CFTR (EC50: 0.1μM)
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), specifically F508del mutant CFTR [1][2]

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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In CFBE41o- cells (human bronchial epithelial cells expressing F508del-CFTR) and primary human cystic fibrosis (CF) airway epithelial cells, Lumacaftor (VX-809; VRT 826809) (1-10 μM) dose-dependently corrected the processing defect of F508del-CFTR. At 3 μM, it increased mature (band C) CFTR protein expression by 2.5-3.0-fold compared to vehicle control (Western blot). It also enhanced CFTR-mediated chloride transport by 2.0-2.2-fold as measured by Ussing chamber assay[1]
- In Calu-3 cells (human lung epithelial cells with endogenous F508del-CFTR), Lumacaftor (VX-809; VRT 826809) (3 μM) combined with other CFTR correctors (e.g., corr-4a) exhibited synergistic effects. The combination augmented maximal chloride current by 40-50% compared to Lumacaftor (VX-809; VRT 826809) alone, without increasing cellular toxicity[2]
- Lumacaftor (VX-809; VRT 826809) (0.5-10 μM) promoted the folding and trafficking of F508del-CFTR to the cell membrane, as confirmed by immunofluorescence staining showing increased membrane-localized CFTR protein. The effect was saturable at concentrations ≥5 μM[1]

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].

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)]

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).

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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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F508del-CFTR processing correction assay: CFBE41o- cells or primary human CF airway epithelial cells were seeded in 6-well plates or permeable supports. Lumacaftor (VX-809; VRT 826809) (1 μM, 3 μM, 10 μM) was added to the culture medium, and cells were incubated for 48-72 hours. Cell lysates were prepared for Western blot analysis to quantify mature (band C) and immature (band B) CFTR protein levels. Transepithelial chloride current was measured by Ussing chamber to assess functional correction[1]
- Synergy assay with CFTR correctors: Calu-3 cells were seeded in 96-well plates or Ussing chamber supports. Lumacaftor (VX-809; VRT 826809) (3 μM) was combined with gradient concentrations of other correctors (e.g., corr-4a) and incubated for 72 hours. Chloride transport activity was detected by fluorescent dye quenching assay or Ussing chamber to evaluate synergistic efficacy. Cell viability was measured by MTT assay to rule out toxicity[2]
- CFTR membrane localization assay: CFBE41o- cells were seeded on glass coverslips, treated with Lumacaftor (VX-809; VRT 826809) (3 μM, 5 μM) for 48 hours. Immunofluorescence staining was performed using anti-CFTR antibody, and confocal microscopy was used to visualize and quantify membrane-localized CFTR protein[1]

Oral administration; 600 mg once daily or 400 mg every 12 hours
Patients with Cystic Fibrosis Homozygous
Male rats (n=3 per dose group) are orally administered Lumacaftor in a vehicle consisting of 0.5% Tween80/0.5% methylcellulose/water at a dose volume of 5 mL/kg. The concentration of Lumacaftor in plasma samples is determined with a liquid chromatography/tandem MS method. Pharmacokinetic parameters are calculated byusing WinNonlin Professional Edition software.

Absorption, Distribution and Excretion
When a single dose of lumacaftor/ivacaftor is taken with fatty foods, lumacaftor exposure is approximately twice that of an empty stomach. Following multiple oral doses of lumacaftor in combination with ivacaftor, lumacaftor exposure generally increases in a dose-dependent manner, ranging from 200 mg per 24 hours to 400 mg per 12 hours. The median (range) time to peak (tmax) of lumacaftor in the postprandial state is approximately 4.0 hours (2.0; 9.0). Following oral administration of lumacaftor, the majority (51%) is excreted unchanged in feces. Urinary clearance of lumacaftor and its metabolites is minimal (only 8.6% of the total radioactivity is recovered from urine, of which 0.18% is the original drug).
In patients with cystic fibrosis who were eating, the mean (± standard deviation) apparent volume of distribution after oral administration of 200 mg lumacaftor every 24 hours for 28 days was 86.0 (69.8) L.
The typical apparent clearance (CL/F) of lumacaftor was estimated to be 2.38 L/hr.
Metabolism/Metabolites
Lumacaftor is primarily excreted unchanged in the feces and is poorly metabolized. When metabolism does occur, oxidation and glucuronidation are the main processes.
Biological Half-Life
In patients with cystic fibrosis, the half-life of lumacaftor is approximately 26 hours.

Protein Binding
Lumacaftor binds extensively to proteins (99%) in plasma, primarily albumin.

[1]. 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]. 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.

Lumacaftor is an aromatic amide formed by the condensation of the carboxyl group of 1-(2,2-difluoro-1,3-benzodioxane-5-yl)cyclopropane-1-carboxylic acid with the aromatic amino group of 3-(6-amino-3-methylpyridin-2-yl)benzoic acid. It is used to treat cystic fibrosis. It is a CFTR enhancer and an orphan drug. It belongs to the benzoic acid, pyridine, aromatic amide, cyclopropane, benzodioxane, and organofluorine compounds classes. Lumacaftor, in combination with [DB08820], forms the fixed-dose combination preparation Orkambi for the treatment of cystic fibrosis (CF) in patients aged 6 years and older. Cystic fibrosis is an autosomal recessive genetic disorder caused by various mutations in the cystic fibrosis transmembrane transport regulator (CFTR) gene. The CFTR protein is a transmembrane ion channel involved in the transport of chloride and sodium ions across the cell membranes of the lungs, pancreas, and other organs. Mutations in the CFTR gene can lead to altered production, misfolding, or function of the CFTR protein, resulting in abnormal fluid and ion transport across the cell membrane. Consequently, cystic fibrosis patients produce thick mucus that can clog the ducts of the organs that produce mucus, making them more susceptible to infections, lung damage, pancreatic insufficiency, and malnutrition. Lumacaftor improves the symptoms and underlying pathological mechanisms of cystic fibrosis by enhancing the conformational stability of the F508del mutant CFTR protein, preventing its misfolding, and promoting the processing and transport of mature proteins to the cell surface. Clinical trial results indicate that treatment with Orkambi (lumacaftor/ivacaftor) improves lung function, reduces the risk of acute lung exacerbations, increases weight, and improves cystic fibrosis symptoms. However, these data have been rigorously reviewed, and clinical trials have shown that despite Orkambi's high annual cost of $259,000, its efficacy is quite limited. Improvements in lung function (ppFEV1) were statistically significant, but small, increasing only 2.6% to 3.0% from baseline, with over 70% of patients failing to achieve at least a 5% absolute improvement. Multiple CFTR gene mutations are associated with the cystic fibrosis phenotype and vary with disease severity. The most common mutation is F508del-CFTR, or delta-F508 (ΔF508), affecting approximately 70% of cystic fibrosis (CF) patients worldwide. This mutation results in the loss of phenylalanine at position 508, impairing CFTR protein production and significantly reducing the number of ion transporters on the cell membrane. Lumacaftor, in combination with [DB08820], forms the fixed-dose combination Orkambi, specifically for treating CF patients carrying the delta-F508 mutation. Lumacaftor, as a protein folding chaperone, helps maintain the conformational stability of the mutant CFTR protein. Therefore, lumacaftor can improve the generation efficiency of CFTR ion channels and increase the total number of receptors on the cell membrane available for liquid and ion transport. The second most common mutation is G551D, affecting 4-5% of cystic fibrosis (CF) patients worldwide. This mutation is a missense mutation, with sufficient cell surface protein but altered channel opening and closing mechanisms. For patients carrying G551D and other rare missense mutations, [DB08820] (Kalydeco) is often used for treatment because it enhances the probability of CFTR protein channel opening, thereby improving the abnormal gating mechanism. Before the advent of lumacaftor and [DB08820] (Kalydeco), CF treatment mainly focused on controlling infection, nutritional support, clearing mucus, and relieving symptoms, rather than improving the disease itself. Orkambi was approved by the U.S. Food and Drug Administration (FDA) in July 2015 and by Health Canada in January 2016, becoming the first approved combination therapy for the treatment of cystic fibrosis carrying the delta-F508 mutation. Ivacaftor is manufactured and marketed by Vertex Pharmaceuticals. Ivacaftor's mechanism of action is as a cytochrome P450 3A inducer, cytochrome P450 2B6 inducer, cytochrome P450 2C8 inducer, cytochrome P450 2C9 inducer, cytochrome P450 2C19 inducer, cytochrome P450 2C8 inhibitor, cytochrome P450 2C9 inhibitor, P-glycoprotein inducer, and P-glycoprotein inhibitor. See also: Ivacaftor; lumacaftor (ingredient).

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Drug Indications
When used in combination with the drug [lumacaftor] as a product of Orkambi, ivacaftor is indicated for the treatment of cystic fibrosis (CF) in patients aged 1 year and older who are homozygous for the _F508del_ mutation in the CFTR gene. If the patient's genotype is unknown, an FDA-approved CF mutation assay should be used to detect the presence of the _F508del_ mutation in both alleles of the CFTR gene.
FDA Label
Treatment of Cystic Fibrosis

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Mechanism of Action
CFTR protein is a chloride ion channel present on the surface of epithelial cells in various organs. The F508del mutation causes protein misfolding, resulting in defects in cellular processing and transport, leading to protein degradation and a reduction in the amount of CFTR on the cell surface. Compared to wild-type CFTR protein, the small amount of F508del-CFTR protein reaching the cell surface is less stable and has a lower probability of channel opening (gating activity defect). Lumacaftor can improve the conformational stability of F508del-CFTR protein, thereby promoting the processing and transport of mature protein to the cell surface. In vitro studies have shown that in primary human bronchial epithelial cell cultures and other cell lines carrying the F508del-CFTR mutation, lumacaftor can directly act on the CFTR protein, increasing the quantity, stability, and function of F508del-CFTR protein on the cell surface, thereby enhancing chloride ion transport. In vitro responses do not necessarily correlate with in vivo pharmacodynamic responses or clinical benefits. Lumacaftor (VX-809; VRT 826809) is an investigational CFTR corrector for the treatment of cystic fibrosis (CF) patients carrying the F508del-CFTR mutation [1][2]. Its core mechanism involves improving the folding, transport, and maturation of F508del-CFTR, which is normally degraded in the endoplasmic reticulum, thereby promoting its delivery to the cell membrane [1]. It has a synergistic effect with other CFTR correctors, enhancing functional correction beyond monotherapy [2]. This drug specifically targets F508del-CFTR and has no significant effect on the expression or function of wild-type CFTR protein [1].

C24H18F2N2O5

452.41

452.118

C, 63.72; H, 4.01; F, 8.40; N, 6.19; O, 17.68

936727-05-8

Lumacaftor-d4;2733561-44-7

16678941

White to off-white solid

1.5±0.1 g/cm3

653.0±55.0 °C at 760 mmHg

348.7±31.5 °C

0.0±2.1 mmHg at 25°C

1.670

5.77

2

8

5

33

776

0

O=C(C1C=C(C2C(C)=CC=C(NC(C3(CC3)C3C=C4C(OC(O4)(F)F)=CC=3)=O)N=2)C=CC=1)O

UFSKUSARDNFIRC-UHFFFAOYSA-N

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)

3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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: ≥ 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.

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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.

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Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30 mg/mL

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