BIBN4096BS is a potent competitive antagonist
BIBN4096BS is the first non-peptide blocker that antagonizes with high potency the relaxant effects of α-CGRP on rings of human temporal artery. The surmountable antagonism of the effects of CGRP caused by 0.1 – 100 nM BIBN4096BS and linear Schild-plots with slopes not different from one suggest specific interactions with CGRP receptors. The specificity is further supported by the failure of BIBN4096BS to antagonize the relaxant effects of papaverine and SNP, at a concentration (1 μM) four orders of magnitude greater than its KB for the CGRP receptors of the human temporal artery.
Most recently, a non-peptide compound chemically related to BIBN4096BS (patent number WO 98/11128) was reported to antagonize the relaxant effects of CGRP on human lenticulostriatal arteries, obtained >27 h after death (Edvinsson et al., 2001). However, the Schild-plot of data from that report yielded a slope of 0.67 and the affinity estimate for that compound (KB=40 nM) was 500 to 1000 times lower than the affinity estimated for BIBN4096BS from our data of competitive inhibition of CGRP-evoked relaxation of human temporal artery.
Our affinity estimate of BIBN4096BS for the CGRP-receptors of human temporal artery (KB= 40 – 90 pM) was somewhat lower than its affinity estimated from binding (Ki=14 pM) to CGRP-receptors of the SK-N-MC human neuroblastoma cell line (Doods et al., 2000). Reasons for this discrepancy are unknown but it could be that BIBN4096BS did not equilibrate completely with the CGRP-receptor population. This is unlikely though, because our affinity estimate was actually slightly lower with the longer BIBN4096BS incubation (2 h) before the KCl-contracture compared to the incubation (1 h) during the KCl-contracture.
Also for unknown reasons, CGRP(8-37) has been reported to bind with considerably higher affinity for CGRP receptors of the SK-N-MC cell line (Ki=3.6 nM, Doods et al., 2000; Ki=1.3 nM, Edvinsson et al., 2001) than estimated from its affinity for CGRP receptors of the human temporal artery (KB= 200 – 280 nM – this work), human middle meningeal artery (KB=150 nM, Edvinsson et al., 1998) and larger branches of lenticulostriate arteries obtained 24 h postmortem (KB∼300 nM, Sams et al., 2000). Unlike these discrepancies, Edvinsson et al. (2001) found that the affinity of CGRP(8-37) as antagonist of the relaxant effects of CGRP on lenticulostriatal arteries (KB=15 nM) agreed with its affinity as antagonist of CGRP-evoked increases in cyclic AMP in SK-N-MC cells (KB=16 nM). However, it is not clear why Edvinsson et al. (2001) estimated a 12 times higher affinity of CGRP(8-37) from binding assays than from cyclic AMP assays. Futhermore, it also unclear why Edvinsson et al. (2001) reported a KB=15 nM for CGRP(8-37) on lenticulostriatal arteries while they estimated a 20 times lower affinity (KB∼300 nM) on large branches (550 – 1727 μm diameter) of these arteries (Sams et al., 2000). One important difference that could also account for differences between the blocking potencies of CGRP(8-37) in arteries, is that the temporal arteries of our study were freshly obtained from patients undergoing surgery, whereas the lenticulostriatal arteries of Edvinsson et al. (2001) were obtained 27 – 32 h after death.
The relaxant potency of α-CGRP on the human temporal artery (EC50∼400 pM) was also lower than its binding affinity on the neuroblastoma cells (Ki=32 pM, Doods et al., 2000; 15 pMEdvinsson et al., 2001). It may be argued that the relaxant potency of α-CGRP found by us for the temporal artery was relatively low due to our experimental conditions of partially depolarized membranes produced by small KCl elevations. However, the relaxant potency α-CGRP against KCl-precontracted arterial rings agrees with that found for human temporal artery preparations precontracted with the prostaglandin F2α (EC50∼400 pM, Jansen-Olesen et al., 1995). Nanomolar concentrations of CGRP hyperpolarize the membrane of arterial smooth muscle cells (Nelson et al., 1990) through activation of ATP-dependent K+ current (Quayle et al., 1994) and this mechanism contributes to vascular relaxation. Large-conductance, Ca2+-activated K+ (BKCa) channels hyperpolarize the membrane of smooth muscle cells, when activated through phosphorylation by cyclic AMP-dependent protein kinase (PKA) (Schubert & Nelson, 2001). CGRP appears to produce PKA-mediated BKCa channel activation in porcine coronary arterial cells (Miyoshi & Nakaya, 1995), thereby contributing to relaxation, and we suggest that this pathway may also play a role in human temporal artery. The lower arterial relaxant potency than neuroblastoma binding affinity of CGRP could be related to the partial depolarization of the vascular cells under our conditions.
Both PKA and cyclic GMP-dependent protein kinase (GPK) phosphorylate BKCa channels, thereby causing sensitization to Ca2+-induced activation, hyperpolarization and relaxation of vascular smooth cells (Schubert & Nelson, 2001). It is likely that papaverine and SNP relax the temporal artery at least in part through activation of PKA and GPK respectively, via BKCa activation. Because BIBN4096BS (1 μM) failed to modify the relaxations produced by both papaverine and SNP, the antagonist does not interfere with the PKA and PKG pathways. Furthermore, the partially insurmountable antagonism of the CGRP effects by 1 μM BIBN4096BS is therefore unlikely to be due to non-specific depression of the CGRP effects, but possibly related to the slow dissociation of BIBN4096BS from the CGRP receptors.
The lower blocking potency of the two antagonists and lower agonist potency of α-CGRP on the temporal artery compared to the corresponding binding affinities in neuroblastoma cells, could also be due to differences in CGRP receptors of the two systems or different regulation by endogenous modulators. Receptor activity modifying proteins, RAMPs, are modulators that determine the pharmacology of CGRP and other peptides (McLatchie et al., 1998). In addition, an accessory protein, CGRP-receptor component protein (RCP), has been found to facilitate coupling of the receptor to the Gs-protein/cyclic AMP pathway (Evans et al., 2000). We speculate that RAMP modulation of the receptor that interacts with α-CGRP and/or RCP modulation of its coupling could be different in human temporal artery and neuroblastoma cells.
Possible therapeutic relevance
Up to one third of migraine sufferers undergo dilatation of extracranial arteries (Lance, 1992), such as the temporal artery. Our results, showing that BIBN4096BS is a potent antagonist of the relaxant effects of α-CGRP in the temporal artery suggest that CGRP can be involved in migraine. CGRP, released from the sensory nerves of the temporal artery, would interact with CGRP receptors, enhance cyclic AMP levels in the smooth muscle (Jansen-Olesen et al., 1996) thereby inducing vasodilatation that in turn activates trigeminal fibres and migraine pain (Uddman et al., 1986). Blockade of the CGRP receptors by BIBN4096BS would be expected to prevent the harmful effects of CGRP. Clinical trials will be required to test this hypothesis.
We conclude that BIBN4096BS is the first potent non-peptide and competitive antagonist of CGRP-evoked relaxation of a human extracranial artery. The ability of BIBN4096BS to block CGRP-evoked vasodilatation in a human extracranial artery might lead to therapeutic benefits.