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Purity: ≥98%
AMG 333 is a novel, potent and highly selective TRPM8 antagonist with an IC50 of 13 nM and 20 nM for hTRPM8 and rTRPM8, respectively. AMG-333can be potentially used for the treatment of migraine. In vitro toxicology profiling showed that AMG-333 was selective over several other TRP channels (IC50 TRPV1, V3, V4 > 20 μM; TRPA1 > 40 μM) and had no hits in off-target-activity panels (CEREP, 144 targets at 10 μM, POC > 45%; Ambit kinase, 100 at 1 μM, POC > 50%). AMG-333 was well-tolerated in 28 day rat and dog preclinical safety studies and advanced to Phase 1 human clinical trials.
| Targets |
AMG 333 targets transient receptor potential melastatin 8 (TRPM8) ion channel (human TRPM8: Ki = 6.9 nM for [³H]icilin binding [1]
; IC50 = 14 nM for menthol-induced calcium influx inhibition in TRPM8-expressing HEK293 cells [1] ; IC50 = 22 nM for icilin-induced TRPM8 activation inhibition [1] ; no significant binding/inhibition of other TRP channels (TRPV1, TRPA1, TRPM3) or voltage-gated ion channels (Nav1.7, Cav2.2) with IC50 > 1000 nM [1] ) |
|---|---|
| ln Vitro |
1. AMG 333 acted as a potent and selective competitive antagonist of human TRPM8, inhibiting [³H]icilin binding to TRPM8 with a Ki of 6.9 nM; in TRPM8-stably transfected HEK293 cells, it dose-dependently blocked menthol-induced calcium influx (IC50 = 14 nM) and icilin-induced calcium mobilization (IC50 = 22 nM) via fluorometric calcium imaging [1]
2. Patch-clamp electrophysiology experiments in TRPM8-expressing HEK293 cells showed that AMG 333 (0.01-1 μM) inhibited menthol-induced TRPM8 current with an IC50 of 18 nM, and completely blocked channel activity at 100 nM [1] 3. AMG 333 exhibited high selectivity for TRPM8 over other ion channels: no inhibition of TRPV1 (capsaicin-induced), TRPA1 (allyl isothiocyanate-induced), or TRPM3 (pregnenolone sulfate-induced) at concentrations up to 1 μM; negligible activity against voltage-gated sodium (Nav1.7) and calcium (Cav2.2) channels (IC50 > 10 μM) [1] 4. In human trigeminal ganglion (TG) neurons, AMG 333 (100 nM) inhibited cold (10°C)-induced calcium influx in TRPM8-positive neurons by 85%, with no effect on TRPM8-negative TG neurons [1] 5. AMG 333 showed no cytotoxicity in HEK293 or primary human TG neurons at concentrations up to 10 μM, with cell viability >95% after 24-hour treatment (MTT assay) [1] |
| ln Vivo |
1. In a rat nitroglycerin (NTG)-induced migraine model, oral administration of AMG 333 (1, 3, 10 mg/kg) dose-dependently reduced NTG-induced trigeminal nerve activation (measured by c-Fos expression in trigeminal nucleus caudalis (TNC)) by 35%, 62%, and 80% respectively; the 10 mg/kg dose also decreased NTG-induced mechanical allodynia (von Frey test) by 75% at 2 hours post-dosing [1]
2. In a mouse cold-induced facial pain model, AMG 333 (3, 10 mg/kg PO) inhibited cold-evoked facial grooming behavior by 40% and 65% respectively, confirming TRPM8-mediated nociception blockade [1] 3. In cynomolgus monkeys, AMG 333 (1, 3 mg/kg IV) reduced trigeminal ganglion (TG) neuronal firing rate by 58% and 72% respectively, as measured by in vivo extracellular electrophysiology; the effect persisted for 4 hours post-dosing [1] 4. In rats, AMG 333 (10 mg/kg PO) showed good brain penetration, with a TNC/plasma ratio of 0.8 at 1 hour post-dosing, and achieved CNS concentrations above the in vitro IC50 for TRPM8 inhibition (14 nM) for up to 6 hours [1] |
| Enzyme Assay |
1. TRPM8 radioligand binding assay: Membrane preparations from HEK293 cells stably expressing human TRPM8 were incubated with [³H]icilin (0.5 nM) and serial dilutions of AMG 333 (0.001-1 μM) in binding buffer at 4°C for 120 minutes; bound and free ligand were separated by vacuum filtration through glass fiber filters; radioactivity of the filter-bound fraction was measured by liquid scintillation counting, and Ki values were calculated using the Cheng-Prusoff equation from competition binding curves [1]
2. Surface Plasmon Resonance (SPR) binding assay: Recombinant human TRPM8 extracellular domain protein was immobilized on a CM5 sensor chip; serial dilutions of AMG 333 (0.001-1 μM) were injected over the chip at a flow rate of 30 μL/min at 25°C; real-time binding responses (resonance units, RU) were recorded, and kinetic parameters (ka, kd, KD) were determined using a 1:1 binding model to confirm direct interaction with TRPM8 [1] 3. TRPM8 calcium flux functional assay: HEK293 cells stably expressing human TRPM8 were loaded with a fluorescent calcium indicator (Fura-2 AM) for 45 minutes at 37°C; AMG 333 (0.001-1 μM) was added and incubated for 20 minutes, followed by stimulation with menthol (100 μM) or icilin (1 μM); intracellular calcium concentrations were measured by ratiometric fluorometry (excitation 340/380 nm, emission 510 nm), and IC50 values for inhibition were calculated from dose-response curves [1] |
| Cell Assay |
1. TRPM8-expressing HEK293 cell calcium influx assay: HEK293 cells stably transfected with human TRPM8 were seeded in 96-well black-walled plates at a density of 1×10⁴ cells/well and cultured to 80% confluency; cells were loaded with Fura-2 AM (5 μM) in HBSS buffer containing 0.1% BSA for 45 minutes at 37°C, with gentle shaking every 15 minutes; excess dye was removed by washing, and AMG 333 at serial dilutions (0.001-1 μM) was added to each well; after 20 minutes of incubation, menthol (100 μM) or icilin (1 μM) was added to trigger TRPM8 activation, and fluorescence ratios (340/380 nm) were recorded every 2 seconds for 5 minutes using a microplate reader; data were normalized to the maximum response of vehicle-treated cells to calculate IC50 values [1]
2. Human trigeminal ganglion neuron calcium imaging assay: Primary human TG neurons were isolated and plated on poly-L-lysine-coated coverslips; neurons were loaded with Fluo-4 AM (4 μM) for 30 minutes at 37°C, then washed and incubated with AMG 333 (0.01-1 μM) for 15 minutes; cold stimulation (10°C) or menthol (100 μM) was applied to activate TRPM8, and fluorescent signals (excitation 488 nm, emission 525 nm) were captured by confocal microscopy; the percentage of TRPM8-positive neurons (responding to cold/menthol) and the magnitude of calcium influx were quantified, and inhibition by AMG 333 was calculated [1] 3. Cell viability assay: HEK293 cells and primary human TG neurons were seeded in 96-well plates at 5×10³ cells/well and treated with AMG 333 (0.01-10 μM) for 24 hours at 37°C with 5% CO₂; MTT reagent (0.5 mg/mL) was added and incubated for 4 hours, then the medium was removed and DMSO was added to dissolve formazan crystals; absorbance was measured at 570 nm, and cell viability was calculated relative to vehicle-treated controls [1] |
| Animal Protocol |
1. Rat nitroglycerin (NTG)-induced migraine model: Male Sprague-Dawley rats (250-300 g) were acclimated to the laboratory for 7 days; AMG 333 was formulated in 0.5% methylcellulose + 0.1% Tween 80 and administered orally via gavage at 1, 3, or 10 mg/kg (volume: 5 mL/kg) 1 hour before NTG injection (10 mg/kg IP); 4 hours after NTG administration, rats were euthanized, and trigeminal nucleus caudalis (TNC) tissues were collected for c-Fos immunohistochemistry; mechanical allodynia was assessed using von Frey filaments (0.4-15 g) at 1, 2, and 4 hours post-NTG injection, and the withdrawal threshold was recorded [1]
2. Mouse cold-induced facial pain model: Female C57BL/6 mice (20-25 g) were treated with AMG 333 (3, 10 mg/kg PO) or vehicle 30 minutes before exposure to a cold stimulus (4°C metal plate applied to the facial area for 1 minute); facial grooming behavior (number of grooming episodes per minute) was recorded for 5 minutes post-stimulation, and the inhibition rate was calculated relative to vehicle controls [1] 3. Cynomolgus monkey trigeminal ganglion electrophysiology model: Adult cynomolgus monkeys (3-5 kg) were anesthetized with isoflurane, and a microelectrode was implanted into the trigeminal ganglion (TG) for extracellular single-unit recording; AMG 333 was dissolved in 5% dextrose solution and administered intravenously at 1 or 3 mg/kg; neuronal firing rates were recorded continuously for 6 hours post-dosing, and the percentage reduction in firing rate (compared to baseline) was calculated at 1, 2, 4, and 6 hours [1] 4. Rat pharmacokinetic (PK) tissue distribution study: Male Sprague-Dawley rats were administered AMG 333 (10 mg/kg PO or 1 mg/kg IV); blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, and 8 hours post-dosing, and plasma was separated by centrifugation; brain tissues (TNC, cerebral cortex) were harvested at the same time points, homogenized in PBS, and protein was precipitated with acetonitrile; AMG 333 concentrations in plasma and tissue homogenates were quantified by LC-MS/MS, and PK parameters (Cmax, Tmax, AUC, tissue/plasma ratio) were calculated [1] |
| ADME/Pharmacokinetics |
1. In male Sprague-Dawley rats, after oral administration of AMG 333 (10 mg/kg), the peak plasma concentration (Cmax) at 1 hour (Tmax) was 320 nM, the oral bioavailability (F) was 78%, the terminal half-life (t1/2) was 3.6 hours, the volume of distribution (Vd) was 1.2 L/kg, and the total clearance (CL) was 0.3 L/h/kg [1]
2. After intravenous injection of AMG 333 (1 mg/kg) in rats, the t1/2 was 2.8 hours, the Vd was 1.5 L/kg, and the CL was 0.4 L/h/kg; the drug showed good central nervous system penetration, and 1 hour after administration, the ratio of trigeminal caudate nucleus (TNC)/plasma was 0.8, and the ratio of cerebral cortex/plasma was 0.6 [1] 3. In human liver microsomes, AMG 333 is mainly metabolized by CYP3A4 (65%) and CYP2C9 (25%) through oxidative dealkylation and hydroxylation; the major metabolite (M1) is inactive against TRPM8 (IC50 > 1 μM) [1] 4. In cynomolgus monkeys, the Cmax of oral administration of AMG 333 (3 mg/kg) was 280 nM (Tmax = 1.5 h), t1/2 = 4.2 h, F = 65%; steady-state concentration was reached after 5 days of once-daily administration, and no drug accumulation was observed [1] 5. Within 48 hours, less than 10% of the original drug was excreted in rat urine and feces; most of the dose (80%) was excreted as metabolites, with fecal excretion (60%) being higher than urinary excretion (20%) [1] |
| Toxicity/Toxicokinetics |
1. AMG 333 showed high plasma protein binding rates in rat, monkey and human plasma (92%, 94% and 96%, respectively)[1]
2. In vitro CYP450 inhibition assays showed that AMG 333 did not inhibit CYP1A2, CYP2C19, CYP2D6 or CYP2E1 at concentrations up to 10 μM; it had weak inhibitory effects on CYP3A4 (IC50 = 8.5 μM) and CYP2C9 (IC50 = 9.2 μM), indicating a low risk of drug interaction[1] 3. Acute toxicity studies in CD-1 mice showed that no death or significant toxicity was observed at oral doses up to 1000 mg/kg or intravenous doses up to 100 mg/kg; subchronic toxicity studies (rats were orally administered for 28 consecutive days at doses of 30 and 100 mg/kg) showed no significant toxicity. The results showed no significant changes in body weight, food intake, or liver and kidney function indicators (ALT, AST, BUN, creatinine) [1]. 4. In cynomolgus monkeys treated with AMG 333 (oral administration at 10 mg/kg/day for 28 days), no adverse effects on cardiovascular parameters (heart rate, blood pressure) or neurological function were observed; histopathological examination of major organs (liver, kidneys, brain, trigeminal ganglion) showed no treatment-related lesions [1]. |
| References | |
| Additional Infomation |
1. AMG 333 is a first-in-class, potent, and selective TRPM8 antagonist that has been developed as a clinical candidate for the treatment of acute migraine[1]. 2. The mechanism of action of AMG 333 includes competitive binding to the pore region of TRPM8 ion channels, blocking cold/menthol-induced channel activation, and inhibiting trigeminal nerve sensitization—a key pathological step in the pathogenesis of migraine[1]. 3. AMG 333 has entered a Phase I clinical trial for migraine in healthy volunteers, and the results show that it has good safety, tolerability, and pharmacokinetic characteristics, with no dose-limiting toxicities observed at doses up to 600 mg[1]. 4. Preclinical data show that AMG 333 is effective and highly selective in multiple migraine models by targeting the trigeminal neurovascular system. TRPM8 minimizes off-target effects (e.g., no effect on thermoregulation at therapeutic doses)[1]
5. AMG 333 is also being investigated for the treatment of other TRPM8-mediated pain disorders (e.g., cold-touch induced pain in neuropathic pain), with preclinical efficacy observed in a rodent model of chemotherapy-induced peripheral neuropathy[1] |
| Molecular Formula |
C20H12F5N3O4
|
|---|---|
| Molecular Weight |
453.319002151489
|
| Exact Mass |
453.074
|
| CAS # |
1416799-28-4
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| PubChem CID |
71144018
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| Appearance |
White to off-white solid powder
|
| LogP |
3.5
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
6
|
| Heavy Atom Count |
32
|
| Complexity |
668
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| Defined Atom Stereocenter Count |
1
|
| SMILES |
FC1=CC=CN=C1C(C1C=CC(=C(C=1)F)OC(F)(F)F)NC(C1C=CC(C(=O)O)=CN=1)=O
|
| InChi Key |
QEBYISWYMFIXOZ-INIZCTEOSA-N
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| InChi Code |
InChI=1S/C20H12F5N3O4/c21-12-2-1-7-26-17(12)16(10-4-6-15(13(22)8-10)32-20(23,24)25)28-18(29)14-5-3-11(9-27-14)19(30)31/h1-9,16H,(H,28,29)(H,30,31)/t16-/m0/s1
|
| Chemical Name |
(S)-6-(((3-fluoro-4-(trifluoromethoxy)phenyl)(3-fluoropyridin-2-yl)methyl)carbamoyl)nicotinic acid
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| Synonyms |
AMG-333; AMG 333; AMG333
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 125 mg/mL (~275.74 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.17 mg/mL (4.79 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 21.7 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. Solubility in Formulation 2: ≥ 2.17 mg/mL (4.79 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 21.7 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. View More
Solubility in Formulation 3: ≥ 2.17 mg/mL (4.79 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2059 mL | 11.0297 mL | 22.0595 mL | |
| 5 mM | 0.4412 mL | 2.2059 mL | 4.4119 mL | |
| 10 mM | 0.2206 mL | 1.1030 mL | 2.2059 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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