons and cultured them for seven days, added the compounds, and assessed survival one week later. In this screen, 27 compounds boosted survival more than three standard deviations above that of untreated cells. Half of these compounds targeted the Src/c-Abl signaling pathway. These cytosolic tyrosine kinases participate in numerous cellular processes and are implicated in cancer. To confirm these enzymes mediated the drug effect, the authors knocked down Src and c-Abl with short interfering RNAs, and again saw improved motor neuron survival.

Among the hits, the authors selected bosutinib for follow up. This drug is approved to treat chronic myelogenous leukemia, directly inhibits Src and c-Abl, and acts at lower doses than the other compounds in the screen. Bosutinib normalized autophagy in the diseased motor neurons. Compounds that blocked autophagy weakened the protective benefits of bosutinib, suggesting this was its mechanism of action. In keeping with this, other known autophagy boosters, such as rapamycin, also improved motor neuron survival. As might be expected, revving up autophagy cleaned up deposits of misfolded, toxic SOD1. The authors did not detail how inhibition of Src and c-Abl stimulated autophagy, but other work provides clues. Moussa and colleagues have reported that c-Abl inhibition activates the ubiquitin ligase parkin, which then interacts with autophagy proteins such as beclin-1 to stimulate degradation of proteins including Aβ and α-synuclein (see Lonskaya et al., 2013; Lonskaya et al., 2014; Wenqiang et al., 2014). A sister compound to bosutinib, nilotinib, is currently in Phase 2 trials for PD and AD that Moussa and colleagues at Georgetown are running (see Nov 2015 conference news). 

Only 2 percent of people with ALS carry SOD1 mutations. What about other forms of the disease? To expand their study, the authors generated motor neurons from three ALS patients with TDP-43 mutations, three with C9ORF72 expansions, and three with sporadic disease. Most people with ALS, regardless of their mutation status, accumulate misfolded TDP-43, and C9ORF72 is the most common familial mutation. In this study, bosutinib lowered levels of misfolded TDP-43 and poly dipeptide repeats formed from the C9ORF72 expansion; it also improved survival in all cell lines save for one from a sporadic case.

Next, the authors tested bosutinib in the SOD1-G93A mouse model of ALS. These animals become paralyzed at four and die by six months of age. The authors injected a single dose, 5 mg/kg/day, intraperitoneally for six weeks beginning at two months of age. Src and c-Abl activity in the spinal cord was cut in half, indicating target engagement. Treated mice accumulated slightly less misfolded SOD1 and had about three times as many surviving motor neurons in their spinal cords as untreated ones. Nevertheless, treatment delayed disease onset by only 11 days and extended survival by just eight days.

Why didn’t the drug work better in mice, given the promising in vitro data? Nonneuronal cells such as astrocytes contribute to ALS pathology, but Inoue’s screen did not test for effects of bosutinib on these cells (e.g. Oct 2014 news; Nov 2014 news). In an email to Alzforum, Inoue also suggested that bosutinib could be optimized to better enter the brain and avoid potential off-target effects. Peter Davies at the Feinstein Institute for Medical Research in Manhasset, New York, pointed out that tyrosine kinase inhibitors such as bosutinib are typically not specific for c-Abl. “I would like to see pharma make more specific compounds, because then we would learn if the key factor really is c-Abl, rather than another kinase, and there would be fewer off-target effects,” Davies wrote to Alzforum. He acknowledged that making specific c-Abl inhibitors is a challenging task, and that companies have tried and abandoned some past efforts for lack of success.

The findings from bosutinib and nilotinib complement those for masitinib. This veterinary drug seems to act mostly on immune cells. Preclinical studies suggested masitinib inhibits the tyrosine kinases CSF-1R and C-kit in microglia, macrophages, and mast cells, circulating white blood cells that trigger allergic and inflammatory reactions. In animal models, masitinib prevents microgliosis and astrogliosis in the spinal cord, as well as the infiltration of mast cells and macrophages into neuromuscular junctions (see Trias et al., 2016). “This provides a rational basis for the protective effects of masitinib in delaying neuromuscular junction denervation. However, more research is needed to understand the detailed mechanism of action of the drug,” Luis Barbeito at the Pasteur Institute of Montevideo, Uruguay, wrote to Alzforum. Barbeito presented preclinical data on masitinib at ENCALS.

In the Phase 3 trial, 394 patients from nine countries took either 4.5 mg/kg masitinib, 3 mg/kg, or placebo for nearly a year. By prespecified plan, the researchers stratified participants into fast progressors (those who declined more than 1.1 point per month on the revised ALS Functional Rating Scale) and normal progressors. About 85 percent of the participants were normal progressors. Among this group, those taking 4.5 mg/kg masitinib declined 3.4 points less on the ALSFRS-R than the placebo group over the course of the study. This translated to 27 percent less functional decline over this time period, a clinically meaningful difference, according to Jesus Mora at Hospital Carlos III in Madrid, who presented the clinical trial findings at ENCALS. Treated participants maintained greater lung capacity and reported better quality of life than the placebo group. They lasted 20 months before their disease progressed nine points or more on the ALSFRS-R, compared with 16 months for those on placebo. Participants who took the lower 3 mg/kg dose also reported better quality of life, but their trend toward slower functional decline did not reach significance.

Other data hinted that the drug was most effective when given at an early stage of disease. When normal and fast progressors were combined, the 4.5 mg/kg dose only slowed decline in those who had had the disease for less than two years. “Fast progressors may need earlier treatment,” Mora suggested.

The safety profile was acceptable, with no surprises cropping up, the researchers said. The treatment group experienced more serious adverse events than the placebo group. These were scattered across different organ systems and did not fall into any pattern. For oncology use, tyrosine kinase inhibitors are normally given at higher doses, from 6 to 12 mg/kg, with no serious safety issues, the researchers noted.—Madolyn Bowman Rogers

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References

Therapeutics Citations

1. Masitinib
2. Riluzole

News Citations

1. FDA Approves Edaravone for Treatment of ALS 8 May 2017
2. Potential Parkinson’s Treatments Target α-Synuclein, Cell Replacement 12 Nov 2015
3. In ALS, Astrocytes Use Ion Pump to Blast Motor Neurons 31 Oct 2014
4. Geriatric Astrocytes Fail Motor Neurons in ALS Model 7 Nov 2014

Research Models Citations

1. SOD1-G93A (hybrid) (G1H)

Paper Citations

1. Lonskaya I, Hebron ML, Desforges NM, Franjie A, Moussa CE. Tyrosine kinase inhibition increases functional parkin-Beclin-1 interaction and enhances amyloid clearance and cognitive performance. EMBO Mol Med. 2013 Aug;5(8):1247-62. PubMed.
2. Lonskaya I, Hebron ML, Desforges NM, Schachter JB, Moussa CE. Nilotinib-induced autophagic changes increase endogenous parkin level and ubiquitination, leading to amyloid clearance. J Mol Med (Berl). 2014 Apr;92(4):373-86. Epub 2013 Dec 13 PubMed.
3. Wenqiang C, Lonskaya I, Hebron ML, Ibrahim Z, Olszewski RT, Neale JH, Moussa CE. Parkin-mediated reduction of nuclear and soluble TDP-43 reverses behavioral decline in symptomatic mice. Hum Mol Genet. 2014 Sep 15;23(18):4960-9. Epub 2014 May 8 PubMed.
4. Trias E, Ibarburu S, Barreto-Núñez R, Babdor J, Maciel TT, Guillo M, Gros L, Dubreuil P, Díaz-Amarilla P, Cassina P, Martínez-Palma L, Moura IC, Beckman JS, Hermine O, Barbeito L. Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis. J Neuroinflammation. 2016 Jul 11;13(1):177. PubMed.

External Citations

1. Phase 3
2. Phase 3
3. Phase 2 trials for PD
4. AD 

Further Reading

News

1. Mexiletine Banishes Muscle Cramps in Phase 2 Trial of ALS 7 Mar 2016
2. Supportive Stem Cells Safe for ALS 14 Jan 2016
3. Help for Speech, Swallowing, and Salivation Problems in ALS 23 Dec 2015
4. Scientists Eager to Test ALS Gene Therapy 12 Jun 2015
5. Helper Drug Boosts Response to Riluzole in ALS Mice 5 Dec 2014