By increasing the expression of the chloride transporter Kcc2 (K-Cl cotransporter 2), researchers at Duke University have reduced chronic pain in mouse models of nerve pain and bone cancer.

In their experiments, the authors used both the glycogen synthase kinase 3-beta (GSK-beta) inhibitor kenpaullone, named after the first director of the National Cancer Institute, and gene therapy delivering the transcription factor regulator delta-catenin to increase Kcc2 expression.

Co-corresponding author Wolfgang Liedtke, who is now chair of neurology, psychiatry, pain medicine and sensory systems at Regeneron Pharmaceuticals but was a professor at Duke University when the studies were performed, told BioWorld Science that while gene therapy is clearly not meant to be an alternative to over-the-counter analgesics, "there [are] also patients who are much more ill...whose lives are destroyed."

He added that "pain, in a way, has something about it like a malignant disorder."

Certainly, although the opioid crisis is testament to a toxic brew of pharmaceutical marketing and physician hubris, its root cause on the side of patients is a widespread and often desperate need for pain relief that current therapeutic options are not addressing, and sometimes making worse.

Back surgery, for example, is effective for only about 25% of patients in relieving pain.

For others, scar formation after back surgery and other "desperation measures" like nerve coagulation make the problem worse.

Liedtke and his co-corresponding authors Michele Yeo, Yong Cen and Ru-Rong Ji, all at Duke University, published their work in the October 27, 2021, issue of Nature Communications. Their goal was to reset what Liedtke called the "genetic malprogramming" that short-circuits the effectiveness of GABA and glycine, the major inhibitory neurotransmitters of the nervous system. In spinal cord pain circuits, those transmitters tamp down pain.

Sometimes.

GABA and glycine "are inhibitory only when chloride is low," Liedtke explained.

Chloride is one of the ions that keep neuronal cell membranes charged, enabling action potentials. Its levels affect how responsive neurons are to external input.

How easily chloride can be kept low depends on KCC2, an ion channel that is specifically expressed in neuronal cell membranes.

In chronic pain, KCC2 expression levels are decreased via epigenetic mechanisms. And with that, Liedtke said, "a fundamental circuit that has been corrupted," because chloride levels within cells are too high to react to inhibitory inputs that would decrease pain signaling.

In their work, the team first screened a compound library to find ways to switch Kcc2 gene expression back on. They found several compounds that were capable of doing so, including kenpaullone.

Mechanistically, kenpaullone inhibited the kinase GSK-3beta. GSK-3beta phosphorylates multiple targets and its activity has been implicated in both cancer and Alzheimer's disease. In the work published by Liedtke and his colleagues, its key target was the transcription factor regulator delta-catenin.

In a further set of experiments, the team tested whether delivering a transgene for delta-catenin could achieve the same effect as inhibiting GSK-3beta, and found that its temporary overexpression in the spinal cord could also increase Kcc2 expression and reduce pain.

Liedtke noted that the work took a long time to complete, which shows in the paper's methods. For the gene therapy, starting today, the team would use a vector that targets spinal cord neurons, rather than a neuronal-specific promoter.

And for screening, a more up-to-date approach would be the use of iPSC-derived neurons and CRISPR screens.

"We did this with nerve cells that grow in a dish that were derived from the cortex of a mouse," he said, a method that made the screening of slightly more than a thousand compounds in their initial experiments a "truly heroic" effort.

Translational possibilities

Liedtke argued that gene therapy delivered via intrathecal injection "is way less invasive than [surgical] desperation measures" to treat debilitating pain.

There are also efforts to target KCC2 through the development of enhancers, pioneered by Yves de Koninck at the University of Laval.

Liedtke likened the KCC2 channel to a revolving door. "Yves has developed compounds that makes the revolving door run faster," he said.

Another possibility could be kenpaullone itself – though the compound is off-patent, making its successful commercialization more of a challenge -- or a derivative.

An advantage of that approach could be that in the bone cancer model, kenpaullone could conceivably treat both metastases and the pain they are causing.

In the metastatic bone pain animal models now published in Nature Communications, kenpaullone did not affect the tumor growth itself.

But "you can probably give kenpaullone at a higher dose" and affect the tumor itself as well as pain, Liedtke said. From what is known of the drug's toxicity profile, the highest dose the team tested, 30 mg, "is not the end of the line."