Anti-migraine CGRP receptor antagonists worsen cerebral ischemic outcome in mice

ABSTRACT
Objective: CGRP pathway inhibitors are emerging treatments for migraine. CGRP-mediated vasodilation is, however, a critical rescue mechanism in ischemia. We, therefore, investigated whether gepants, small molecule CGRP receptor antagonists, worsen cerebral ischemia. Methods: Middle cerebral artery was occluded for 12-60 minutes in mice. We compared infarct risk and volumes, collateral flow and neurological deficits after pretreatment with olcegepant (single or 10 daily doses of 0.1-1 mg/kg) or rimegepant (single doses of 10-100 mg/kg) versus vehicle. We also determined their potency on CGRP-induced relaxations in mouse and human vessels, in vitro. Results: Olcegepant (1mg/kg, single dose) increased infarct risk after 12- to 20-minute occlusions mimicking transient ischemic attacks (14/19 versus 6/18 with vehicle, relative risk 2.21, p<0.022), and doubled infarct volumes (p<0.001) and worsened neurological deficits (median score 9 versus 5 with vehicle, p=0.008) after 60-minute occlusion. Ten daily doses of 0.1-1mg/kg olcegepant yielded similar results. Rimegepant 10mg/kg increased infarct volumes by 60% after 20-minute ischemia (p=0.03); 100 mg/kg caused 75% mortality after 60-minute occlusion. In familial hemiplegic migraine type 1 mice, olcegepant 1 mg/kg increased infarct size after 30-minute occlusion (1.6-fold, p=0.017). Both gepants consistently diminished collateral flow and reduced reperfusion success. Olcegepant was 10-fold more potent than rimegepant on CGRP-induced relaxations in mouse aorta. Interpretation: Gepants worsened ischemic stroke in mice via collateral dysfunction. CGRP pathway blockers might thus aggravate coincidental cerebral ischemic events. The cerebrovascular safety of these agents must therefore be better delineated, especially in patients at increased risk of ischemic events or on prophylactic CGRP inhibition.

INTRODUCTION
Migraine is a common paroxysmal neurovascular disorder, typically characterized by disabling attacks of headache, associated autonomic features and, in one third of patients, aura. Calcitonin-gene-related peptide (CGRP) is an important neurotransmitter within the migraine headache generating trigeminovascular system and believed to play a crucial role in migraine pathophysiology.1 Gepants, short-acting small molecule CGRP receptor antagonists, and long- acting monoclonal antibodies targeting CGRP or the CGRP receptor have recently emerged as promising acute and prophylactic therapeutic options for migraine.2 Analysts expect these drugs to be used by millions of migraineurs by 2027 in the G7 countries alone.3CGRP is also among the most potent vasodilators in animals and humans.4 It induces endothelium-independent vasodilation via direct action on vascular smooth muscle cells in cerebral and coronary vascular beds. Cerebral blood flow (CBF) autoregulation is in part mediated by CGRP.5 Several lines of evidence suggest that CGRP-mediated vasodilation is a rescue mechanism in brain ischemia.6 CGRP released from trigeminal perivascular nerves7 is a potent direct vascular smooth muscle dilator counteracting vasoconstriction and hypoperfusion in the cerebrovascular bed.5, 8, 9 Low pH- and ischemia-induced CGRP release from perivascular C-fibers results in coronary and cerebrovascular vasodilation and restoration of circulation.10-12 Treatment with CGRP improves and genetic ablation of CGRP worsens blood flow and outcomes after focal cerebral arterial occlusion in experimental animals.13-15 Administration of CGRP has shown beneficial effects in peripheral and coronary artery disease16-18 and ameliorated experimental cerebral and cardiac ischemia by preserving blood flow and the blood-brain barrier.15, 19-21 Consequently, inhibition of the CGRP system withgepants or monoclonal antibodies might theoretically worsen the outcome of coincidental coronary and cerebral ischemic events.6Despite these obvious concerns, remarkably few studies have addressed the cardiovascular safety of inhibitors of CGRP or its receptor.

As far as we know, their cerebrovascular safety has never been studied. Patients with important vascular risk factors were even excluded from most clinical trials.22, 23 This is all the more surprising since migraine with aura is an established risk factor for ischemic stroke, especially in women.24 The higher stroke risk is likely due to increased risk of experiencing cerebral ischemic events25 in combination with enhanced susceptibility of brain tissue to infarction if and when such events occur.26, 27 We, therefore, investigated whether gepants worsen cerebral ischemic outcomes, and the mechanism of this effect, in established mouse models of transient ischemic attack (TIA) and stroke.Experiments were approved by the MGH Institutional Animal Care and Use Committee and carried out in accordance with the Guide for Care and Use of Laboratory Animals (NIH Publication No. 85-23, 1996). In addition to wild-type mice (C57BL/6J), we also used heterozygous transgenic knock-in mice expressing the human familial hemiplegic migraine type 1 (FHM1) S218L missense mutation in the α1A subunit of CaV2.1 voltage-gated Ca2+ channels and their wild-type littermates.28 Mice were housed with their littermates, in cages with standard embedding and enrichment, reversed light/dark cycle and with food and waterad libitum. While migraine is most prevalent among women under age 50, the vast majority of non-migraine related strokes occur in the elderly, including men. We, therefore, studied both young and aged mice (2-16 months), and both males and females. A priori sample size determinations aimed to achieve 80% power to detect a 33% effect size on infarct volume with 20% standard deviation of the mean (=0.05, =0.20). Animals were randomized to treatment arms using an online tool (CoinTosser, ©Hello Heydays Inc. and Microsoft® Office Excel 2016). Each treatment group had its own control group. Group sizes, exclusions, early mortality, age and sex distributions for each experiment are shown in the Supplemental Table. All investigators were blinded to the treatment group during the surgical procedures, data collection and analysis.

TreatmentsAnimals were treated with the small molecule CGRP receptor antagonists olcegepant (BIBN4096, 0.1 or 1 mg/kg, 97.5% purity, intravenous in acute and intraperitoneal in chronic treatment; Tocris Bioscience, Bristol, UK) or rimegepant (BMS-927711, 10 or 100 mg/kg, intraperitoneal; 99.08% purity; MedChemExpress, Monmouth Junction, NJ, USA) either with a single dose 10 minutes before ischemia onset (olcegepant and rimegepant), or with 10 once- daily doses over 2 weeks followed by a single dose 10 minutes before ischemia onset (olcegepant only). These commercial vendors were selected based on previous literature.29-31 Stock solutions were prepared in dimethyl sulfoxide and diluted as needed in saline (0.02-10 mg/ml to administer the target dose in 4-10 l volume per gram of body weight. Control groups received the same volume of identical vehicle via the same route. The selected olcegepant doses were within the previously reported dose range (1 µg/kg – 30 mg/kg) in experimental modelsin rodents.29, 30, 32-37 In rats, 1 mg/kg olcegepant that we used herein has yielded a peak plasma concentration of 0.8 M compared with peak plasma concentrations of approximately 0.15 M after a 10 mg dose in clinical studies.34, 38-41 Unfortunately, plasma concentrations ofrimegepant has not been reported in rodents or in humans. Rimegepant doses were selected based on a single report in the marmoset,42 taking into account (a) the significantly lower affinity of small molecule CGRP receptor antagonists in rodents compared with primates,43 as we confirmed herein using isolated vessels, (b) in vitro data showing significantly lower binding affinity for rimegepant than olcegepant in isolated vessels,31 and (c) the 7-fold higher efficacious human plasma concentrations of rimegepant compared with olcegepant in clinical trials.

Buprenorphine (0.08 mg/kg, s.c.; Patterson Veterinary, Devens, MA, USA) was given as analgesic prior to surgery.All experiments were carried out under isoflurane anesthesia (3% induction, 1.5% maintenance in 70% N2O and 30% O2). Rectal temperature was maintained at 36.7±0.3°C using a thermostatic heating pad (FHC, Bowdoinham, ME, USA). Focal ischemia was induced using transient middle cerebral artery occlusion (MCAO) by a nylon monofilament (701723Re, Doccol Corporation, Sharon, MA, USA). After a ventral midline neck incision (1-2 cm), common carotid artery bifurcation was gently dissected free of connected tissue taking care to preserve the vagus nerve. A closed system around the bifurcation was created, the filament was inserted into the external carotid artery, retrogradely guided into the internal carotid artery and advanced until the origin of the MCA. After 12-60 minutes of occlusion, the filament was removed to achieve reperfusion. Cerebral blood flow (CBF) was monitored (% of baseline)using laser Doppler flowmetry in ischemic core (PeriFlux System 5000; Perimed, Järfälla- Stockholm, Sweden). In experiments with short occlusion times mimicking TIA in wild-type mice, and with 30-minute ischemia in FHM1 mutant mice, CBF was recorded throughout the MCAO and reperfusion. In 60-minute ischemia experiments, animals were allowed to recover from anesthesia shortly after MCAO, and re-anesthetized for the reperfusion procedure; therefore, in this cohort CBF monitoring was discontinued after MCAO. After the surgical procedure, mice were placed in a temperature-controlled heating chamber (~32°C) for 2 hours. At 24 hours after MCAO, we quantified the sensorimotor deficits using a scale ranging from 3 (normal) to 18 as previously described with minor modifications.46 We then harvested the brain to measure the infarct volume (mm3) by integrating the infarct area (ImageJ, NIH) on 1-mm thick coronal slices stained with 2,3,5-triphenyltetrazolium chloride (TTC), while correcting for ischemic swelling using the following formula:10