Biotechs have been investigating the therapeutic potential of drugs called calcium/calmodulin-dependent protein kinase II inhibitors, or CaMKII inhibitors, for at least a decade. But while the drugs have shown some promise in preclinical and early-stage clinical trials, questions remain about whether they have deleterious off-target effects on the brain.
Now, the results of a new study published June 21 in Science Translational Medicine by researchers from the University of Chicago and Johns Hopkins University may begin to put some of those concerns to rest. They also suggest that the cancer drug ruxolitinib, a JAK1/2 inhibitor commercialized by Novartis as Jakavi, could be a viable treatment against atrial fibrillation and a dangerous pediatric cardiac arrhythmia called catecholaminergic polymorphic ventricular tachycardia (CPVT).
“Our study was able to show for the first time that you can actually block the ‘bad’ CaMKII in the heart and completely spare the brain version of CaMKII with a small molecule, so you get the benefits without the side effects,” first author Oscar Reyes Gaido, an M.D.-Ph.D. student in the Johns Hopkins lab of Mark Anderson, M.D., Ph.D., told Fierce Biotech Research in an interview. “And we found that ruxolitinib behaves that way—it doesn’t cross into the brain very well, so this could be a really good repurposing candidate.”
While CaMKII, an enzyme, has some functions in the heart, experiments in animals have shown that it isn’t essential, Reyes Gaido and his team noted in their paper. On top of that, there’s increasingly abundant evidence that excessive CaMKII activity contributes to heart disease and arrhythmias, as the team explained in a January 2023 review article.
There have been but a handful of attempts to develop a CaMKII inhibitor for heart disease, and so far, such drugs have failed to translate from preclinical models to humans. Scientists are only just learning how many different conditions may be precipitated by excess CaMKII activity, so it makes sense that innovation on the front hasn’t yet picked up. But there’s another reason for stifled progress, too: There are four different isoforms, or types, of CaMKII found throughout the body. Hitting the wrong one—or all of them—could cause serious harm.
“They basically look identical, so it’s really challenging to inhibit one version without inhibiting all four,” Reyes Gaido explained. “That’s kept a lot of people from even trying.”
Given the inherent difficulty of developing a new CaMKII inhibitor, the Anderson lab wondered whether any drugs already approved by the U.S. FDA might have activity against the molecule. Their proven safety profile would mean the toxicity hurdle had already been surmounted, and they could potentially be fast-tracked to clinical trials.
“If we start with only molecules that are safe for humans, then you’ve completely eliminated that one deal breaker that all these other molecules have fallen to,” Reyes Gaido said.
But first, the researchers would need a better way to visualize the effects of the drugs on the enzyme’s activity. To that end, Reyes Gaido developed what’s known as a reporter molecule that could enable the researchers to monitor CaMKII levels in cells. Using a jellyfish-derived protein called green fluorescent protein—one that emits green light, just as it sounds—he created CaMKII Activity Reporter, or CaMKAR. The reporter molecule is injected into the tissue being studied, where it glows bright green whenever CaMKII is activated.
The team’s experiments showed CaMKAR to be far more sensitive to CaMKII levels than other biosensors used by the industry. It could detect CaMKII activity directly, rather than relying on proxy signals. That would be vital for figuring out which drugs were truly efficacious against CaMKII itself and weren’t instead targeting similar molecules, a problem that might explain why earlier inhibitors that seemed promising in preclinical studies failed to pan out in clinical trials. It would also lower the cost of high-throughput screening, as the researchers could draw conclusions from a smaller number of cells.
Using CaMKAR, the researchers analyzed how 4,475 different FDA-approved drugs affected CaMKII levels in human heart cells. They found five: baricitinib, a JAK1/2 inhibitor commercialized by Lilly for rheumatoid arthritis as Olumiant; the CK2 inhibitor silmitasertib, from Senhwa Biosciences, which has orphan-drug status for biliary tract cancer; crenolanib, a tyrosine kinase inhibitor by Arog Pharmaceuticals that was fast-tracked by the FDA in 2017 for acute myeloid leukemia; Lilly’s breast cancer drug abemaciclib, trade name Verzenio; and ruxolitinib.
Out of the five hits, ruxolitinib was the most potent CaMKII inhibitor. To see whether it had efficacy in the context of arrhythmia, the team first looked at whether it could hinder the enzyme in cells with a genetic mutation that caused CPVT. Seeing positive results, they moved to testing the drug in a mouse model of the same condition. Ten minutes after injecting the mice with ruxolitinib, CaMKII levels plunged and the arrhythmia was extinguished.
“It was surprising how well this drug worked,” Reyes Gaido said. “We could see CaMKII activity go to basically zero in 10 minutes, which was quite striking.”
The researchers saw similar results in two mouse models of acquired afib. Hyperglycemic mice with different forms of arrhythmia were given the same dose of ruxolitinib as the CPVT models. Ten minutes later, the arrhythmia could no longer be induced in one model and its frequency had fallen from 95% to 33% of heartbeats in the other.
But did the benefits come at the cost of cognition? To find out, the researchers ran mice through various types of learning and memory tests after giving them a single dose of ruxolitinib. They also ran longer studies where mice received daily doses of the drug before being trained on how to get through a maze, which would show any impacts on long-term spatial memory formation. The treated mice performed just as well as the control animals.
One concern going forward is that while ruxolitinib may not damage the brain, it does have known side effects including thrombocytopenia, a condition where blood platelets are too low. But for patients with conditions like CPVT—where as many as 30% of patients are resistant to treatment, and a full 31% die before reaching age 30—the benefits may outweigh the downsides.
Editor's note: This story has been updated to clarify a comment from Oscar Reyes Gaido.