CGRP receptor ( IC50 = 0.12 nM )
CGRP receptor (human CGRP receptor: Ki = 0.024 ± 0.001 nM in SK-N-MC cells; Ki = 0.022 ± 0.002 nM on recombinant human receptor; rhesus monkey CGRP receptor: Ki = 0.024 ± 0.001 nM; canine CGRP receptor: Ki = 10 nM; rat CGRP receptor: Ki = 10 ± 1.2 nM) [1]
Human adrenomedullin AM1 receptor (CLR/RAMP2): Ki = 16,500 nM [1]
Human adrenomedullin AM2 receptor (CLR/RAMP3): Ki = 156 ± 17 nM [1]
Human calcitonin receptor (CTR): Ki = 1.9 ± 0.58 μM [1]
Human amylin AMY1 receptor (CTR/RAMP1): Ki = 0.75 ± 0.13 nM [1]
Human amylin AMY3 receptor (CTR/RAMP3): Ki = 128 ± 25 nM [1]

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MK-3207 potently blocked human α-CGRP-stimulated cAMP responses in human CGRP receptor-expressing HEK293 cells, with IC50 = 0.12 ± 0.02 nM; addition of 50% human serum resulted in IC50 = 0.17 ± 0.02 nM [1]
Increasing concentrations of MK-3207 caused parallel rightward shifts in CGRP dose-response curve with no reduction in maximal agonist response; Schild regression yielded pA2 = 10.3 and KB = 0.05 nM, consistent with competitive antagonism [1]
MK-3207 displayed significantly lower potency on rat CGRP receptor with pIC50 = 7.31 ± 0.09 [1]
MK-3207 is a substrate for human and mouse P-glycoprotein (P-gp) at 1 and 5 μM as indicated by B-to-A/A-to-B transport ratios of 33 and 50 in L-MDR1 and L-mdr1a cell lines, respectively; MK-3207 has high passive permeability of 24 × 10⁻⁶ cm/s [1]

In vitro activity: MK-3207 exhibits a rhesus monkey receptor affinity (Ki) of 0.024±0.001 nM; n=14) that is comparable to that of humans, but its affinity for the canine and rat receptors is >400-fold lower, at 10 nM and 10±1.2 nM, respectively. With Ki values of 16,500 nM and 156±17 nM, respectively, MK-3207 exhibits high selectivity against the human AM1 (CLR/RAMP2) and AM2 (CLR/RAMP3) receptors. At 1.9±0.58 μM, MK-3207 exhibits a high level of selectivity against human CTR. With a Ki value of 128±25 nM, MK-3207 demonstrates good selectivity against the AMY3 (CTR/RAMP3) receptor as well. However, its Ki value of 0.75±0.13 nM indicates less selectivity against the AMY1 (CTR/RAMP1) receptor. MK-3207 potently inhibits 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 exhibits a significantly lower potency for the rat CGRP receptor, with a pIC50=7.31±0.09.

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In rhesus monkey pharmacodynamic assay measuring capsaicin-induced dermal vasodilation (CIDV), MK-3207 produced concentration-dependent inhibition of dermal blood flow. Plasma exposures ranged from <1 nM to approximately 260 nM. MK-3207 had EC50 = 0.8 ± 0.3 nM and Emax = 81 ± 5% for inhibition of CIDV; EC90 value was approximately 7 nM [1]

MK-3207 profoundly activates central receptors because it is a CNS-penetrant. Following a 10 mg/kg oral dose of MK-3207, the ratio of CSF to plasma is 2 to 3%[1].

MK-3207 and 40 pM rat amylin are combined in amylin binding assays, and then 25 μg of CTR/RAMP1 or 25 μg of CTR/RAMP3 membranes are added. The mixture is then incubated for three hours at room temperature in binding buffer (10 mM HEPES, 5 mM MgCl₂, and 0.2% bovine serum albumin) in a volume of one milliliter. As the radioligand in calcitonin binding assays, 25 μg of CTR membranes are used with 30 pM ¹²⁵I-human calcitonin. Filtration through GF/B 96-well filter plates blocked with 0.5% polyethylenimine ends the incubation process. Prism is utilized for data analysis, and the equation Ki=IC50/1 + ([ligand]/K_D is used to calculate the Ki value. Saturation binding experiments are used to calculate the K_D value for each receptor.

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Competitive binding experiments were carried out to determine the relative affinity of MK-3207 for human, rhesus monkey, rat, and canine CGRP receptors. Membranes (SK-N-MC cells, recombinant human, rhesus cerebellum, dog brain, rat brain) were incubated with ¹²⁵I-hCGRP and varying concentrations of MK-3207. Incubations were terminated by filtration through GF/B 96-well filter plates blocked with 0.5% polyethylenimine. Ki values were calculated using the equation Ki = IC50/(1+[ligand]/KD) [1]
Competitive binding experiments for adrenomedullin, calcitonin, and amylin receptors: MK-3207 was combined with ⁴⁰ pM ¹²⁵I-rat amylin (for CTR/RAMP1 or CTR/RAMP3 membranes), or ³⁰ pM ¹²⁵I-human calcitonin (for CTR membranes), or appropriate radioligands for AM1/AM2 receptors, with 25 μg membranes in binding buffer (10 mM HEPES, 5 mM MgCl₂, 0.2% bovine serum albumin) in 1 ml total volume. Incubations lasted 3 h at room temperature, terminated by filtration [1]
[³H]MK-3207 saturation binding: increasing concentrations of [³H]MK-3207 (specific activity 73.7 Ci/mmol) were combined with 1 μM compound 25 for nonspecific binding and 50 μg/well SK-N-MC membrane in 1 ml binding buffer, incubated overnight (18 h) at room temperature, terminated by filtration. KD = 60 pM, Bmax = 350 fmol/mg protein [1]
Association kinetics: 60 pM [³H]MK-3207 with 50 μg/well SK-N-MC membranes in binding buffer, incubated at room temperature for 1 to 300 min; apparent kon = 1.5 × 10⁹ M⁻¹·min⁻¹ [1]
Dissociation kinetics: 60 pM [³H]MK-3207 with 50 μg/well SK-N-MC membranes incubated for 3 h at room temperature, then 1 μM compound 25 added, dissociation monitored for 1 to 390 min; koff = 0.012 min⁻¹, t1/2 = 59 min [1]

Functional assays using HEK293 cells stably expressing human CGRP receptor (CLR/RAMP1). Cells were plated at 85,000 cells/well in 96-well poly-D-lysine-coated plates ~19 h before assay. Cells were washed with PBS and preincubated with various concentrations of MK-3207 in presence or absence of 50% human serum for 30 min at 37°C in CO₂ incubator. 300 μM isobutyl-methylxanthine was added, incubated 30 min at 37°C, then stimulated with 0.3 nM α-CGRP for 5 min at 37°C. Intracellular cAMP measured. IC50 = 0.12 ± 0.02 nM (without serum), 0.17 ± 0.02 nM (with 50% human serum) [1]
Functional assays with HEK293 cells transiently expressing hybrid human/rat CGRP receptors: cells were transfected with human CLR + rat RAMP1, human CLR + human W74A RAMP1, or rat CLR + rat RAMP1. After 48 h, cells were dissociated and resuspended in assay buffer with 300 μM isobutyl-methylxanthine, added to 384-well plates at 2000 cells/well containing antagonist, incubated 30 min at room temperature, then EC50 concentration of CGRP added, incubated 20 min, then HTRF cAMP detection. pIC50 values: hCLR/hRAMP1 = 9.75 ± 0.03; hCLR/rRAMP1 = 8.12 ± 0.1; hCLR/hW74A = 8.66 ± 0.18; rCLR/rRAMP1 = 7.31 ± 0.09 [1]

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Rhesus monkey pharmacodynamic assay for capsaicin-induced dermal vasodilation (CIDV). Six adult rhesus monkeys (either sex, 4.8–12.7 kg) were anesthetized with ketamine (5–30 mg/kg i.m.), intubated, anesthesia maintained with isoflurane (1–2% in 100% oxygen). Four rubber O-rings (8 mm inner diameter) placed on ventral forearm. Dermal vasodilation induced by topical capsaicin (2 mg in 30% ethanol, 30% Tween 20, 40% water). Laser Doppler imager used for quantitation. Three sequential infusions of vehicle or MK-3207 treatment regimen after no-treatment capsaicin baseline challenge. Vehicle: dose 1: i.v. bolus 0.5 ml 50% DMSO, 50% water followed by 25-min continuous i.v. infusion 0.025 ml/min 50% DMSO, 50% water; dose 2: same; dose 3: i.v. bolus 0.5 ml 100% DMSO followed by 25-min infusion 0.025 ml/min 100% DMSO. MK-3207 dosing regimens to target plasma exposures 1-300 nM: 1) 0.3 μg/kg i.v. bolus + 0.008 μg/kg/min, 0.6 μg/kg i.v. bolus + 0.025 μg/kg/min, then 2.1 μg/kg + 0.084 μg/kg/min; 2) 0.6+0.025, 2.1+0.084, then 9.1+0.25; 3) 9.1+0.25, 21.2+0.84, then 60.6+2.5. Dermal blood flow measured at 20 min after capsaicin application (25 min after start of infusion). Blood samples for plasma concentration taken at same time point [1]
Cisterna magna-ported rhesus monkey model for CSF collection: a flexible silicone catheter suspended in cisterna magna, anchored on atlanto-occipital membrane, tunneled subcutaneously to midscapular region into a surgically implanted port body. CSF accessed by needle through skin and membrane. After oral administration of MK-3207 at 10 mg/kg (0.5% methylcellulose, pH ~3), CSF and plasma samples collected at 0.5, 1, 2, 4, 8, and 24 h (n=3) [1]
In vitro P-glycoprotein transport studies: cells plated on 96-well filters at 85,000 cells/0.17 ml/well 5 days before study. Media replaced with 10 mM HEPES-buffered HBSS pH 7.4, aspirated after 30 min equilibration. Transport initiated by adding 0.15 ml HBSS to receiver side and 0.15 ml HBSS containing 1 or 5 μM MK-3207 to donor side (apical for A-to-B, basolateral for B-to-A). Directional transport of 1 μM verapamil as positive control. Incubated 3 h at 37°C, samples collected and quantitated via LC/MS/MS [1]

A specially made flexible silicone catheter is fed into a surgically implanted port body in the midscapular region after being fully suspended in the cisterna magna and securely anchored on both sides of the atlanto-occipital membrane. The catheter is tunneled subcutaneously to this location. Via the skin and membrane covering the port, a needle is aseptically inserted to access the CSF; peripheral venipuncture is used to obtain blood samples. 0.5, 1, 2, 4, 8, and 24 hour CSF and plasma samples are taken and subjected to compound level analysis following oral administration of MK-3207 at 10 mg/kg (0.5% methylcellulose, with an adjusted pH of approximately 3) to rhesus monkeys with port-implanted cisterna magna catheters.

MK-3207 displayed good oral bioavailability in rats (74%), dogs (67%), and rhesus monkeys (41%) [1]
In rhesus monkeys, clearance was 15 ml/min/kg and intravenous half-life was 1.5 h [1]
After oral dosing at 10 mg/kg in cisterna magna-ported rhesus monkeys, CSF Cmax = 20.0 ± 13.5 nM, plasma Cmax = 979 ± 570 nM; CSF AUC0-24h = 96.4 ± 41.8 nM·h, plasma AUC0-24h = 3285 ± 1205 nM·h; CSF/plasma ratio = 2.0% (based on Cmax) and 2.9% (based on AUC) [1]
CSF/plasma ratio is approximately 30% of the unbound fraction (9.4%) in plasma, indicating central and peripheral compartments are not freely equilibrating [1]

MK-3207 is a substrate for human and mouse P-glycoprotein (P-gp) (B-to-A/A-to-B transport ratios of 33 and 50 in L-MDR1 and L-mdr1a cell lines at 1 and 5 μM) [1]
Unbound fraction in plasma is 9.4% [1]
In a screen of 169 enzyme and binding assays, MK-3207 was highly selective for CGRP receptor with no IC50 < 5 μM in any assay except human calcitonin receptor (IC50 = 1.75 μM) [1]

[1]. Pharmacological properties of MK-3207, a potent and orally active calcitonin gene-related peptide receptor antagonist. J Pharmacol Exp Ther. 2010 Apr;333(1):152-60.

MK-3207 is a potent and orally active CGRP receptor antagonist being developed for acute treatment of migraine. It has demonstrated efficacy in a phase II adaptive dose-ranging trial, significantly improving migraine pain relief 2 h after dosing compared with placebo [1]
In vitro autoradiography on rhesus monkey brain slices using [³H]MK-3207 (0.045 nM) showed highest binding densities in cerebellum (gray matter), brainstem (regions processing nociceptive stimuli), and meninges; minimal binding in cerebral cortex and caudate putamen [1]
The species selectivity of MK-3207 is primarily driven by RAMP1; mutation of tryptophan 74 to alanine in human RAMP1 resulted in 10- to 40-fold reduction in potency [1]
The slow dissociation kinetics of MK-3207 (t1/2 = 59 min) compared to telcagepant (t1/2 = 1.3 min) is a consequence of its greatly enhanced affinity for the CGRP receptor [1]
MK-3207 is being evaluated as a potential new therapy for migraine sufferers, with the advantage of lacking vasoconstrictor effects (a liability of triptans) [1]

C31H29F2N5O3.HCL

594.05

593.2

957116-20-0

MK-3207;957118-49-9

49867927

Off-white to pink solid powder

5.052

4

7

4

42

1040

2

C1CCC2(C1)C(=O)N([C@@H](CN2)C3=CC(=CC(=C3)F)F)CC(=O)NC4=CC5=C(C[C@@]6(C5)C7=C(NC6=O)N=CC=C7)C=C4.Cl

VWKNXYACOMQRBX-KZCKSIIFSA-N

InChI=1S/C31H29F2N5O3.ClH/c32-21-10-19(11-22(33)13-21)25-16-35-31(7-1-2-8-31)29(41)38(25)17-26(39)36-23-6-5-18-14-30(15-20(18)12-23)24-4-3-9-34-27(24)37-28(30)40;/h3-6,9-13,25,35H,1-2,7-8,14-17H2,(H,36,39)(H,34,37,40);1H/t25-,30+;/m0./s1

2-[(8R)-8-(3,5-difluorophenyl)-10-oxo-6,9-diazaspiro[4.5]decan-9-yl]-N-[(2R)-2'-oxospiro[1,3-dihydroindene-2,3'-1H-pyrrolo[2,3-b]pyridine]-5-yl]acetamide;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.21 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)