Inhibition of stimulated meningeal blood flow by a calcitonin gene-related peptide binding mirror-image RNA oligonucleotide

Abstract

Calcitonin gene-related peptide (CGRP) released from trigeminal afferents is known to play an important role in the control of intracranial blood flow. In a rat preparation with exposed cranial dura mater, periods of electrical stimulation induce increases in meningeal blood flow. These responses are due to arterial vasodilatation mediated in part by the release of CGRP. In this preparation, the effect of a CGRP-binding mirror-image oligonucleotide (Spiegelmer NOX-C89) was examined. Spiegelmer NOX-C89 applied topically at concentrations between 10(-7) and 10(-5) M to the exposed dura mater led to a dose-dependent inhibition of the electrically evoked blood flow increases. The highest dose reduced the mean increases in flow to 56% of the respective control levels. A nonfunctional control Spiegelmer (not binding to CGRP) was ineffective in changing blood flow increases. Intravenous injection of NOX-C89 (5 mg kg(-1)) reduced the evoked blood flow increases to an average of 65.5% of the control. The basal blood flow was not changed by any of the applied substances. In addition, an ex vivo preparation of the hemisected rat skull was used to determine CGRP release from the cranial dura mater caused by antidromic activation of meningeal afferents. In this model, 10(-6) M of NOX-C89 reduced the evoked CGRP release by about 50%. We conclude that increases in meningeal blood flow due to afferent activation can be reduced by sequestering the released CGRP and thus preventing it from activating vascular CGRP receptors. Moreover, the Spiegelmer NOX-C89 may inhibit CGRP release from meningeal afferents. Therefore, the approach to interfere with the CGRP/CGRP receptor system by binding the CGRP may open a new opportunity for the therapy of diseases that are linked to excessive CGRP release such as some forms of primary headaches.

Figure 1

(a) Stimulation and recording window in the parietal bone showing bipolar electrodes (±) and the recording site (ring) over a branch of the MMA; cortical veins (CV). (b) Sections from a continuous recording of meningeal blood flow showing increases in flow upon electrical stimulation (bars) and their changes after topical application of PBS and Spiegelmer NOX-C89 (10⁻⁷–10⁻⁵ M).

Figure 2

Mean increases in flow evoked by electrical stimulation before (control) and after topical application of PBS, Spiegelmer NOX-C89 and control Spiegelmer onto the exposed dura. (a) Flow increases (normalised to the mean of the control responses) and their variation after application of PBS, NOX-C89 (10⁻⁷–10⁻⁵ M) and control Spiegelmer (10⁻⁵ M); ^*significant difference to all three control values (P<0.05, ANOVA). (b) Normalised and averaged flow responses of six stimulation intervals after application of PBS, NOX-C89 and control Spiegelmer, respectively; ^*significant difference to PBS.

Figure 3

Mean increases in flow evoked by electrical stimulation before (control) and after i.v. injection of PBS or CGRP-binding Spiegelmer NOX-C89 onto the exposed dura. (a) Flow increases (normalised to the mean of the control responses) and their variation after administration of PBS or NOX-C89 (5 mg kg⁻¹); ^*significant difference to all three control values (P<0.05, ANOVA). (b) Normalised and averaged flow responses of six stimulation intervals after administration of PBS or NOX-C89; ^*significant difference to PBS.

Figure 4

(a) Mean concentrations of iCGRP in the eluate of skull cavities collected over 5 min intervals before, during and after electrical stimulation of the trigeminal ganglion in the presence of buffer (vehicle) or Spiegelmer NOX-C89 (10⁻⁶ M), measured with an ELISA; ^*significant difference between vehicle and Spiegelmer samples (P<0.01, U-test). (b) Mean iCGRP concentration measured in solutions of α-CGRP (10⁻¹⁰ and 10⁻¹¹ M) without and in the presence of 10⁻⁶ M NOX-C89.