Abstract Body:

Introduction: Migraine is a chronic, incapacitating neurovascular disorder accompanied by attacks of moderate to severe headache and autonomic nervous system dysfunction. The trigeminovascular system (TGVS) is the main anatomic substrate for the pathophysiology of migraine¹. The activation of migraine pain begins peripherally when nociceptive neurons that innervate the dura mater are stimulated and release vasoactive neuropeptides such as calcitonin gene-related peptide (CGRP), known to modulate dural blood flow ^(2,3). Capsaicin, a potent transient Receptor Potential Vanilloid 1 (TRPV1) channel agonist, has been used extensively in human pain models to induce experimental pain and capsaicin-induced neurogenic inflammation shows striking similarities with the neurogenic inflammation of the trigeminovascular system, which is thought to be of central importance during a migraine attack and predominantly driven by CGRP release from TRPV1 rich nociceptive neurons. Here we show how laser doppler imaging of capsaicin induced increase in dermal blood flow may be utilized to assess efficacy of compounds, such as TRPV1 antagonists that could reverse capsaicin driven release of vasoactive peptides from peripheral nerves and subsequent increase in dermal blood flow. This assay can be further utilized for the development of novel migraine therapeutics.

Methods: On the day of the experiment, mice were anesthetized with 2.5-3% isoflurane flowing at 2 L/min 10 minutes prior to and during laser doppler imaging on a temperature-controlled water circulating blanket. Dermal blood flow was assessed from the dorsal ear area. The mouse was positioned on its ventral (abdominal) side down so that the dorsal side of the ears was accessible for the laser recordings. One hour before the topical application of Capsaicin, an oral dose of 10 mg/kg of the TRPV1 antagonist (AMG517) was given via oral gavage. After a baseline scan, 25 μL of vehicle was applied on the contralateral ear and 25 μL of capsaicin on ipsilateral ear, so that each animal represents its own control. The time course of the blood flow response to each dose of capsaicin was measured at 5, 10, 20, 30, 40, 50, and 60 minutes post capsaicin application. After obtaining the last laser Doppler scan, the animals were left to recover from anesthesia in their home cages placed on the heating pad, until they fully regained reflex and looked alert and responsive. At the end of the last experiment, all animals were euthanized in accordance with the approved AUP and tissue (blood, skin) was harvested.

Results: The model adjusted mean percent change from baseline was significantly decreased in the AMG517 group compared to the Vehicle group as early as 10 minutes post capsaicin application (Figure 1D). The AMG517 treated mice had reduced blood flow over 60 minutes recording compared to the Vehicle group at every timepoint except 40 minutes post capsaicin. The maximum peak % decrease of 55% in mean flux occurred at 20 minutes post capsaicin application.

Conclusions: The oral dosing of the TRPV1 antagonist AMG517 one hour prior to the topical application of capsaicin resulted in a significant decrease of the dermal blood flow response compared to the capsaicin treated vehicle control group with a peak decrease of 55% occurring at 20 minutes post capsaicin treatment. Laser doppler imaging offers a clinically translatable imaging technique to assess the early efficacy of compounds such as TRPV1 antagonists, that could indirectly reverse capsaicin induced effects on dermal blood flow in the clinic to provide a valuable tool for early efficacy evaluations in migraine therapeutic development.

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