Highly Sensitive Assays for detection of headache inducing neuropeptides, Pituitary adenylate cyclase-activating polypeptide (PACAP) and Calcitonin gene-related peptide (CGRP)

Abstract

Background: Migraine is one of the most disabling diseases that continues to pose a significant societal burden. Although there are now treatment options for people with migraine, it remains challenging to identify them as clinical features are diverse and complex, and there are no validated diagnostic or treatment prediction biomarkers. Identification is based on either diagnostic coding or the use of certain acute headache abortive treatments. However, socioeconomic disparities can contribute to under-diagnosis and under-treatment of migraine. Thus, efforts to find biomarkers to identify individuals with migraine and which variables could explain migraine-related chronification and disability are warranted. We aimed to investigate the levels of migraine inducing neuropeptides; calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in peripheral blood samples as potential biomarkers of migraine.

Methods: We developed highly sensitive assays for CGRP and PACAP on the MSD S-PLEX assay platform and used them for bioanalysis of preclinical and clinical samples. Wildtype and neuropeptide challenged mice and rats were profiled using the developed assay. To follow-up, commercially obtained plasma samples from healthy controls and migraineurs were initially profiled. Subsequently, we profiled plasma samples from people with migraine (during and after a headache attack and healthy controls. Both MSD S-PLEX assays were transferred to Celerion where they were validated for analysis of clinical samples.

Results: Using the highly sensitive PACAP assay, we were able to reliably measure circulating levels of endogenous and administrated PACAP38in mouse and rat plasma. Additionally, using the highly sensitive CGRP assay, we were able to reliably measure circulating levels of endogenous and administrated CGRP in mouse and rat plasma. Furthermore, in the initial human samples, circulating CGRP and PACAP levels were not significantly different in healthy controls compared to people with migraine patients. However, ≥50% people with migraine showed increased circulating CGRP and PACAP levels during their attack period compared to post attack. Overall, people with migraine showed a 3 - 396% increase in one or both neuropeptides during their attack period compared to post attack. Circulating plasma CGRP and PACAP levels in healthy control subjects were consistent with previously measured levels.

Conclusion: Our highly sensitive PACAP and CGRP assays were successful in measuring circulating levels of endogenous PACAP38 and CGRP in mouse and rat plasma. Our highly sensitive PACAP and CGRP assays were qualified for measurement of human CGRP and PACAP in healthy control and migraine samples. Plasma CGRP and PACAP levels are elevated in migraineurs during an attack period, and the increased plasma neuropeptide levels during an attack may help the differentiation of migraineurs from non-Migraineurs, or amongst people with migraines to help identify the best treatment for each patient.