Therapeutic targeting of endosomal GTP-binding (G protein)-coupled receptors (GPCRs) is a novel means of drug delivery that may offer more effective and selective treatment for chronic pain and possibly other conditions, a study by Australian researchers at Monash University in Melbourne has concluded.

Safer and more effective pain treatments are urgently needed, given the limitations of current medications, noted corresponding author Meritxell Canals, a research fellow in the Monash Institute of Pharmaceutical Sciences.

"Current pain treatments have major clinically limiting side effects and are not always effective," Canals told BioWorld. "For example, opioid-based analgesics have major side effects such as respiratory depression, constipation and dependence. Therefore we need better, safer and more effective ways of dealing with this debilitating condition."

Normally considered to be cell surface sensors of extracellular signaling, GPCRs control many different pathophysiological processes and are involved in a number of diseases. They are also the target of approximately 30 percent to 40 percent of all modern drugs.

Activated GPCRs move to endosomes, which are membrane-bound cellular compartments of the endocytic membrane transport pathway. However, scientists have yet to explore whether endosomal receptors generate signals that control complex physiological processes in vivo and therefore might represent viable therapeutic targets.

Therefore researchers co-led by Nigel Bunnett, formerly at Monash but now professor of pharmacology and medicine at Columbia University, professor Christopher Porter and Canals performed the first ever study to investigate targeting endosomal GCPRs, rather than those on the cell surface.

They demonstrated that the substance P (SP) neurokinin 1 receptor (NK1R) signals from endosomes, which then induces sustained excitation of spinal neurons and pain transmission, they reported in the May 31, 2017, edition of Science Translational Medicine.

A neuropeptide acting as a neurotransmitter in the brain and spinal cord, SP and the closely related molecule, neurokinin A, are released from the terminals of certain sensory nerves and are associated with inflammatory processes and pain.

"We demonstrated sustained NK1R activation by specifically stimulating the receptor with its ligand, [namely] SP, then measuring the firing of action potentials electrophysiologically," Canals explained.

"We injected capsaicin, complete Freund's adjuvant [an antigen solution acting as an immunopotentiator] or formalin into the paws of laboratory rats or mice," she said. "These noxious stimuli trigger the release of SP in primary order spinal neurons; SP activates the NK1R, which mediates pain transmission."

Pain transmission was then assessed using Von Frey hair testing, which measures the time taken for the animal to withdraw its paw when prodded with filaments of different strengths.

Importantly, the researchers also showed that specific antagonism of the NK1R in endosomes with membrane-anchored drug conjugates provided more effective and sustained pain relief than conventional plasma membrane–targeted antagonists.

Because NK1R antagonists have so far proven to be ineffective for pain control, "we conjugated two known SP/NK1R antagonists, called spantide and L-733,066, to a lipid molecule, cholestanol, which anchors the drug into cellular membranes to promote endosomal targeting," said Canals.

"Cholestanol conjugation proved to be effective at treating pain in our animal models, amplifying and prolonging the [pain killing] activity of NK1R antagonists. Conjugated NK1R antagonists were also shown selectively to inhibit endosomal signaling and sustained neuronal excitation," she added.

The Monash researchers then investigated the pharmacological and genetic disruption of clathrin, dynamin and beta-arrestin, which are proteins involved in endocytosis. This blocked SP-induced NK1R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as gene transcription.

Based on these results, "we suggest that these are the signaling events that mediate pain transmission," Canals told BioWorld.

Endocytosis inhibitors were also shown to prevent sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuate nociception, the ability to feel pain, in vivo.

"This illustrates that internalization of the receptor is necessary, in order to mediate sustained neuronal excitation and to mediate nociception," said Canals, noting that attenuation of nociception was shown by injecting internalization inhibitors into mice and assessing pain transmission.

These results reveal a critical role for endosomal signaling of the NK1R in pain pathophysiology and demonstrate the efficacy of endosomally targeted GPCR antagonists in pain control. They also suggest that targeting endosomal GPCRs is an effective and selective treatment for chronic pain and possibly other conditions.

"We suggest that endosomal signaling is more general than previously anticipated and that other GPCRs may continue to signal once internalized. This may help us to refine the therapeutic targeting of this important family of receptors," concluded Canals.

"It seems we may have been targeting the right receptor but in the wrong location," she said. In future, "we will attempt to demonstrate that this [endosomal] signaling also occurs with other receptors and try to achieve translation of these study findings into clinical trials."