CFTR/cystic fibrosis transmembrane conductance regulator

Galicaftor (ABBV-2222; GLPG-2222)has an EC50 <10 nM and strong in vitro functional activity in primary patient cells expressing F508del/F508del CFTR[2].
ABBV-2222 Promotes Maturation of F508delCFTR as Assessed by Western Blot. ABBV-2222 Stabilizes the Nucleotide Binding Domain 1/Membrane-Spanning Domain Interface. ABBV-2222 Corrects F508delCFTR through Action on Membrane-Spanning Domain 1. [2]

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Compound 22 [Galicaftor (ABBV-2222; GLPG-2222)] was highly potent (5 nM) and efficacious in cells from multiple CF patient donors who have F508del homozygous mutation. In comparison with Lumacaftor, 22 was significantly more potent (>25-fold) and exhibited comparable efficacy. It demonstrated low clearance across multiple preclinical species, did not inhibit CYP enzymes, was not a CYP3A4 inducer, and therefore presented a low DDI perpetrator liability[1].

Galicaftor (ABBV-2222; GLPG-2222; 1 mg/kg, iv; 1 mg/kg, po) is used in the rat pharmacokinetic experiments to demonstrate its pharmacokinetic characteristics. T1/2 is equal to 2.7 hours (iv). Additionally, the bioavailability (%F) for intragastric injection is 74%[1].

Patch-Clamp Electrophysiological Assay.[2]
To examine the open probability (Po) of F508delCFTR after pretreatment with Galicaftor (ABBV-2222; GLPG-2222), CHO cells transiently transfected with CFTR-cDNA [pcDNA 3.1 Zeo (+) vector; Invitrogen] and GFP encoding pEGFP-C3 were incubated with Galicaftor (ABBV-2222; GLPG-2222) overnight before patch-clamp experiments. To avoid repetition, detailed experimental methods, materials, and data analysis can be found in our latest publication (Yeh et al., 2019).

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Trans-epithelial Current Clamp on Human Bronchial Epithelial Cells Conductance Assay[1]
A cell based assay using the primary human bronchial epithelial cells (hBE) was used as a secondary assay to test novel F508del CFTR correctors for their activity on primary hBE cells with F508del/F508del CFTR mutation. Primary human bronchial epithelial (hBE) cells from F508del/F508del CFTR patients were expanded from 1 × 106 to 250 × 106 cells. For this purpose, cells isolated from CF patients with the homozygous mutation were seeded onto 24 well Corning filter plates that were coated with 3T3 conditioned media and grown at an air–liquid interface for 35 days using an Ultroser G supplemented differentiation media. Apical surface mucus was removed 72 h before the experiment by incubating the apical surface of the cells for 30 min with 3 mM dithiothreitol (DTT) prepared in the differentiation media, followed by aspiration of the mucus along with the media. The apical surface is washed again with phosphate buffered saline (PBS) incubated for 30 min followed with aspiration. The cells were then incubated with the desired dose of the corrector compounds 18–24 h at 37 °C, 5% CO2. The corrector compounds were prepared as 10 mM stocks, and the desired concentrations were prepared in differentiation media and were always applied on the basolateral side of the epithelial cells.

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CYP3A4 Induction: [1]
Cryopreserved primary human hepatocytes were thawed and cultured overnight prior to treatment. Cultured hepatocytes were treated with either test compounds (10 µM), vehicle control (0.1% v/v DMSO), or prototypical inducer of CYP3A4 (rifampin 10 µM) for 48 hours, with culture medium being refreshed every 24 hours. Following the 48 hour treatment, CYP3A4 mRNA levels measured in compound treated hepatocytes were expressed as a percentage of the response of positive control (Rifampin 10 µM). In test compound treated hepatocytes, CYP3A4 mRNA level increase by less than 20% of the response of positive control (Rifampin) is considered low risk for CYP3A4 induction.

Western Blot Analysis of F508delCFTR Band C/B in Baby Hamster Kidney Cells Expressing Suppressor Mutations.[2]
Briefly, the BHK suppressor mutant cells were seeded at 1 × 106 cells per well onto six-well dishes overnight and then treated with either 0.2% DMSO or 1 µM Galicaftor (ABBV-2222; GLPG-2222) for 18–24 hours at 37°C in a 5% CO2 humidified incubator. Just prior to lysis, cell monolayers were rinsed twice with cold PBS to remove serum and medium.

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Cell Surface Expression-Horse Radish Peroxidase (CSE-HRP) Assay[1]
A cellular assay for measuring the F508del CFTR cell surface expression after correction with test compounds was developed in the human lung derived epithelial cell line (CFBE41o-). This was achieved by expressing the F508del CFTR mutation along with a horseradish peroxidase (HRP) in the fourth exofacial loop and then measuring the HRP activity using luminescence readout from these cells, CFBE41o-F508del CFTR-HRP, that were incubated overnight with the test corrector compounds. Briefly, for this primary assay, the CFBE41o-F508del CFTR-HRP cells were plated in 384-well plates at 4000 cells/well along with 0.5 μg/mL doxycycline to induce the F508del CFTR-HRP expression and further incubated at 37 °C, 5% CO2 for 72 h. The test compounds were then added at the required concentrations and further incubated for 18–24 h at 33 °C. The highest concentration tested was 20 μM with an 8-point concentration response curve using a 3-fold dilution. Three replicate plates were run to determine one EC50. All plates contained negative controls (dimethyl sulfoxide, DMSO) and positive controls (3 μM of 3-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-methoxy-3,4-dihydro-2H-chromen-2-yl]benzoic acid) (Compound 15) as well as the on-plate concentration response of the positive control. Post-incubation, the plates were washed 5× times with Dulbecco’s phosphate buffered saline (DPBS), followed by the addition of the HRP substrate, luminol (50 μL), and measuring the HRP activity using luminescence readout on EnVision Multilabel Plate Reader (PerkinElmer; product number 2104-0010). The raw counts from the experiment are analyzed using Accelrys Assay Explorer v3.3. The % activity measured at each of the eight test concentrations of the test compound was normalized to the on-plate positive control using the following formula: The maximum % activity achieved for the test compound at any tested concentration is presented in tables along with the EC50 calculated using the general sigmoidal curve with a variable Hill slope equation.

Pharmacokinetic studies were performed using rats.

The pharmacokinetic profile of Galicaftor (ABBV-2222; GLPG-2222) is provided in Table 7. Galicaftor (ABBV-2222; GLPG-2222) is primarily cleared via glucoronidation in human plasma, presenting a low DDI victim liability. Thorough preclinical characterization including DMPK, and safety pharmacology and toxicology of Galicaftor (ABBV-2222; GLPG-2222) in rat and dog support this compound as a candidate for clinical development. In a first-in-human phase I study, Galicaftor (ABBV-2222; GLPG-2222) was given to healthy volunteers at single doses up to 800 mg and multiple doses up to 600 mg daily for 14 days. The drug was well tolerated and had a favorable safety profile, with no early discontinuations of study drug and no severe adverse events.[1]

[1]. Discovery of 4-[(2R,4R)-4-({[1-(2,2-Difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic Acid (ABBV/GLPG-2222), a Potent Cystic Fibrosis Transmembrane Conductance Regulator.

[2]. Biological Characterization of F508delCFTR Protein Processing by the CFTR Corrector ABBV-2222/GLPG2222. J Pharmacol Exp Ther. 2020 Jan;372(1):107-118.

Galicaftor is under investigation in clinical trial NCT03540524 (A Study Looking at the Safety, Tolerability and Efficacy of the Combination of the Study Drugs GLPG2451 and GLPG2222 With or Without GLPG2737 in Patients With Cystic Fibrosis.).

C28H21F4NO7

559.462462186813

559.125

C, 60.11; H, 3.78; F, 13.58; N, 2.50; O, 20.02

1918143-53-9

121301049

White to off-white solid powder

1.6±0.1 g/cm3

701.1±60.0 °C at 760 mmHg

377.8±32.9 °C

0.0±2.3 mmHg at 25°C

1.640

6.05

2

11

7

40

957

2

FC1(OC2=CC=C(C=C2O1)C1(C(N[C@H]2C3C=CC(=CC=3O[C@@H](C3C=CC(C(=O)O)=CC=3)C2)OC(F)F)=O)CC1)F

QVDYQHXNAQHIKH-TZIWHRDSSA-N

InChI=1S/C28H21F4NO7/c29-26(30)37-17-6-7-18-19(13-21(38-22(18)12-17)14-1-3-15(4-2-14)24(34)35)33-25(36)27(9-10-27)16-5-8-20-23(11-16)40-28(31,32)39-20/h1-8,11-12,19,21,26H,9-10,13H2,(H,33,36)(H,34,35)/t19-,21-/m1/s1

4-((2R,4R)-4-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1-carboxamido)-7-(difluoromethoxy)chroman-2-yl)benzoic acid

GLPG-2222; Galicaftor; 1918143-53-9; ABBV-2222; GLPG2222; Galicaftor [INN]; Galicaftor [USAN]; GLPG 2222; J0IIT8QSQS; ABBV-2222; Galicaftor; ABBV 2222; GLPG2222; ABBV2222.

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)

DMSO : ~50 mg/mL (~89.37 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.47 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 (4.47 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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 (Please use freshly prepared in vivo formulations for optimal results.)