Periodic Reporting for period 1 - PAIN-CARE (Chronic PAIN eradiCAtion by taRgeting Schwann cElls)
Periodo di rendicontazione: 2023-11-01 al 2025-04-30
Two observations led us to propose the PAIN-CARE project: the first focused on the clinical burden, and the second on the socioeconomic impact of chronic pain. In fact, chronic pain incurs costs running into billions of Euros annually and represents 3–10% of the European GDP. Current pain treatments primarily include non-steroidal anti-inflammatory drugs (NSAIDs) and opioids, whose frequent use is associated with life-threatening adverse events and physical dependence. Some new effective drugs, including anti-calcitonin gene-related peptide (anti-CGRP) drugs and the sodium channel 1.8 selective blocker, suzatrigine, have been approved for migraine and post-surgical pain, respectively. However, 30% of migraine patients are resistant to anti-CGRP drugs, and the clinical impact of suzatrigine remains to be determined. Therefore, the widespread use of NSAIDs and opioids highlights the urgent need for safer and more effective analgesic medications.
The SCOPE project, from which the PAIN-CARE initiative is derived, was based on our discovery of the unexpected role of Schwann cells in mechanical allodynia associated with neuropathic pain. Schwann cells engaged by macrophages, via the transient receptor potential ankyrin 1 (TRPA1) channel, amplify the proalgesic oxidative stress burden to signal mechanical allodynia. During the SCOPE project, we found that Schwann cells express receptors that may function similarly to macrophages. Specifically, CGRP binds to its receptor on Schwann cells and is internalized into endosomes, where cyclic AMP facilitates TRPA1-mediated amplification of oxidative stress, leading to pain signaling. The PAIN-CARE project aims to develop Schwann Cell–selective Analgesic Drugs (SCADs) for improved and safer pain treatment, surpassing the efficacy and safety of NSAIDs and opioids.
The SCOPE project, from which the PAIN-CARE initiative is derived, was based on our discovery of the unexpected role of Schwann cells in mechanical allodynia associated with neuropathic pain. Schwann cells engaged by macrophages, via the transient receptor potential ankyrin 1 (TRPA1) channel, amplify the proalgesic oxidative stress burden to signal mechanical allodynia. During the SCOPE project, we found that Schwann cells express receptors that may function similarly to macrophages. Specifically, CGRP binds to its receptor on Schwann cells and is internalized into endosomes, where cyclic AMP facilitates TRPA1-mediated amplification of oxidative stress, leading to pain signaling. The PAIN-CARE project aims to develop Schwann Cell–selective Analgesic Drugs (SCADs) for improved and safer pain treatment, surpassing the efficacy and safety of NSAIDs and opioids.
The PAIN-CARE project investigated the activity of bifunctional chimeric molecules (BCM) composed of an antagonist of the CGRP receptor (CLR/RAMP1), olcegepant, and a derivative of the AM class of TRPA1 antagonists, called AM-0902. Initial results did not demonstrate an evident improved efficacy of BCM in inhibiting responses to the selective TRPA1 agonist allyl isothiocyanate (AITC), which increases intracellular calcium, or to CGRP, which increases cyclic AMP. To explore the reasons for the failure, in collaboration with Prof. Delia Preti and colleagues at the University of Ferrara, Prof. Romina Nassini and colleagues at the University of Florence, and Prof. Dane Jensen at the New York University, the CGRP receptor antagonist olcegepant was conjugated to a fluorescent dye, creating olcegepant-BODIFY. Fluorescence of olcegepant-BODIFY was observed inside the cells but not in endosomes, suggesting a non-specific passive passage across the plasma membrane. Next, we developed a pegylated, fluorescent olcegepant. In this case, fluorescence was confined to the plasma membrane of HEK-293 cells, which do not express the CGRP receptor, whereas it was found inside human Schwann cells, which do express the CGRP receptor (see enclosed Figures 1-4). From these findings, we concluded that: (1) olcegepant, unexpectedly being a peptoid molecule, easily enters the plasma membrane via a non-specific mechanism; (2) bifunctional molecules may share this ability to cross the plasma membrane of various cell types, thus not necessarily offering advantages in terms of efficacy and selectivity; and (3) to achieve receptor-dependent internalization of olcegepant, increasing molecular hindrance—such as with pegylation—appears necessary, as demonstrated by the pegylated compound.
Based on these considerations, two points led us to reorient the PAIN-CARE project. First, constructing a suitable BMC based on olcegepant proved more complex than anticipated. Second, data obtained under the SCOPE project on inflammatory pain provided unexpected and innovative results that are amenable to drug development. NSAIDs are considerably less safe than anti-CGRP drugs, as they carry risks of gastrointestinal, cardiovascular, and kidney toxicity—particularly in the increasing target population of the elderly. We reasoned that developing better and safer analgesic for inflammatory pain is a much more important task that optimizing anti-migraine treatments. Therefore, we combined the efforts of the PAIN-CARE and SCOPE projects to study inflammatory mechanical allodynia induced by two classical proinflammatory agents: carrageenan and complete Freund’s adjuvant (CFA). This response is known to be mediated by prostaglandin E2 (PGE2) and is inhibited by approximately 70% with NSAIDs.
Based on these considerations, two points led us to reorient the PAIN-CARE project. First, constructing a suitable BMC based on olcegepant proved more complex than anticipated. Second, data obtained under the SCOPE project on inflammatory pain provided unexpected and innovative results that are amenable to drug development. NSAIDs are considerably less safe than anti-CGRP drugs, as they carry risks of gastrointestinal, cardiovascular, and kidney toxicity—particularly in the increasing target population of the elderly. We reasoned that developing better and safer analgesic for inflammatory pain is a much more important task that optimizing anti-migraine treatments. Therefore, we combined the efforts of the PAIN-CARE and SCOPE projects to study inflammatory mechanical allodynia induced by two classical proinflammatory agents: carrageenan and complete Freund’s adjuvant (CFA). This response is known to be mediated by prostaglandin E2 (PGE2) and is inhibited by approximately 70% with NSAIDs.
We provided in vitro evidence using human Schwann cells and in vivo evidence in mice that pharmacological antagonism or selective silencing of EP2 receptors in Schwann cells—achieved through adeno-associated virus—abolished PGE2-dependent mechanical allodynia, but did not affect inflammation, induced by carrageenan or CFA.
Thus, we challenged two prevailing dogmas: first, that inflammatory mechanical pain is mediated by EP4 receptors in DRG neurons; and second, that the analgesic effect of NSAIDs is dependent on their anti-inflammatory action. We conclude that EP2 inhibition in Schwann cells represents a novel, safe, and effective strategy to alleviate inflammatory pain without impairing the protective and healing functions of inflammation. Furthermore, we observed that while CGRP-mediated cyclic AMP increase—responsible for migraine pain—results from the internalization of the agonist/receptor complex into the endosomal compartment, the cyclic AMP signaling pathway initiated by EP2 is entirely dependent on a nanodomain associated with the plasma membrane of Schwann cells. Additionally, Schwann cell EP4 which internalizes in endosomes does not mediate pain.
These crucial, innovative findings—decoupling pain from inflammation and distinguishing the intracellular pathways of migraine pain (caused by neurogenic inflammation and mediated by CGRP) from inflammatory pain (generated by tissue inflammation and mediated by PGE2) highlight new opportunities for the treatments of inflammatory pain by targeting Schwann cell pathways. Consequently, the PAIN-CARE project provided crucial findings for the development of SCADs (Schwann Cell-Associated Drugs) that selectively inhibit/silence the EP2 receptor in Schwann cells by diverse strategies, including BMC or viral vectors.
Thus, we challenged two prevailing dogmas: first, that inflammatory mechanical pain is mediated by EP4 receptors in DRG neurons; and second, that the analgesic effect of NSAIDs is dependent on their anti-inflammatory action. We conclude that EP2 inhibition in Schwann cells represents a novel, safe, and effective strategy to alleviate inflammatory pain without impairing the protective and healing functions of inflammation. Furthermore, we observed that while CGRP-mediated cyclic AMP increase—responsible for migraine pain—results from the internalization of the agonist/receptor complex into the endosomal compartment, the cyclic AMP signaling pathway initiated by EP2 is entirely dependent on a nanodomain associated with the plasma membrane of Schwann cells. Additionally, Schwann cell EP4 which internalizes in endosomes does not mediate pain.
These crucial, innovative findings—decoupling pain from inflammation and distinguishing the intracellular pathways of migraine pain (caused by neurogenic inflammation and mediated by CGRP) from inflammatory pain (generated by tissue inflammation and mediated by PGE2) highlight new opportunities for the treatments of inflammatory pain by targeting Schwann cell pathways. Consequently, the PAIN-CARE project provided crucial findings for the development of SCADs (Schwann Cell-Associated Drugs) that selectively inhibit/silence the EP2 receptor in Schwann cells by diverse strategies, including BMC or viral vectors.