Characterisation of the effects of a non-peptide CGRP receptor antagonist in SK-N-MC cells and isolated human cerebral arteries

Abstract

The cerebral circulation is innervated by calcitonin gene-related peptide (CGRP) containing fibers originating in the trigeminal ganglion. During a migraine attack, there is a release of CGRP in conjunction with the head pain, and triptan administration abolishes both the CGRP release and the pain at the same time. In the search for a novel treatment of migraine, a non-peptide CGRP antagonist has long been sought. Here, we present data on a human cell line and human and guinea-pig isolated cranial arteries for such an antagonist, Compound 1 (4-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acid [1-(3,5-dibromo-4-hydroxy-benzyl)-2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-amide). On SK-N-MC cell membranes, radiolabelled CGRP binding was displaced by both CGRP-(8–37) and Compound 1, yielding pK_i values of 8.9 and 7.8, respectively. Functional studies with SK-N-MC cells showed that CGRP-induced cAMP production was antagonised by both CGRP-(8–37) and Compound 1 with pA₂ values of 7.8 and 7.7, respectively. Isolated human and guinea pig cerebral arteries were studied with a sensitive myograph technique. CGRP induced a concentration-dependent relaxation in human cerebral arteries which was antagonized by both CGRP-(8–37) and Compound 1 in a competitive manner. In guinea pig basilar arteries, CGRP-(8–37) antagonised the CGRP-induced relaxation while Compound 1 had a weak blocking effect . The clinical studies of non-peptide CGRP antagonists are awaited with great interest.

Introduction

The cerebral circulation is innervated by calcitonin-gene related peptide (CGRP)-containing nerve fibres originating in the trigeminal ganglion Edvinsson, 1985, Uddman et al., 1985. These fibres, which release CGRP following activation by electrical stimulation (Goadsby et al., 1988) or by capsaicin (Jansen-Olesen et al., 1996), are activated in primary headaches and following subarachnoid haemorrhage (Goadsby et al., 1990; Goadsby and Edvinsson, 1994; Juul et al., 1990, Juul et al., 1995). Trigeminal fibres can mediate dilatation of brain vessels (Edvinsson et al., 1986) and increase cerebral blood flow (Goadsby, 1993) and they may also mediate the trigemino-vascular reflex, thereby counterbalancing cerebrovascular contraction McCulloch et al., 1986, Edvinsson et al., 1987. In migraineurs, elevated jugular blood levels of CGRP are found during an attack and these levels are normalised on administration of sumatriptan concomitant with relief from headache pain (Edvinsson and Goadsby, 1998). CGRP is a potent vasodilator and through this action may be the key in the pathogenesis of migraine headache. A non-peptide CGRP antagonist has long been sought as an attractive and novel approach in treating migraine.

mRNAs encoding CGRP receptors and accessory proteins (RAMPs) required for CGRP receptor functionality have been detected in various cerebral and cranial arteries Edvinsson et al., 1997, Sams and Jansen-Olesen, 1998. Pharmacological characterisation of CGRP receptors in isolated cerebral vessels has relied largely on testing a limited number of CGRP analogues and on the antagonistic properties of the CGRP-(8–37) fragment. To date, a limited number of non-peptide CGRP receptor antagonists have been reported. Examples include quinine analogues (Daines et al., 1997) which have relatively weak activity; displacement of ¹²⁵I-CGRP from SK-N-MC membranes revealed IC₅₀ values in the micromolar concentration range (Daines et al., 1997). This result contrasts with nanomolar affinity of CGRP-(8–37) (Aiyar et al., 1996). More recently, Doods et al. (2000) described BIBN4096BS (a Lys–Tyr dipeptide derivative) which had high affinity (picomolar) for CGRP receptors endogenously expressed in SK-N-MC cells and inhibited neurogenic vasodilation evoked by trigeminal stimulation in marmosets.

The aim of the present study was therefore to evaluate a second CGRP receptor antagonist (see patent number WO 98/11128, coded Compound 1: 4-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acid [1-(3,5-dibromo-4-hydroxy-benzyl)-2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-amide, see Fig. 1) and use it as a research tool to characterise CGRP-mediated responses in cranial arteries. Compound 1 was characterised initially in the neuroblastoma cell line, SK-N-MC, which expresses an endogenous CGRP receptor. The effects of Compound 1 on CGRP-evoked relaxation in human and guinea-pig cranial arteries were studied next and compared with the antagonistic effects of CGRP-(8–37).