CFTR/cystic fibrosis transmembrane conductance regulator

In order to restore Phe508del CFTR protein function, elexacaftor (VX-445) is a next-generation cystic fibrosis transmembrane regulator (CFTR) corrector. It may be possible to treat cystic fibrosis with elexacaftor (VX-445). When employed in tandem, VX-445-Tezacaftor-VX-770 greatly enhanced the Phe508del CFTR protein's processing, transport, and chloride ion transport while also increasing the quantity of each chemical to the degree that it was combined [2].
In vitro, VX-445–tezacaftor–ivacaftor significantly improved Phe508del CFTR protein processing, trafficking, and chloride transport to a greater extent than any two of these agents in dual combination[2].

In patients with cystic fibrosis, VX-445–tezacaftor–ivacaftor had an acceptable safety and side-effect profile. Most adverse events were mild or moderate. The treatment also resulted in an increased percentage of predicted FEV1 of up to 13.8 points in the Phe508del–MF group (P<0.001). In patients in the Phe508del–Phe508del group, who were already receiving tezacaftor–ivacaftor, the addition of VX-445 resulted in an 11.0-point increase in the percentage of predicted FEV1 (P<0.001). In both groups, there was a decrease in sweat chloride concentrations and improvement in the respiratory domain score on the Cystic Fibrosis Questionnaire–Revised. CONCLUSIONS: The use of VX-445–tezacaftor–ivacaftor to target Phe508del CFTR protein resulted in increased CFTR function in vitro and translated to improvements in patients with cystic fibrosis with one or two Phe508del alleles. This approach has the potential to treat the underlying cause of cystic fibrosis in approximately 90% of patients [2].

Ussing chamber assay[2]
Ussing chamber techniques were used to record the short-circuit transepithelial current due to CFTR-mediated chloride transport in HBE cells. HBE cells were incubated for 18 to 24 hours with compounds before recording chloride transport. CFTR-mediated chloride transport was measured in the presence of amiloride, forksolin, and CFTR inhibitors as previously described.

CFTR processing and trafficking was assessed by immunoblotting techniques. Briefly, HBE cells were washed with HBSS containing 0.4% sodium bicarbonate once for 3 hours at 37°C, then washed with warm PBS twice before incubation for 24 hours at 37°C with DMSO, ivacaftor and tezacaftor, VX-445 alone, or a combination of VX-445-tezacaftor-ivacaftor in HBE culture media. After incubation, cells were lysed in ice-cold CHAPS lysis buffer containing EDTA-free protease inhibitors. Lysates were spun for 15 minutes at 10,000 × g at 4°C to pellet nuclei and insoluble material. Approximately 12 μg of total protein was mixed with 2x Laemmli sample buffer containing 5% β-Mercaptoethanol, and loaded onto a 3% to 8% Tris-acetate gel. The gel was transferred to a nitrocellulose membrane and processed for Western blotting by using a 1:1000 dilution of monoclonal CFTR antibody 596 and a 1:5000 dilution of calnexin rabbit monoclonal antibody. The secondary antibody used for CFTR was a 1:5000 dilution of donkey anti-mouse-HRP antibody and for calnexin was a 1:5000 dilution of donkey anti-rabbitHRP antibody. Blots were developed by SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA) before being visualized by myECL Imager. Quantification of the relative amounts of band C, band B, and calnexin was performed using Image Studio Lite. To quantify CFTR maturation, the relative amount of CFTR C-band protein was normalized to calnexin measured in the identical protein sample, and these levels were used for subsequent calculations[2].

Clinical Development[2]
After a phase 1 trial involving healthy volunteers (not reported here), a three-part, randomized, double-blind, placebo- or active-controlled, parallelgroup, dose-ranging, phase 2 trial was conducted from July 2017 through March 2018. Patients 18 years of age or older with cystic fibrosis were enrolled at 38 sites in the United States, the Netherlands, Belgium, and Australia. The trial design and conduct were similar to those presented in the companion trial of VX-6591 (see page 7 in the Supplementary Appendix for details).
Patients with Phe508del–MF genotypes were randomly assigned to receive 4 weeks of active treatment — with VX-445 at a dose of 50, 100, or 200 mg orally once daily in triple combination with tezacaftor (100 mg per day) and ivacaftor (150 mg every 12 hours) — or a triple placebo control. Patients with the Phe508del–Phe508del genotype received a 4-week run-in with tezacaftor and ivacaftor and were randomly assigned to receive 4 weeks of treatment with either VX-445 (200 mg per day orally) plus tezacaftor (100 mg per day) and ivacaftor (150 mg every 12 hours) or matched placebo plus tezacaftor and ivacaftor. In addition, the trial included patients with Phe508del–MF genotypes treated with VX-445 in triple combination with tezacaftor and VX-561, a deuterated form of ivacaftor taken once daily, or triple placebo. For details regarding trial design and oversight, including a description of VX-561, trial participants, and assessments, see the accompanying article on VX-659 by Davies et al.1 and the Supplementary Appendix of this article (pages 7 through 14, Fig. S1, and Table S1).

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Clinical Efficacy[2]
Clinical efficacy was evaluated on the basis of the change in forced expiratory volume in 1 second (FEV1) from baseline and a disease-specific health-related quality-of-life instrument, the Cystic Fibrosis Questionnaire–Revised (CFQ-R). Each CFQ-R domain is scored on a 100-point scale, with higher scores indicating a lower effect of symptoms on the patient’s quality of life. A minimal clinically important difference of 4 points has been determined for the respiratory symptoms domain.

Absorption, Distribution and Excretion
The absolute oral bioavailability of elexacaftor is approximately 80%. The steady-state AUC0-24h and Cmax following once daily dosing with elexacaftor 200mg are 162 mcg∙h/mL and 8.7 mcg/mL, respectively, and the median Tmax is 6 hours. The AUC of elexacaftor is increased 1.9-2.5-fold following a moderate-fat meal - for this reason, it is recommended to give TrikaftaTM with fat-containing food.
Approximately 87.3% of an administered radio-labeled dose of elexacaftor was found in the feces, mostly as metabolites, while only 0.23% of that same dose was found excreted in the urine.
The apparent volume of distribution of elexacaftor is 53.7 L.
The mean apparent clearance of elexacaftor is 1.18 L/h.

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Metabolism / Metabolites
The metabolism of elexacaftor is extensive and primarily catalyzed via CYP3A4/5. Its main active metabolite, M23-ELX, carries a similar potency as the parent drug. The precise metabolic pathway of elexacaftor has not yet been elucidated in published research.

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Biological Half-Life
The mean terminal half-life of elexacaftor is approximately 24.7 hours.

Protein Binding
Elexacaftor is >99% protein bound in plasma, primarily to albumin.

[1]. MODULATOR OF THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR , PHARMACEUTICAL COMPOSITIONS , METHODS OF TREATMENT , AND PROCESS FOR MAKING THE MODULATOR. US 20180162839 A1.

[2]. VX-445-Tezacaftor-VX-770 in Patients with Cystic Fibrosis and One or Two Phe508del Alleles. N Engl J Med. 2018 Oct 25;379(17):1612-1620.

Elexacaftor (previously VX-445) is a small molecule, next-generation corrector of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. It received FDA approval in October 2019 in combination with [tezacaftor] and [ivacaftor] as the combination product TrikaftaTM. Elexacaftor is considered a next-generation CFTR corrector as it possesses both a different structure and mechanism as compared to first generation correctors like tezacaftor. While dual corrector/potentiator combination therapy has proven useful in the treatment of a subset of CF patients, their use is typically limited to patients who are homozygous for the F508del-CFTR gene. Elexacaftor, along with [VX-659], was designed to fill the need for an efficacious CF therapy for patients who are heterozygous for F508del-CFTR and a gene that does not produce protein or produces proteins unresponsive to ivacaftor or tezacaftor. The triple combination product TrikaftaTM, manufactured by Vertex Pharmaceuticals, is the first product approved for the treatment of CF in individuals who are either homo- or heterozygous for the F508del-CFTR gene - this represents approximately 70-90% of all CF patients.

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Drug Indication
Elexacaftor, in combination with [ivacaftor] and [tezacaftor] as the combination product TrikaftaTM, is indicated for the treatment of cystic fibrosis (CF) in patients 12 years of age and older who have at least one _F508del_ mutation in the CTFR gene.

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Mechanism of Action
Cystic fibrosis (CF) is the result of a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR proteins produced by this gene are transmembrane ion channels that move sodium and chloride across cell membranes - water follows the flow of chloride ions to the cell surface, which consequently helps to hydrate the surface of the cell and thin the secretions (i.e. mucous) around the cell. Mutations in the CFTR gene produce CFTR proteins of insufficient quantity and/or function, leading to defective ion transport and a build-up of thick mucous throughout the body that causes multi-organ disease involving the pulmonary, gastrointestinal, and pancreatic systems (amongst others). The most common CFTR mutation, the _F508del_ mutation, is estimated to account for 70 to 90% of all CFTR mutations and results in severe processing and trafficking defects of the CFTR protein. Elexacaftor is a CFTR corrector that modulates CFTR proteins to facilitate trafficking to the cell surface for incorporation into the cell membrane. The end result is an increase in the number of mature CFTR proteins present at the cell surface and, therefore, improved ion transport and CF symptomatology. Elexacaftor is used in combination with tezacaftor, another CFTR corrector with a different mechanism of action, and ivacaftor, a CFTR potentiator that improves the function of CFTR proteins on the cell surface - this multi-faceted, triple-drug approach confers a synergistic effect beyond that seen in typical corrector/potentiator dual therapy regimens.

C26H34F3N7O4S

597.6529

597.23

C, 52.25 H, 5.73 F, 9.54 N, 16.41 O, 10.71 S, 5.36

2216712-66-0

(R)-Elexacaftor;2229860-99-3;Elexacaftor-d3;Elexacaftor-13C,d3

134587348

White to off-white solid powder

4.9

1

11

8

41

1050

1

C[C@H]1CC(N(C1)C2=C(C=CC(=N2)N3C=CC(=N3)OCC(C)(C)C(F)(F)F)C(=O)NS(=O)(=O)C4=CN(N=C4C)C)(C)C

MVRHVFSOIWFBTE-INIZCTEOSA-N

InChI=1S/C26H34F3N7O4S/c1-16-12-25(5,6)35(13-16)22-18(23(37)33-41(38,39)19-14-34(7)31-17(19)2)8-9-20(30-22)36-11-10-21(32-36)40-15-24(3,4)26(27,28)29/h8-11,14,16H,12-13,15H2,1-7H3,(H,33,37)/t16-/m0/s1

(S)-N-((1,3-dimethyl-1H-pyrazol-4-yl)sulfonyl)-6-(3-(3,3,3-trifluoro-2,2-dimethylpropoxy)-1H-pyrazol-1-yl)-2-(2,2,4-trimethylpyrrolidin-1-yl)nicotinamide

Elexacaftor, WHO 11180; WHO 11180; RRN67GMB0V; UNII-RRN67GMB0V; Elexacaftor (USAN); ELEXACAFTOR [MI]; WHO11180; VX-445; VX 445; VX445; Trikafta

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 : ~125 mg/mL (~209.15 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.48 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 20.8 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: ≥ 2.08 mg/mL (3.48 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 20.8 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.)