In vitro activity: MK 3207 exhibits significantly higher affinity for both native and recombinant human CGRP receptor, as well as rhesus monkey CGRP receptor with Ki of 24 pM, ~24 pM and 22 pM, respectively, as compared to CGRP receptors from other species, including canine and rodent (Ki values of ~10 nM). Although has affinity for AMY1 (CTR/RAMP1) receptor with a Ki value of 0.75 nM, MK 3207 displays marked selectivity for human CGRP receptor versus related human AM1 (CLR/RAMP2) receptor, AM2 (CLR/RAMP3) receptor, AMY3 (CTR/RAMP3) receptor, and CTR with Ki values of 16.5 μM, 0.156 μM, 0.128 μM and 1.9 μM, respectively. MK 3207 potently inhibits human α-CGRP-induced cAMP production in HEK293 cells stably expressing human CLR/RAMP1 with an IC50 of 0.12 nM, and maintains similar potency in the presence of 50% human serum with an IC50 of 0.17 nM, indicating that the activity of MK 3207 would not be dramatically affected by plasma protein binding in vivo. MK 3207 exhibits approximately 65-fold more potent in the human serum-shifted in vitro functional assay than telcagepant with an IC50 of 10.9 NM.

Kinase Assay: Amylin binding assays are conducted by combining MK-3207 and 40 pM 125I-rat amylin, followed by 25 μg of CTR/RAMP1 or 25 μg of CTR/RAMP3 membranes and incubated for 3 h at room temperature in binding buffer (10 mM HEPES, 5 mM MgCl2, and 0.2% bovine serum albumin) in a total volume of 1 mL. Calcitonin binding assays are with 25 μg of CTR membranes and 30 pM 125I-human calcitonin as the radioligand. Incubations are terminated by filtration through GF/B 96-well filter plates that has been blocked with 0.5% polyethylenimine. Data are analyzed using Prism, and the Ki value is determined using the equation Ki=IC50/1 + ([ligand]/KD). The KD value for each receptor is determined by saturation binding experiments.

Cell Assay: MK-3207 displays a similar affinity (Ki) for the rhesus monkey receptor (0.024±0.001 nM; n=14) as for human, but it displays>400-fold lower affinity for the canine and rat receptors, with values of 10 nM and 10±1.2 nM, respectively. MK-3207 is highly selective versus the human AM1 (CLR/RAMP2) and AM2 (CLR/RAMP3) receptors, with Ki values of 16,500 nM and 156±17 nM, respectively. MK-3207 maintains a high degree of selectivity versus human CTR, with a Ki value of 1.9±0.58 μM. MK-3207 also displays good selectivity versus the AMY3 (CTR/RAMP3) receptor, with a Ki value of 128±25 nM, but it is less selective versus the AMY1 (CTR/RAMP1) receptor, with a Ki value of 0.75±0.13 nM. MK-3207 potently blocks human α-CGRP-stimulated cAMP responses in human CGRP receptor-expressing HEK293 cells, with an IC50 value of 0.12±0.02 nM. MK-3207 displays significantly lower potency for the rat CGRP receptor, with a pIC50=7.31±0.09.