The identification of potent, orally bioavailable tricyclic CGRP receptor antagonists.

A series of tricyclic CGRP receptor antagonists was optimized in order to improve oral bioavailability. Attenuation of polar surface area and incorporation of a weakly basic indoline nitrogen led to compound 5, a potent antagonist with good oral bioavailability in three species.

Laser Doppler imaging of reactive hyperemia exposes blood flow deficits in a rat model of experimental limb ischemia.

The primary pathophysiology of peripheral artery occlusive disease is associated with impaired perfusion to the lower extremities. The lack of effective pharmacologic agents to treat this disease emphasizes the need for well-characterized animal models that can be used to evaluate the efficacy of emerging therapies. A major limitation with the current animal models of peripheral artery occlusive disease is that the variety of surgical methods employed to reduce peripheral blood flow produce differences in the severity and time course of the resting and reserve blood flow deficits. Furthermore, the methods used to evaluate the restoration of peripheral flow are often not suitable for serial measurements. This study used laser Doppler imaging to serially evaluate resting blood flow and the development of a functional collateral circulation after the induction of hind limb ischemia in the rat. Reserve blood flow was assessed by measuring hyperemic blood flow in the hind paw after temporary arterial occlusion. The magnitude of the hyperemic response was found to be dependent upon both the duration of arterial occlusion and the measurement time after release of the occluder. After ligation of the common iliac artery, but at a time when resting blood flow was reestablished, hyperemic tests unmasked a sustained deficit in reserve blood flow capacity that persisted for at least 14 days. Therefore, the use of a noninvasive vascular occluder and laser Doppler imaging represents a sensitive and consistent technique to measure peripheral blood flow status to assess the development of functional collateral vessels. These findings will enhance the ability to effectively study pharmacologic therapies aimed at promoting the growth and development of collateral vessels.

Potent benzimidazolone-based CGRP receptor antagonists.

The previously disclosed spirohydantoin-based CGRP receptor antagonists were optimized for potency through modification of the benzimidazolone substituents. Compounds were identified which had minimal shift in the cAMP functional assay containing 50% human serum. Blockade of CGRP-mediated vasodilation was observed with these compounds in a rhesus pharmacodynamic assay and the in vivo potency correlated with the in vitro activity in the serum-shifted functional assay.

Pharmacological characterization of MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide], a potent and orally active calcitonin gene-related peptide receptor antagonist for the treatment of migraine.

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide that plays a key role in the pathophysiology of migraine headache. CGRP levels in the cranial circulation are increased during a migraine attack, and CGRP itself has been shown to trigger migraine-like headache. The correlation between CGRP release and migraine headache points to the potential utility of CGRP receptor antagonists as novel therapeutics in the treatment of migraine. Indeed, clinical proof-of-concept in the acute treatment of migraine was demonstrated with an intravenous formulation of the CGRP receptor antagonist BIBN4096BS (olcegepant). Here we report on the pharmacological characterization of the first orally bioavailable CGRP receptor antagonist in clinical development, MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide]. In vitro, MK-0974 is a potent antagonist of the human (K(i) = 0.77 nM) and rhesus (K(i) = 1.2 nM) CGRP receptors but displays >1500-fold lower affinity for the canine and rat receptors as determined via (125)I-human CGRP competition binding assays. A rhesus pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging was utilized to determine the in vivo activity of CGRP receptor antagonism. MK-0974 produced a concentration-dependent inhibition of dermal vasodilation, generated by capsaicin-induced release of endogenous CGRP, with plasma concentrations of 127 and 994 nM required to block 50 and 90% of the blood flow increase, respectively. In conclusion, MK-0974 is a highly potent, selective, and orally bioavailable CGRP receptor antagonist, which may be valuable in the acute treatment of migraine.

Investigation of the species selectivity of a nonpeptide CGRP receptor antagonist using a novel pharmacodynamic assay.

The recent discovery of several nonpeptide CGRP antagonists have led to significant advances in our understanding of CGRP receptor pharmacology. Specifically, these antagonists have demonstrated a clear species selectivity with >100-fold greater affinity for human CGRP receptor compared to receptors from other species, such as rat, rabbit and guinea pig. Therefore, nonhuman primate models are required to accurately assess the in vivo activity of these antagonists. The commonly used model in marmosets involves electrical stimulation of the trigeminal ganglia and is a technically difficult and terminal procedure. In this report, we describe a noninvasive pharmacodynamic model in which topical application of capsaicin is utilized to induce the release of endogenous CGRP and a vasodilatory response which can be measured using laser Doppler imaging. Using the potent and selective CGRP antagonist Compound 3, which is an analog of the well-characterized compound BIBN4096BS, we demonstrated 62% inhibition with 300 microg/kg, i.v., in the rat. When tested in the rhesus monkey, only 30 microg/kg of Compound 3 was needed to produce complete inhibition, suggesting that the rhesus CGRP receptor shares a pharmacological profile similar to marmoset and human receptors. Two separate measurements were obtained in this model to provide an indication of both the acute inhibitory effect as well as the prophylactic effect of the CGRP antagonist. At the doses studied, Compound 3 was equally effective on both the acute and prophylactic inhibition of CGRP-mediated vasodilation in rat and rhesus. In conclusion, this is the first report to describe and validate a noninvasive model in nonhuman primates that allows rapid evaluation of CGRP antagonist activity against endogenous CGRP.

Enhanced hindlimb collateralization induced by acidic fibroblast growth factor is dependent upon femoral artery extraction.

UNLABELLED: Recent investigations have established the feasibility of using exogenously delivered angiogenic growth factors to increase collateral artery development in animal models of myocardial and hindlimb ischemia. OBJECTIVE: Our aim was to evaluate the ability of a stabilized form of acidic fibroblast growth factor (aFGF-S117) to stimulate collateralization and arteriogenesis in the rabbit hindlimb following the surgical induction of ischemia by femoral artery extraction. A secondary objective was to examine angiogenic and arteriogenic effects of aFGF-S117 in the absence of a peripheral blood flow deficit. METHODS AND RESULTS: Five days after femoral artery removal, aFGF-S117 (1, 3, or 30 microg/kg) was intramuscularly delivered into the hindlimb, three times per week for 2 consecutive weeks. End-point measurements performed on day 20 found that hindlimb reserve blood flow was significantly improved in rabbits that received 3 or 30 microg/kg of aFGF-S117, with no difference in efficacy between these two doses. These hemodynamic results were supported by angiographic evidence showing enhanced density of collateral vessels in the medial thigh region and histological findings of increased capillary density within the gastrocnemius muscle from rabbits treated with aFGF-S117. When an efficacious dose of 3 microg/kg of aFGF-S117 was administered to sham-operated rabbits with intact femoral arteries, there was no change in any of the blood flow, angiographic or histological parameters measured. CONCLUSIONS: These findings demonstrate that a stabilized form of aFGF stimulated the development of functional collateral arteries in the rabbit hindlimb, an effect which was dependent upon removal of the femoral artery. These results suggest that aFGF-S117 may have therapeutic potential for the treatment of arterial occlusive disorders.

Vascular endothelial growth factor stimulates angiogenesis without improving collateral blood flow following hindlimb ischemia in rabbits.

This study was designed to test the ability of adenovirus-delivered vascular endothelial growth factor (Ad-VEGF) to stimulate angiogenesis and arteriogenesis in the rabbit hindlimb following the induction of ischemia and to evaluate the functional changes in the collateral circulation. Ten days after the surgical induction of hindlimb ischemia, either a control virus (1 x 10(9) pfu) or an adenovirus containing the gene for VEGF(165) (1 x 10(6), 1 x 10(7), 1 x 10(8), or 1 x 10(9) pfu) was administered intramuscularly into the ischemic limb. Thirty days after administration of the adenoviral vectors, skeletal muscle capillary density was assessed and angiography was performed as markers of angiogenesis and arteriogenesis, respectively. Hindlimb blood flow was directly measured and hyperemic tests were performed to evaluate the functional improvements in collateral blood flow. Animals treated with Ad-VEGF at 1 x 10(8) and 1 x 10(9) pfu showed elevated levels of circulating VEGF and dose-dependent hindlimb edema. These doses also led to a robust angiogenic response (i.e., increase in capillary density), but failed to improve collateral blood flow. Consistent with the lack of a functional response, there was no angiographic evidence of enhanced arteriogenesis with any dose of Ad-VEGF. Following the induction of hindlimb ischemia, administration of Ad-VEGF stimulated capillary sprouting (i.e., angiogenesis), but did not increase the growth and development of larger conduit vessels (i.e., arteriogenesis) or improve collateral blood flow. These results support the concept that VEGF may not be expected to have therapeutic utility for the treatment of peripheral or myocardial ischemia.