CRMP2

Researchers studied the effects of their compound, CBD3063, in multiple models of acute and chronic pain. A small molecule alleviates four different types of acute and chronic pain in rodents, so far with none of the side effects associated with widely prescribed non-opioid painkiller gabapentin. In an article published Nov. 13 in Proceedings of the National Academy of Sciences, researchers from New York University described how they discovered their compound, dubbed CBD3063, and tested it in models of injury- and chemotherapy-induced neuropathy, trigeminal nerve pain and inflammatory pain. They hope to eventually take it to the clinic. “The compound can be given [via] multiple routes, it works in different species—mice and rats—and in males and females,” senior author Rajesh Khanna, Ph.D., who is also working on a gene therapy for chronic pain, told Fierce Biotech Research in an interview. “No toxicity has been seen, and it works across an array of pain conditions. That’s really the crux of this paper.” CBD3063, gabapentin and pregabalin all ultimately exert their effects through their interactions with a calcium channel called Cav2.2 on pain-sensing neurons, but they do so via different mechanisms. Where gabapentin acts on the outside of the channel, CBD3063 alters it from the inside by stopping a protein called CRMP2 from binding to it. Decoupling the two keeps calcium from entering the channel, which ultimately leads to less neurotransmitter release—and pain reduction. Khanna’s lab first discovered the peptide that would become the basis for CBD3063 back in 2011. Since then, the team has worked on formulating it into a small molecule, figuring out that they could take the number of necessary amino acids down from 15 to just two and pairing those with a protein taken from the HIV virus to help it get into cells. As part of the new study, they worked with the University of Pittsburgh to run a large screen of 27 million existing drug compounds to see whether any had the same composition. The results led them to CBD3063. After a barrage of cell studies to show that the compound did indeed inhibit CRMP2 and was highly selective for Cav2.2—an important consideration, as drugs for arrhythmias and hypertension target similar channels—the researchers tested it in double-blinded experiments on a mouse model of nerve injury, assessing it head-to-head with gabapentin. They found that mice who received the compound were less debilitated by pain than untreated mice. On top of that, CBD3063 worked at a fraction of gabapentin’s effective dose.  “We were getting the same amount of relief [with 1 to 10 milligrams per kilogram of CBD3063] that the clinically available compound gabapentin was producing at 30 milligrams per kilogram,” Khanna said. “That’s very important, because now we are comparing it to a clinical benchmark.” The researchers then conducted experiments on rodents that had nerve damage caused by treatment with paclitaxel, a chemotherapy drug. After giving four doses of paclitaxel over eight days, the researchers waited for the animals to develop nerve damage and an aversion to cold, another common symptom experienced by people who undergo chemotherapy. When the animals developed symptoms two or three weeks later, the team gave them single doses of CBD3063. “We saw a beautiful reversal of both nerve pain and cold aversion,” Khanna said. Next, the researchers looked at how well the drug could control orofacial pain. Until this set of experiments, the scientists had been giving the drug through the animals’ abdomens via what’s known as intraperitoneal delivery. This time, they tried giving it intranasally, thinking that a nasal spray formulation would be ideal for this kind of scenario. “It also bypasses the blood-brain barrier, and it goes right into the regions it needs to,” Khanna said. Once again, the drug worked: It relieved pain consistent with the expected half-life of the compound in such a formulation, he said, which is a little more than three hours. His team then moved to their final models. An assessment of whether CBD3063 could relieve chronic pain from inflammatory conditions like arthritis showed that single injections of the drug into mice with paw inflammation relieved their pain for at least four hours afterward. And an experiment on rats with a chronic nerve injury showed that they didn’t develop tolerance to the drug even with multiple injections over a two-week period. But the researchers weren’t done yet: They wanted to be sure that CBD3063 didn’t block out unrelated types of pain or replicate gabapentin’s side effects, which include memory loss, mood changes, lethargy and more. Experiments on uninjured mice showed that the drug had no effect on types of pain that were unrelated to the type of neuropathy it was designed to treat. Meanwhile, gabapentin seemed to make mice less responsive to important acute pain signals. As for side effects, the scientists found that abdominal injections of CBD3063 had no impact on rodents’ memory or cognition—unlike gabapentin, which dramatically diminished their ability to recognize objects. Their compound also had no effects on the animals’ cardiovascular function, which case studies suggest may be impacted by gabapentin. “This demonstrates that CBD3063 preserves that adaptive role of pain without causing any kind of neurological side effects,” Khanna said. “That’s really the benefit.” There’s still a long road to the clinic. The researchers will need to do many more experiments to understand the drug’s long-term effects as well as whether tolerance eventually arises. And while CBD3063 has never been used for pain relief, it is not technically a new compound. Thus, Khanna’s team will need to substantially transform and optimize it before they can patent it, then will need to partner with a pharmaceutical company to take it into development, he said. “I don’t want to provide any false hope, because the path to humans is three to five years away at the very least,” Khanna said. “In order to get there, there’s a huge journey ahead.”

A compound -- one of 27 million screened in a library of potential new drugs -- reversed four types of chronic pain in animal studies, according to new research. The small molecule, which binds to an inner region of a calcium channel to indirectly regulate it, outperformed gabapentin without troublesome side effects, providing a promising candidate for treating pain. A compound -- one of 27 million screened in a library of potential new drugs -- reversed four types of chronic pain in animal studies, according to new research led by NYU College of Dentistry's Pain Research Center and published in the Proceedings of the National Academy of Sciences (PNAS). The small molecule, which binds to an inner region of a calcium channel to indirectly regulate it, outperformed gabapentin without troublesome side effects, providing a promising candidate for treating pain. Calcium channels play a central role in pain signaling, in part through the release of neurotransmitters such as glutamate and GABA -- "the currency of the pain signal," according to Rajesh Khanna, director of the NYU Pain Research Center and professor of molecular pathobiology at NYU Dentistry. The Cav2.2 (or N-type) calcium channel is the target for three clinically available drugs, including gabapentin (sold under brand names including Neurontin) and pregabalin (Lyrica), which are widely used to treat nerve pain and epilepsy. Gabapentin mitigates pain by binding to the outside of the Cav2.2 calcium channel, affecting the channel's activity. However, like many pain medications, gabapentin use often comes with side effects. "Developing effective pain management with minimal side effects is crucial, but creating new therapies has been challenging," said Khanna, the senior author of the PNAS study. "Rather than directly going after known targets for pain relief, our lab is focused on indirectly targeting proteins that are involved in pain." Inside the channel Khanna has long been interested in a protein called CRMP2, a key regulator of the Cav2.2 calcium channel that binds to the channel from the inside. He and his colleagues previously discovered a peptide (a small region of amino acids) derived from CRMP2 that could uncouple CRMP2 from the calcium channel. When this peptide -- dubbed the calcium channel-binding domain 3, or CBD3 -- was delivered to cells, it acted as a decoy, blocking CRMP2 from binding to the inside of the calcium channel. This resulted in less calcium entering the calcium channel and less neurotransmitter release, which translated to less pain in animal studies. Peptides are difficult to synthesize as drugs because they are short-acting and easily degrade in the stomach, so the researchers sought to create a small molecule drug based on CBD3. Starting with the 15 amino acids that make up the CBD3 peptide, they honed in on two amino acids that studies showed were responsible for inhibiting calcium influx and mitigating pain. "At that point, we realized that these two amino acids could be the building blocks for designing a small molecule," said Khanna. From 27 million to one In collaboration with colleagues at the University of Pittsburgh, the researchers ran a computer simulation that screened a library of 27 million compounds to look for a small molecule that would "match" the CBD3 amino acids. The simulation narrowed the library down to 77 compounds, which the researchers experimentally tested to see if they lessened the amount of calcium influx. This further pared the pool down to nine compounds, which were assessed using electrophysiology to measure decreases in electrical currents through the calcium channels. One compound, which the researchers named CBD3063, emerged as the most promising candidate for treating pain. Biochemical tests revealed that CBD3063 disrupted the interaction between the CaV2.2 calcium channel and CRMP2 protein, reduced calcium entering the channel, and lessened the release of neurotransmitters. "Many scientists have screened the same library of compounds, but have been trying to block the calcium channel from the outside. Our target, these two amino acids from CRMP2, is on the inside of the cell, and this indirect approach may be the key to our success," said Khanna. Four labs, four types of pain Khanna's lab then tested CBD3063 with mouse models for pain related to injury. The compound was effective in alleviating pain in both male and female mice -- and notably, in a head-to-head test with the drug gabapentin, the researchers needed to use far less CBD3063 (1 to 10 mg) than gabapentin (30 mg) to reduce pain. To explore whether CBD3063 helped with different types of chronic pain, Khanna partnered with researchers at Virginia Commonwealth University, Michigan State University, and Rutgers University. Collaborators ran similar studies administering CBD3063 to treat animal models of chemotherapy-induced neuropathy, inflammatory pain, and trigeminal nerve pain -- all successfully reversing pain, similar to gabapentin. But unlike gabapentin, the use of CBD3063 did not come with side effects, including sedation, changes to cognition such as memory and learning, or changes to heart rate and breathing. What's next The researchers are continuing to study CBD3063, refining its chemical composition and running additional tests to study the compound's safety and assess if tolerance develops. Long-term, they hope to bring a CBD3063-derived drug to clinical trials in an effort to offer new options for safe and effective pain relief. "Identifying this first-in-class small molecule has been the culmination of more than 15 years of research. Though our research journey continues, we aspire to present a superior successor to gabapentin for the effective management of chronic pain," said Khanna. Additional authors include Kimberly Gomez, Tyler Nelson, Heather Allen, Aida Calderon-Rivera, Sara Hestehave, Erick Rodríguez Palma, Paz Duran, Santiago Loya-Lopez, Samantha Perez-Miller, and May Khanna of NYU Dentistry's Pain Research Center; Elaine Zhu and Jing Wang of NYU Grossman School of Medicine; Handoko and Paramjit Arora of NYU's Department of Chemistry; Ulises Santiago and Carlos Camacho of the University of Pittsburgh; Yuan Zhou, Angie Dorame, and Aude Chefdeville of the University of Arizona; Upasana Kumar, Rory Shields, Wanhong Zuo, Huijuan Hu, and Olga Korczeniewska of Rutgers University; Eda Koseli, Bryan McKiver, and M. Imad Damaj of Virginia Commonwealth University; Denise Giuvelis and Tamara King of the University of New England; Kufreobong Inyang and Geoffroy Laumet of Michigan State University; Dongzhi Ran, Yi Lu, and Xia Liu of Chongqing Medical University; Marcel Patek of Bright Rock Path LLC; and Aubin Moutal of St. Louis University. The research labs are supported in part by the National Institutes of Health (NS098772, NS120663, DA042852, NS119263, GM115384, NS121776, NS121259, P30GM145497, and R01CA219637).

The gene therapy indirectly targets the sodium ion channel NaV1.7, long-established to be a driver of pain in humans. Researchers have identified another potential gene therapy for treating chronic pain, this time by indirectly targeting a key sodium ion channel. In a study published July 27 in Proceedings of the National Academy of Sciences, researchers from New York University (NYU) described how they developed a gene therapy that relieved chronic neuropathic pain in mice by regulating a previously uncharacterized part of the sodium ion channel NaV1.7. The treatment was also effective against chemotherapy-induced peripheral neuropathy, a type of pain experienced by some cancer patients who undergo chemo. “Our study represents a major step forward in understanding the underlying biology of the NaV1.7 sodium ion channel, which can be harnessed to provide relief from chronic pain,” Rajesh Khanna, Ph.D., the study's senior author and director of the NYU Pain Research Center, said in a press release. Scientists have known since 2006 that the NaV1.7 sodium ion channel—found on the ends of pain-sensing nerve cells—is key to experiencing pain. But the pharma industry’s attempts to drug it directly with small molecules have failed. Khanna’s former lab at the University of Arizona previously developed its own small molecule against NaV1.7, dubbed 194. The drug worked differently from its predecessors; rather than directly inhibiting NaV1.7, it inhibited a protein called CRMP2, which regulates the sodium ion channel’s activity. After finding that 194 was effective in controlling chronic neuropathic pain in mice, the researchers licensed it out for development by Khanna’s startup, Regulonix. But Khanna still wondered why regulation by CRMP2 was specific to NaV1.7 and not to one of the other seven sodium ion channels of the same family. His lab at NYU addressed that question as part of their new study. Using a technique called peptide mapping, they discovered a unique binding domain for CRMP2 in NaV1.7 that wasn’t present in other sodium ion channels. They also found that if they removed the binding domain, CRMP2 could no longer regulate NaV1.7. Armed with this discovery, the researchers set out to see whether they could leverage it to relieve pain for the long run. They created an AAV9 viral vector that encoded a genetic sequence that would inhibit CRMP2, then injected it into male and female mice that already had sciatic nerve injury. The treatment appeared to reverse pain in the mice for the duration of the 30-day study period, compared to no pain relief in mice that received a control virus. Next, the researchers tested whether the treatment could prevent chronic pain. In this case, they injected male and female mice with the gene therapy before performing spared nerve injury surgery, a technique used to induce sciatic nerve injury. None of the mice that received the therapy showed symptoms of pain following recovery, while the mice in the control group did. “Collectively, these findings reveal that the [CRMP2 binding domain] can be targeted to prevent the initiation and maintenance of chronic neuropathic pain in both male and female mice,” the scientists wrote in their paper. “Therefore, the [treatment] could be used as a genetic therapy to provide long-lasting pain relief.” In their next set of experiments, the researchers investigated whether the therapy could be used to treat chemotherapy-induced peripheral neuropathy. They injected male and female mice with a chemo drug called paclitaxel, most commonly known as Taxol, and assessed their pain responses seven days later. On Day 8 following the treatment, they also tested whether the animals were hypersensitive to cold, another symptom of chemotherapy-induced peripheral neuropathy. After confirming that the mice had developed both heightened pain and hypersensitivity to cold, the researchers injected one group of them with the treatment and another with a control virus.  Fourteen, 16 or 21 days after treatment, they assessed the mice's pain responses again; on Days 15, 17 and 22, they tested their cold sensitivity. By Days 16 and 21, the treatment had reversed pain levels in the mice. It worked even sooner on cold hypersensitivity, which dropped by Day 15. The researchers noted that in both the studies on sciatic nerve injury and chemotherapy-induced peripheral neuropathy, motor function remained intact, with no signs of off-target effects. Going a step further, the researchers studied the gene therapy’s effects on spinal cord neurons from macaque monkeys, which are genetically similar to the same type of cell in humans. Their findings showed that the treatment reduced NaV1.7 function, suggesting it might work against pain in nonhuman primates—and perhaps in humans—as well. The study has some limitations. For one, CRMP2 may not be the only protein that interacts with the newly identified receptor binding domain, the researchers pointed out. Furthermore, they know that other proteins do get involved when CRMP2’s interaction with NaV1.7 is inhibited; exactly what those proteins are and how they exert their effects remains unknown. And, of course, mice aren’t men—indeed, studies have shown that the NaV1.7-mediated pain responses in the two species aren’t exactly the same. Finally, there are drawbacks to gene therapy, the scientists noted. Some reports have suggested the possibility of sensory neuron toxicity from AAV9 vectors like the one used in the study. Long-term NaV1.7 suppression might have deleterious side effects that weren’t apparent in the new study, they wrote. Still, the team said they “expect that the targeting of the [CRMP2 binding] domain can be used as a viable strategy for developing future therapeutics that are highly specific and safe for treating chronic pain.” The long-term goal is to take the gene therapy to the clinic, Khanna said in the press release. The findings are the latest to validate gene therapy as a potential solution for chronic pain. In 2021, a team from startup Navega, a spinout from the University of California, San Diego, reported that it had successfully used CRISPR and zinc fingers—both gene editing techniques—to completely suppress the gene that encodes NaV1.7 in mice. Before it shuttered in March, Coda Biotherapeutics was developing an approach that involved shutting down neuronal pain circuits by combining gene therapy with an oral small-molecule agonist.