CFTR (EC50: 0.1μM)

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].

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.

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.

Oral administration; 600 mg once daily or 400 mg every 12 hours

Patients with Cystic Fibrosis Homozygous

Absorption, Distribution and Excretion
When a single dose of lumacaftor/ivacaftor was administered with fat-containing foods, lumacaftor exposure was approximately 2 times higher than when taken in a fasting state. Following multiple oral dose administrations of lumacaftor in combination with ivacaftor, the exposure of lumacaftor generally increased proportionally to doses over the range of 200 mg every 24 hours to 400 mg every 12 hours. The median (range) tmax of lumacaftor is approximately 4.0 hours (2.0; 9.0) in the fed state.
Following oral administration of lumacaftor, the majority of lumacaftor (51%) is excreted unchanged in the feces. There was minimal elimination of lumacaftor and its metabolites in urine (only 8.6% of total radioactivity was recovered in the urine with 0.18% as the unchanged parent drug).
Following oral administration of 200 mg of lumacaftor every 24 hours to cystic fibrosis patients in a fed state for 28 days, the mean (+/-SD) for apparent volumes of distribution was 86.0 (69.8) L.
The typical apparent clearance, CL/F (CV), of lumacaftor was estimated to be 2.38 L/hr.

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Metabolism / Metabolites
Lumacaftor is mostly excreted unchanged in the feces and is not extensively metabolized. When metabolism does occur, oxidation and glucuronidation are the main processes involved.

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Biological Half-Life
The half-life of lumacaftor is approximately 26 hours in patients with cystic fibrosis.

Protein Binding
Lumacaftor is extensively protein bound in the plasma (99%), and binds primarily to 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 obtained by formal condensation of the carboxy group of 1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropane-1-carboxylic acid with the aromatic amino group of 3-(6-amino-3-methylpyridin-2-yl)benzoic acid. Used for the treatment of cystic fibrosis. It has a role as a CFTR potentiator and an orphan drug. It is a member of benzoic acids, a member of pyridines, an aromatic amide, a member of cyclopropanes, a member of benzodioxoles and an organofluorine compound.
Lumacaftor is a drug used in combination with [DB08820] as the fixed dose combination product Orkambi for the management of Cystic Fibrosis (CF) in patients aged 6 years and older. Cystic Fibrosis is an autosomal recessive disorder caused by one of several different mutations in the gene for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, a transmembrane ion channel involved in the transport of chloride and sodium ions across cell membranes of the lungs, pancreas, and other organs. Mutations in the CFTR gene result in altered production, misfolding, or function of the CFTR protein and consequently abnormal fluid and ion transport across cell membranes. As a result, CF patients produce thick, sticky mucus that clogs the ducts of organs where it is produced making patients more susceptible to infections, lung damage, pancreatic insufficiency, and malnutrition. Lumacaftor improves CF symptoms and underlying disease pathology by aiding the conformational stability of F508del-mutated CFTR proteins, preventing misfolding and resulting in increased processing and trafficking of mature protein to the cell surface. Results from clinical trials indicated that treatment with Orkambi (lumacaftor/ivacaftor) results in improved lung function, reduced chance of experiencing a pulmonary exacerbation, increased weight gain, and improvements in CF symptoms. This data has been heavily scrutinized, however, with clinical trials showing only modest improvements despite a hefty yearly cost of $259,000 for Orkambi. Improvements in lung function (ppFEV1) were found to be statistically significant, but minimal, with only a 2.6-3.0% change from baseline with more than 70% of patients failing to achieve an absolute improvement of at least 5%. A wide variety of CFTR mutations correlate to the Cystic Fibrosis phenotype and are associated with differing levels of disease severity. The most common mutation, affecting approximately 70% of patients with CF worldwide, is known as F508del-CFTR, or delta-F508 (ΔF508), in which a deletion in the amino acid phenylalanine at position 508 results in impaired production of the CFTR protein, thereby causing a significant reduction in the amount of ion transporter present on cell membranes. When used in combination with [DB08820] as the fixed dose combination product Orkambi, lumacaftor is specific for the management of CF in patients with delta-F508 mutations as it acts as a protein-folding chaperone, aiding the conformational stability of the mutated CFTR protein. Consequently, lumacaftor increases successful production of CFTR ion channels and the total number of receptors available for use at the cell membrane for fluid and ion transport. The next most common mutation, G551D, affecting 4-5% of CF patients worldwide, is characterized as a missense mutation, whereby there is sufficient amount of protein at the cell surface, but opening and closing mechanisms of the channel are altered. Treatment of patients with G551D and other rarer missense mutations is usually managed with [DB08820] (Kalydeco), as it aids with altered gating mechanisms by potentiating channel opening probability of CFTR protein. Prior to the development of lumacaftor and [DB08820] (Kalydeco), management of CF primarily involved therapies for the control of infections, nutritional support, clearance of mucus, and management of symptoms rather than improvements in the underlying disease process. Approved for use by the Food and Drug Administration in July 2015 and by Health Canada in January 2016, Orkambi was the first combination product approved for the management of Cystic Fibrosis with delta-F508 mutations. Ivacaftor is manufactured and distributed by Vertex Pharmaceuticals.
The mechanism of action of lumacaftor is as a Cytochrome P450 3A Inducer, and Cytochrome P450 2B6 Inducer, and Cytochrome P450 2C8 Inducer, and Cytochrome P450 2C9 Inducer, and Cytochrome P450 2C19 Inducer, and Cytochrome P450 2C8 Inhibitor, and Cytochrome P450 2C9 Inhibitor, and P-Glycoprotein Inducer, and P-Glycoprotein Inhibitor.
See also: Ivacaftor; lumacaftor (component of).

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Drug Indication
When used in combination with the drug [lumacaftor] as the product Orkambi, ivacaftor is indicated for the management of CF in patients aged one year and older who are homozygous for the _F508del_ mutation in the CFTR gene. If the patient’s genotype is unknown, an FDA-cleared CF mutation test should be used to detect the presence of the _F508del_ mutation on both alleles of the CFTR gene.
FDA Label
Treatment of cystic fibrosis

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Mechanism of Action
The CFTR protein is a chloride channel present at the surface of epithelial cells in multiple organs. The F508del mutation results in protein misfolding, causing a defect in cellular processing and trafficking that targets the protein for degradation and therefore reduces the quantity of CFTR at the cell surface. The small amount of F508del-CFTR that reaches the cell surface is less stable and has a low channel-open probability (defective gating activity) compared to wild-type CFTR protein. Lumacaftor improves the conformational stability of F508del-CFTR, resulting in increased processing and trafficking of mature protein to the cell surface. In vitro studies have demonstrated that lumacaftor acts directly on the CFTR protein in primary human bronchial epithelial cultures and other cell lines harboring the F508del-CFTR mutation to increase the quantity, stability, and function of F508del-CFTR at the cell surface, resulting in increased chloride ion transport. In vitro responses do not necessarily correspond to in vivo pharmacodynamic responses or clinical benefits.

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.)