Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

Abstract

Protease activated receptor 2 (PAR2) is a G-protein coupled receptor expressed in meningeal neurons, fibroblasts and mast cells that may be targeted to treat migraine. MEDI0618, a fully humanized PAR2 monoclonal antibody, engineered to enhance FcRn-dependent recycling and currently in clinical development, was evaluated in human and rodent in vitro assays, in multiple murine in vivo migraine models and in a model of post-traumatic headache. MEDI0618 bound specifically and with high affinity to cells expressing human PAR2 (hPAR2) and prevented matriptase-induced increase in cytosolic calcium. Similarly, MEDI0618 prevented matriptase-induced calcium in primary fibroblasts and microvascular endothelial cells from human dura mater. MEDI0618 had no effect on hPAR1 receptors. Single-cell calcium imaging of acutely dissociated mouse trigeminal ganglion neurons confirmed expression and functionality of mouse PAR2. Studies in vivo used evoked cutaneous allodynia as a surrogate of headache-like pain and, in some experiments, rearing as a measure of non-evoked headache pain. MEDI0618 was administered subcutaneously to C57BL6/J female mice prior to induction of migraine-like pain with (i) systemic nitroglycerin or compound 48/80 (mast cell degranulator); or (ii) with supradural compound 48/80 or an inflammatory mediator (IM) cocktail. To assess possible efficacy against CGRP receptor (CGRP-R)-independent pain, MEDI0618 was also evaluated in the IM model in animals pretreated with systemic olcegepant (CGRP-R antagonist). Migraine-like pain was also induced by inhalational umbellulone, a TRPA1 agonist, in animals primed with restraint stress in the presence or absence of MEDI0618 as well as in a model of post-traumatic headache pain induced by a mild traumatic brain injury. MEDI0618 prevented cutaneous allodynia elicited by systemic nitroglycerin, compound 48/80 and from supradural compound 48/80 and IM. Systemic olcegepant completely blocked periorbital cutaneous allodynia induced by supradural CGRP but failed to reduce IM-induced cutaneous allodynia. In contrast, MEDI0618 fully prevented IM-induced cutaneous allodynia, regardless of pretreatment with olcegepant. Umbellulone elicited cutaneous allodynia only in restraint stress-primed animals, which was prevented by MEDI0618. MEDI0618 prevented the decrease in rearing behaviour elicited by compound 48/80. However, MEDI0618 did not prevent mild traumatic brain injury-related post-traumatic headache measures. These data indicate that MEDI0618 is a potent and selective inhibitor of PAR2 that is effective in human and rodent in vitro cell systems. Further, blockade of PAR2 with MEDI0618 was effective in all preclinical migraine models studied but not in a model of post-traumatic headache. MEDI0618 may represent a novel therapy for migraine prevention with activity against CGRP-dependent and independent attacks.

Keywords: PAR2; mast cells; migraine prevention; pain; post-traumatic headache; trigeminal.

Figure 1

Specificity and potency of anti-PAR2 monoclonal antibody MEDI0618. (A) Live staining of PAR2-expressing (1321N1-hPAR2.cl8) or non-expressing (1321N1 parental) cell lines with MEDI0618 directly conjugated to Alexa Fluor 647. Scale bar = 20 µm. (B) Flow cytometry of hPAR2 overexpressing cells (1321N1-hPAR2.cl8) or non-expressing cells (1321N1 parental cell line) or A549 cells endogenously expressing hPAR2 live labelled with MEDI0618. (C and D) Calcium imaging from 1321N1-hPAR2 cells pretreated with MEDI0618 hIgG or an isotype control protein. (C) Exemplar raw calcium trace from a single well pretreated with MEDI0618 or isotype control antibody both at 1 nM, followed by PAR2 agonist stimulation with matriptase (10 nM). (D) Antibody titration of the anti-PAR2 antibodies MEDI0618 or PAR650097 or an isotype control antibody. Data show the matriptase (10 nM)-mediated calcium signal after preincubation with antibody and normalized to the matriptase response in the absence of antibody treatment. (E) Calcium imaging from A549 cells pretreated with MEDI0618, isotype control protein or PAR1 inhibitors (ATAP2 + WEDE15 mAbs) followed by 10 nM thrombin (PAR1 agonist) stimulation. Data are presented as mean ± standard error of the mean, n = 4. The concentration of the inhibitor is shown on the x-axis. Data are normalized to the peak thrombin calcium response in the absence of inhibitor pretreatment.

Figure 2

PAR2 functional expression in human and mouse cells relevant to migraine. Whole well calcium imaging from primary human dural fibroblasts (HDuF), human dural microvascular endothelial (HDuMEC) and mouse brain endothelial (bEnd.3) cells. (A, D and G) PAR2 agonists concentration response curve in (A) HDuF, (D) HDuMEC and (G) bEnd.3 cells. (B, E and F) Effect of MEDI0618 and isotype control protein (IgG) on inhibition of matriptase-induced calcium signalling at 30 nM in (B) HDuF, (E) HDuMEC and (H) bEnd.3 cells. (C, F and I) Representative calcium imaging traces of 30 nM matriptase-evoked activity following MEDI0618 or isotype control protein preincubation in (C) HDuF, (F) HDuMEC and (I) bEnd.3 cells. (J and K) Single-cell calcium imaging from mouse trigeminal neuron cultures. (J) Pseudocolour images of fura-2 ratio intensity show a subset of trigeminal neurons activated by treatment with 10 µM LIGRLO in comparison to 20 mM KCl treatment. Scale bar = 20 µm. (K) Representation of fura-2 traces recorded from two individual neurons during acute LIGRLO (10 µM) or KCl (20 mM) treatment.

Figure 3

MEDI0618 prevented evoked and spontaneous migraine-like pain behaviour induced by supradural and systemic mast cell degranulation. Data represent tactile responses from different cohorts of female mice receiving isotype control protein or MEDI0618 prior to supradural (A) or intraperitoneal (C) administration of compound 48/80 and (E and F) count of rearing/vertical episodes as an outcome measurement of spontaneous migraine-like headache behaviour after systemic administration of compound 48/80. (A and C) Tactile frequency of response was collected before treatment at baseline (BL1), followed by subcutaneous administration of isotype control protein or MEDI0618 at 50 mg/kg, 24 h prior to testing. Tactile frequency of response for baseline 2 (BL2) was collected right before (A) supradural administration of compound 48/80 (6.5 nmol/5 µl) or (C) intraperitoneal administration of compound 48/80 (2 mg/kg). Behaviour was evaluated 30 min and 1–5 h after compound 48/80, with cutaneous allodynia revealed by increased frequency of response to tactile stimuli. Area under the curve (AUC) obtained from (B) supradural and (D) systemic compound 48/80-time courses. Female mice receiving intraperitoneal administration of compound 48/80 at 2 mg/kg or vehicle control were immediately individually placed in a Plexiglas activity box arena for 3 h after the treatment for the quantification of (E) vertical episodes. In a separate cohort of animals, MEDI0618 or isotype control protein was administered subcutaneously and 24 h later the mice received intraperitoneal injection of compound 48/80 and were placed in a Plexiglas activity box for the quantification of (F) vertical episodes. Data are presented as mean ± standard error of the mean and were analysed using two-way ANOVA followed by Sidak’s multiple comparison test (A, C, E and F) and unpaired Student’s t-test (B and D). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (n = 7–8) versus control or isotype control protein. Details of the statistical analyses are provided in Supplementary Table 3.

Figure 4

MEDI0618 prevented periorbital cutaneous allodynia induced by migraine-like pain models. Data represent tactile responses from different cohorts of female mice. Inhalational exposure of a subthreshold dose of umbellulone (UMB) on Day 16 after the first restraint stress (RS)-priming, systemic intraperitoneal (i.p.) administration of nitroglycerin (NTG) and supradual injection of inflammatory mediators (IM) were used to induce migraine-like pain behaviour in mice. MEDI0618 or isotype control protein were administered subcutaneously at 50 mg/kg 24 h prior to (A) UMB, (C) NTG or (E) IM time courses. Periorbital tactile frequency of response was collected at baseline (BL, A; BL1, C and E) before and after (BL2) monoclonal antibody (mAb) administration, followed by (A) inhalational UMB (0.01 M/500 µl, each gauze; 30 min), (C) NTG (10 mg/kg, i.p.) or (E) supradural IM (5 µl, each). Area under the curve (AUC) calculation was performed for (B) UMB, (D) NTG and (F) IM time courses. Data are presented as mean ± standard error of the mean and analysed using two-way ANOVA followed by Sidak’s multiple comparison test (A, C and E) and unpaired Student’s t-test (B, D and F). *P < 0.05, **P < 0.01 and ****P < 0.0001 (UMB, n = 18; NTG and IM, n = 8) versus isotype control protein. Details of the statistical analyses are provided in Supplementary Table 3.

Figure 5

MEDI0618 prevented calcitonin gene-related peptide (CGRP)-dependent and -independent mechanisms involved in migraine-like pain. Data represent tactile responses from different cohorts of female mice. (A and C) Periorbital tactile frequency of response was collected prior to (BL1) and 30 min after (BL2) intraperitoneal administration of control or the small molecule CGRP-receptor (CGRP-R) antagonist, olcegepant, at 1 mg/kg followed by supradural injection of (A) CGRP (1 pg/5 µl, each) or (C) inflammatory mediators (IM; 5 µl, each). Cutaneous allodynia was evaluated at indicated times after CGRP or IM. (E) Behaviour was recorded before subcutaneous administration of MEDI0618 or isotype control protein, both at 50 mg/kg (BL1). Twenty-four hours later, the tactile frequency of response was assessed prior to (BL2) and 30 min after (BL3) intraperitoneal administration of olcegepant followed by supradural IM and cutaneous allodynia assessment. Area under the curve (AUC) calculation was performed for (B) CGRP, (D) IM and (F) IM/MEDI0618 time courses. Data are presented as mean ± standard error of the mean and analysed using two-way ANOVA followed by Sidak’s multiple comparison test (A, C and E) and unpaired Student’s t-test (B, D and F). *P < 0.05, **P < 0.01 (CGRP, n = 6–8; IM, n = 6 and IM/MEDI0618, n = 8) versus control or isotype control protein. Details of the statistical analyses are provided in Supplementary Table 3.

Figure 6

MEDI0618 failed to prevent acute and persistent post-traumatic headache-like behaviours induced by mild traumatic brain injury. (A) Periorbital tactile frequency of response was collected before (BL) and on Days 1, 2, 3, 5, 6, 10 and 14 after mild traumatic brain injury (mTBI). MEDI0618 or isotype control protein were administered subcutaneously at 50 mg/kg, 2 h, and on Days 7, 14, 16 and 21 after mTBI induction. On Days 15, 17 and 22 after mTBI (B, C and D, respectively) using the same cohort of animals. The baseline (BL) tactile frequency of response was collected, and mice were immediately (B) exposed to a 1000-lx bright light (bright light stress) for 15 min, (C) received inhalational umbellulone (UMB, 0.01 M/500 µl) for 30 min or (D) intraperitoneal nitroglycerin (NTG) at 10 mg/kg followed by periorbital cutaneous allodynia evaluation. Data are presented as mean ± standard error of the mean and analysed using two-way ANOVA followed by Sidak’s multiple comparison test (n = 8). Details of the statistical analyses are provided in Supplementary Table 3.