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.
