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Purity: ≥98%
Lumacaftor (formerly VX809; VRT-826809; VRT826809; VX-809; Orkambi) is a potent and orally bioavailable CFTR (cystic fibrosis transmembrane conductance regulator) corrector with antifibrotic effects. It acts by correcting CFTR mutations common in cystic fibrosis by increasing mutant CFTR (F508del-CFTR) maturation, with EC50 of 0.1 μM. VX-809 may be useful for treating patients with cystic fibrosis who have a phe508del CFTR mutation. Cystic fibrosis (CF) is a genetic disorder that causes multiorgan morbidity and premature death, most commonly from pulmonary dysfunction.
| Targets |
CFTR (EC50: 0.1μM)
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| ln Vitro |
In fischer rat thyroid (FRT) cells, Lumacaftor improves F508del-CFTR maturation by 7.1±0.3 fold (n=3) compared with vehicle-treated cells (EC50, 0.1±0.1 μM; n=3) and enhances F508del-CFTR-mediated chloride transport by nearly fivefold (EC50, 0.5±0.1 μM; n=3). At Lumacaftor doses larger than 10 μM, the reaction is diminished, resulting in a bell-shaped dose-response relationship with an IC50 of around 100 μM. Lumacaftor is orally accessible in rats and achieved in vivo plasma levels much beyond quantities required for in vitro efficacy[1]. Lumacaftor exhibits a concentration-dependent rise in the HRP luminescence signal after incubation with cells at 37°C or 27°C in both cells lines, with a similar EC50 value of around 0.3 µM. In F508-HRP CFBE41o- cells at 37°C, Lumacaftor enhances the signal maximally to around 250 luminescence arbitrary units (au) over the DMSO control baseline of approximately 60 au, reflecting an approximately 4-fold signal increase. Similarly, with the R1070W-HRP CFBE41o- cells, Lumacaftor enhances the signal maximally to around 220 au over the DMSO control baseline of roughly 85 au, suggesting an approximately 2.5-fold signal increase. Therefore, both cells lines give robust signals with a good dynamic range for high-throughput screening[2].
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| ln Vivo |
In male Sprague-Dawley rats, oral administration of 1 mg/kg Lumacaftor yields a Cmax of 2.4±1.3 μM and a t1/2 of 7.7±0.4 h (mean±SD; n=3). These data suggest that Lumacaftor is orally accessible and can achieve plasma levels that greatly above EC50s for F508del-CFTR correction[1].
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| Enzyme Assay |
Screening Procedures [2].
Screening was carried out using a Beckman Coulter (Fullerton, CA) Biomek FX platform. In one set of assays, R1070W-∆F508-CFTR-HRP (R1070W-HRP)–expressing CFBE41o− cells were incubated with 100 µl medium containing 25 µM test compounds and 0.5 μg/ml doxycycline for 24 hours at 37°C. In a second set of assays, ∆F508-CFTR-HRP (∆F508-HRP)–expressing CFBE41o− cells were incubated with 100 µl medium containing 25 µM test compounds, 2 µM VX-809, and 0.5 μg/ml doxycycline for 24 hours at 37°C. All compound plates contained negative controls [dimethylsulfoxide (DMSO) vehicle] and positive controls [2 µM VX-809]. In both assays, the cells were washed four times with phosphate-buffered saline (PBS), and HRP activity was assayed by the addition of 50 µl/well of HRP substrate (WesternBright Sirius Kit; Advansta Corp, Menlo Park, CA). After shaking for 5 minutes, chemiluminescence was measured using a Tecan Infinite M1000 plate reader (Tecan Groups Ltd, Mannedorf, Switzerland) equipped with an automated stacker (integration time, 100 milliseconds). Z′ is defined as = 1 − [(3 × standard deviation of maximum signal control + 3 × standard deviation of minimum signal control)/absolute (mean of maximum signal control − mean of minimum signal control)] |
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| Cell Assay |
Functional Assays.[2]
A549 cells expressing ∆F508-CFTR YFP were grown at 37°C/5% CO2 for 18–24 hours after plating. The cells were then incubated with 100 μl of medium containing test compounds for 18–24 hours. At the time of the assay, cells were washed with PBS and then incubated for 10 minutes with PBS containing forskolin (20 μM) and genistein (50 μM). Each well was assayed individually for I– influx by recording fluorescence continuously (200 milliseconds per point) for 2 seconds (baseline) and then for 12 seconds after rapid addition of 165 μl PBS in which 137 mM Cl– was replaced by I–. The initial I– influx rate was computed by fitting the final 11.5 seconds of the data to an exponential for extrapolation of initial slope, which was normalized for background-subtracted initial fluorescence. All compound plates contained negative controls (DMSO vehicle) and positive controls (5 µM VX-809). Fluorescence was measured using a Tecan Infinite M1000 plate reader equipped with a dual syringe pump (excitation/emission 500/535 nm). Short-Circuit Current Measurements.[2] Test compounds (without or with 10 μM VX-809) were incubated with primary human CF bronchial epithelial cells from ΔF508-CFTR–homozygous subjects at the basolateral side for 18–24 hours at 37°C prior to measurements. The apical and basolateral chambers contained identical solutions as follows: 130 mM NaCl, 0.38 mM KH2PO4, 2.1 mM K2HPO4, 1 mM MgCl2, 1 mM CaCl2, 25 mM NaHCO3, and 10 mM glucose. Solutions were bubbled with 5% CO2/95% O2 and maintained at 37°C. Hemichambers were connected to a DVC-1000 voltage clamp (World Precision Instruments Inc., Sarasota, FL) via Ag/AgCl electrodes and 1 M KCl agar bridges for recording of short-circuit current. |
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| Animal Protocol |
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| References |
[1]. Van Goor F, et al. Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18843-8.
[2]. Phuan PW, et al. Synergy-based small-molecule screen using a human lung epithelial cell line yields ΔF508-CFTR correctors that augment VX-809 maximal efficacy. Mol Pharmacol. 2014 Jul;86(1):42-51 |
| Molecular Formula |
C24H18F2N2O5
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| Molecular Weight |
452.41
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| Exact Mass |
52.118378
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| Elemental Analysis |
C, 63.72; H, 4.01; F, 8.40; N, 6.19; O, 17.68
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| CAS # |
936727-05-8
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| Related CAS # |
Lumacaftor-d4;2733561-44-7
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| Appearance |
White to off-white solid
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| LogP |
4.4
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| tPSA |
97.8Ų
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| SMILES |
FC1(OC2C([H])=C([H])C(=C([H])C=2O1)C1(C(N([H])C2C([H])=C([H])C(C([H])([H])[H])=C(C3C([H])=C([H])C([H])=C(C(=O)O[H])C=3[H])N=2)=O)C([H])([H])C1([H])[H])F
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| InChi Key |
UFSKUSARDNFIRC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H18F2N2O5/c1-13-5-8-19(27-20(13)14-3-2-4-15(11-14)21(29)30)28-22(31)23(9-10-23)16-6-7-17-18(12-16)33-24(25,26)32-17/h2-8,11-12H,9-10H2,1H3,(H,29,30)(H,27,28,31)
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| Chemical Name |
3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3 mg/mL (6.63 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 30.0 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. Solubility in Formulation 2: ≥ 3 mg/mL (6.63 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. View More
Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30 mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2104 mL | 11.0519 mL | 22.1038 mL | |
| 5 mM | 0.4421 mL | 2.2104 mL | 4.4208 mL | |
| 10 mM | 0.2210 mL | 1.1052 mL | 2.2104 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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