The Effect of Cortical Spreading Depression Events on Anti-Migraine Agent Uptake Via Regulation of Functional Expression of OATP1A2/OATP1A4 at the Blood Brain Barrier

The blood brain barrier (BBB), regulated by the neurovascular unit (NVU), restricts movement of substances to and from the blood to the brain, posing a daunting challenge for drug design where effective uptake is critical to a drug’s efficacy and safety. Previous research has shown that cortical spreading depression (CSD) events may be associated with migraine and other neurological disorders. What has not been well studied is how CSD pathological conditions, including associated changes in potassium ion levels and the release of mediators such as calcitonin gene related peptide (CGRP), may affect BBB integrity at the level of the microvessels, and consequently, uptake of brain-targeting therapeutics such as Triptans and Gepants. Xenobiotic transport occurs by paracellular, transcellular and/or transport-mediated processes, including by the solute carrier organic anion transporter, OATP. OATP1A2 (human)/ Oatp1a4 (rodent) are widely expressed bidirectional sodium-independent solute carriers involved in transport of a wide range of amphipathic substrates. Previous studies have shown OATP expression and activity may be pathologically regulated during inflammatory and/or hypoxic/reoxygenation states. Previously, we showed CSD promotes paracellular leak as determined by sucrose uptake as well as transporter facilitated uptake of Sumatriptan. Herein, we first investigated how high abluminal potassium levels, in vitro, and potassium chloride (KCl)-induced CSD events, in vivo, impact Oatp1a4 functional expression and uptake of Sumatriptan following prophylactic or abortive administration. Oatp1a4 facilitated parenchymal uptake was observed when Sumatriptan was dosed prior, but not after, CSD induction suggesting subclinical insults can rapidly change Oatp1a4 functional expression and its role in Sumatriptan uptake relies on timing of each dosing paradigm. Next, to investigate how CSD events promote paracellular leak, tight junction reorganization and regulation of transporter functional expression, we tested whether CSD elevates local CGRP leading to uptake of Rimegepant via paracellular and Oatp1a4 transport. Several novel findings were identified including: (1) CSD induction with a single cortical injection of KCl did not significantly change CGRP levels in the V1M portion of the occipital cortex (V1M cortex), plasma, Trigeminal nucleus caudalis (Vc) or Periaqueductal grey (PAG) of mice; (2) brief and acute increases in CGRP induced significant decreases in Transendothelial electrical resistance (TEER), but not a functional breach of BEB paracellular integrity; (3) Sustained Rimegepant produced delayed paracellular leak absent in the presence of CGRP; (4) CGRP dose-dependently altered Oatp1a4 expression in bEnd.3 cells; and (5) sucrose uptake increased in KCl- pulsed cells after sustained exposure to Rimegepant, but Rimegepant is not detected after during KCl pulse, suggesting that the size of paracellular leak during CSD as previously reported is < 1200g/mol (MW Rimegepant succinate),, narrowing the paracellular leak size during CSD from 4000g/mol. Finally, we hypothesized that the CGRP receptor could be selectively targeted at the CLR-RAMP1 interaction to reduce side effects and drug-drug interactions. We used in silico molecular modeling techniques and identified potential novel CGRP receptor targeting compounds. Quantitative functional analysis was performed by measuring nitrate levels in bEND.3 cells treated with the top four compounds; however data was inconclusive requiring future studies with other methods to validate these agents.Release after 08/29/202