Altering the chemical properties of an anti-nausea drug enables it to enter the endosomal compartments inside the cell and provide long-lasting pain relief, according to a new study led by researchers at NYU College of Dentistry’s Pain Research Center.

The study results illustrate how pain signaling occurs inside cells rather than just at the surface, highlighting the need for drugs that can reach receptors within cells. “Sustained signaling in endosomes is necessary for the hyperexcitability of pain-sensing neurons involved in chronic pain,” said Nigel Bunnett, PhD, professor and chair of the Department of Molecular Pathobiology at NYU College of Dentistry. “As a result, treating pain may require the development of drugs that penetrate cells, are retained in endosomes, and disrupt signaling inside the cell.”

Bunnett is senior author of the team’s published paper in PNAS, which is titled “Therapeutic antagonism of the neurokinin 1 receptor in endosomes provides sustained pain relief.” In their paper the team concluded, “The results identify criteria that will facilitate the design of antagonists of endosomal receptors and provide evidence for the contribution of endosomal signaling to disease.”

G protein-coupled receptors (GPCRs) are a large family of proteins that regulate many processes in the body and are the target of one third of clinically used drugs. A subset of these receptors plays an important role in pain, including the neurokinin-1 (NK1) receptor (NK1R), which is activated by a pain-transmitting neuropeptide called substance P. “Painful stimuli provoke the release of substance P (SP) from primary sensory neurons, which stimulates endocytosis of the neurokinin 1 receptor (NK1R) in second-order spinal neurons and endothelial cells of postcapillary venules,” the authors explained. “NK1R signals in endosomes mediate sustained activation of spinal neurons and pain transmission.”

Several FDA-approved drugs that target the NK1 receptor are used to prevent nausea and vomiting associated with chemotherapy or surgery. Scientists previously hoped that the NK1 receptor would be a promising target for treating pain—but drugs targeting the receptor failed to control pain in clinical trials in the 1990s and early 2000s.

One reason why drugs targeting the NK1 receptor may not have been effective against pain is that most drugs block receptors at the surface of cells. However, researchers at the NYU Pain Research Center have shown that GPCRs signal pain not from the surface of cells, but from compartments called endosomes inside the cell. “The hypothesis that sustained G protein-coupled receptor (GPCR) signaling from endosomes mediates pain is based on studies with endocytosis inhibitors and lipid-conjugated or nanoparticle-encapsulated antagonists targeted to endosomes,” they wrote. “Optimal therapy requires development of antagonists that penetrate cells, are retained in endosomes, and disrupt endosomal signaling.”

For their study published in PNAS, the researchers generated mice that were genetically modified to express the human NK1 receptor. “Nociception was studied in mice either expressing the human NK1R, which avoided interspecies differences in NK1R antagonist potency,” they noted. The researchers focused on two FDA-approved drugs, aprepitant and netupitant, which are both NK1 receptor antagonists used to prevent nausea and vomiting. Bunnett and colleagues had previously shown that encapsulating aprepitant in nanoparticles could deliver the drug to endosomes to block pain, but in the newly reported study, aprepitant only briefly disrupted endosomal signaling in cellular studies and stopped pain in mice for short periods.

Modifying the second drug, netupitant, was more promising. The researchers changed the chemical properties of the drug to make it more capable of penetrating a cell’s lipid membrane. They also altered the charge on the molecule within an acidic environment so that once the drug entered the acidic environment of an endosome, it would stay trapped inside and accumulate. “To enhance membrane penetration and retention in acidified endosomes, analogs of the neurokinin 1 receptor (NK1R) antagonist netupitant were synthesized with altered lipophilicity and acidity,” the team stated.

These changes allowed the modified netupitant to readily penetrate cells to reach the endosome and block signaling of the NK1 receptor in endosomes with a much more prolonged effect in cells. The altered netupitant also had a more potent and long-lasting analgesic effect in mice than aprepitant and the regular form of netupitant. “When injected intrathecally to target spinal NK1R neurons in knockin mice expressing human NK1R, aprepitant transiently inhibited nociceptive responses to intraplantar injection of capsaicin,” they stated. “Conversely, netupitant analogs had more potent, efficacious, and sustained antinociceptive effects … In summary, lipophilic and acidic NK1R antagonists cause sustained disruption of endosomal signaling and long-lasting antinociception.”

In another experiment, the researchers studied mice with a different type of NK1 receptor on the outer membrane of the cell, rather than inside. These mice were more resistant to pain than those with human NK1 receptors inside the cell, illustrating the importance of endosomes in signaling pain and the need for treatments that can penetrate cells.

“The results identify criteria for the design of GPCR antagonists that are capable of penetrating endosomes and disrupting signalosomes and provide evidence for the contribution of endosomal NK1R signaling in nociception,” the investigators concluded. “The results provide insights into strategies for antagonizing GPCRs in intracellular locations, with implications for designing improved treatments for disease.”

The researchers are continuing this research and other studies in animal models to develop new therapies for pain that block GPCRs in endosomes. “Although we focused on the neurokinin-1 receptor, our findings are likely applicable to many G-protein coupled receptors because many of them show sustained signaling within cells, and therefore require drugs that can enter cells and block the receptors in endosomes,” said Bunnett.

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