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 elexacin is approximately 80%. Following a once-daily dose of 200 mg elexacin, the steady-state AUC 0–24 h and Cmax are 162 mcg∙h/mL and 8.7 mcg/mL, respectively, with a median Tmax of 6 hours. The AUC of elexacin increases 1.9–2.5 times after ingestion of a moderate-fat diet—therefore, it is recommended to take Trikafta™ with fatty foods. Approximately 87.3% of the radiolabeled elexacin dose is found in feces, primarily as metabolites, while only 0.23% of the same dose is found in urine. The apparent volume of distribution of elexacin is 53.7 L. The mean apparent clearance of elexacin is 1.18 L/h.

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Metabolism/Metabolites
Elexacaftor has a broad metabolism, primarily catalyzed by CYP3A4/5. Its main active metabolite, M23-ELX, has similar potency to the parent drug. Published studies have not elucidated the exact metabolic pathway of elexacaftor.

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Biological Half-Life
The average terminal half-life of elexacaftor is approximately 24.7 hours.
Meaning time

Protein Binding
Elexacaftor has a protein binding rate of >99% in plasma, primarily binding to albumin. Warnings and Precautions Drug-Induced Hepatic Injury and Liver Failure TRIKAFTA can cause serious and potentially fatal drug-induced hepatic injury. Liver failure leading to liver transplantation and death has been reported in patients taking TRIKAFTA, regardless of their history of liver disease. Liver injury has been reported within the first month of TRIKAFTA treatment and up to 15 months after treatment initiation. All patients should undergo liver function tests (ALT, AST, alkaline phosphatase, and bilirubin) before starting TRIKAFTA treatment, then monthly for the first 6 months of treatment, every 3 months for the next 12 months, and at least annually thereafter. If signs or symptoms of liver injury occur, TRIKAFTA should be discontinued. These signs or symptoms may include: significantly elevated liver function indicators (e.g., ALT or AST > 5 times the upper limit of normal (ULN), or ALT or AST > 3 times the ULN and bilirubin > 2 times the ULN); or clinical symptoms suggestive of liver injury (e.g., jaundice, right upper quadrant pain, nausea, vomiting, altered mental status, ascites). Refer the patient to a hepatologist and closely monitor the patient with clinical and laboratory surveillance until the abnormalities subside. If symptoms improve and the expected benefits outweigh the risks, TRIKAFTA may be resumed under close monitoring. TRIKAFTA is contraindicated in patients with severe hepatic impairment. TRIKAFTA is not recommended for patients with moderate hepatic impairment and should only be considered when there is a clear medical need and the benefits outweigh the risks. If use is necessary, the dose should be cautiously reduced and the patient closely monitored.
Hypersensitivity reactions, including anaphylactic shock
Hypersensitivity reactions, including angioedema and anaphylactic shock, have been reported in post-marketing surveillance. If signs or symptoms of severe hypersensitivity occur during treatment, TRIKAFTA should be discontinued and appropriate treatment measures should be taken. The benefits and risks for each patient should be weighed to determine whether to resume TRIKAFTA treatment. Concomitant use with CYP3A inducers:
Concomitant use with potent CYP3A inducers will significantly reduce ivacafor exposure and is expected to also reduce elexacaftor and tezacaftor exposure, which may reduce the efficacy of TRIKAFTA. Concomitant use with potent CYP3A inducers is not recommended. Concomitant use with CYP3A inhibitors:
Concomitant use with potent or intermediate-potency CYP3A inhibitors will increase elexacaftor, tezacaftor, and ivacaftor exposure. When used in combination with intermediate- or high-potency CYP3A inhibitors, the dose of TRIKAFTA should be reduced. Cataracts:
Non-congenital lens opacities have been reported in pediatric patients receiving ivacaftor-containing regimens. Baseline and follow-up ophthalmological examinations are recommended for pediatric patients starting TRIKAFTA treatment.
Adverse Reactions
Serious Adverse Reactions
Compared to the placebo group, the more common serious adverse reactions in the TRIKAFTA treatment group were rash (1% vs <1%) and influenza (1% vs 0%). Most Common Adverse Reactions
The most common adverse reactions occurring ≥5% in the TRIKAFTA treatment group and ≥1% higher than in the placebo group were headache, upper respiratory tract infection, abdominal pain, diarrhea, rash, elevated alanine aminotransferase, nasal congestion, elevated serum creatine phosphokinase, elevated aspartate aminotransferase, rhinorrhea, rhinitis, influenza, sinusitis, elevated serum bilirubin, and constipation. Special Populations
Pediatric Use
The safety and efficacy of TRIKAFTA in patients under 2 years of age with cystic fibrosis have not been established.

[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 (formerly known as VX-445) is a small-molecule, next-generation cystic fibrosis transmembrane conductance regulator (CFTR) protein corrector. It was approved by the FDA in October 2019 for use in combination with tezacaftor and ivacaftor, forming the combination formulation Trikafta™. Elexacaftor is considered a next-generation CFTR corrector because it has a different structure and mechanism of action compared to first-generation correctors such as tezacaftor. While dual corrector/enhancer combination therapy has been shown to be effective for some cystic fibrosis (CF) patients, its application is generally limited to patients homozygous for the F508del-CFTR gene. Elexacaftor, along with [VX-659], was designed to meet the need for effective CF therapy in F508del-CFTR heterozygous patients whose genes do not produce the protein or whose produced protein is unresponsive to ivacaftor or tezacaftor. Trikafta™, a triple combination therapy manufactured by Vertex Pharmaceuticals, is the first approved drug for the treatment of patients with cystic fibrosis (CF) who are homozygous or heterozygous for the F508del-CFTR gene—approximately 70-90% of all CF patients. Drug Indications Trikafta™, in combination with [ivacaftor] and [tezacaftor], is indicated for the treatment of cystic fibrosis (CF) in patients aged 12 years and older with at least one mutation in the CFTR gene_F508del_. Mechanism of Action Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane transport regulator (CFTR) gene. The CFTR protein produced by this gene is a transmembrane ion channel responsible for transporting sodium and chloride ions across the cell membrane—water molecules follow chloride ions to the cell surface, helping to retain moisture on the cell surface and dilute secretions around the cell (such as mucus). Mutations in the CFTR gene lead to insufficient quantity and/or abnormal function of the CFTR protein, resulting in ion transport defects and the accumulation of thick mucus throughout the body, causing diseases affecting multiple organ systems, including the lungs, gastrointestinal tract, and pancreas. The most common CFTR mutation is the F508del mutation, estimated to account for 70% to 90% of all CFTR mutations, causing severe defects in the processing and transport of the CFTR protein. Elexacaftor is a CFTR corrector that regulates the CFTR protein, promoting its transport to the cell surface and integration into the cell membrane. The end result is an increase in the number of mature CFTR proteins on the cell surface, thereby improving ion transport and cystic fibrosis symptoms. Elexacaftor is used in combination with tezacaftor, another CFTR corrector with a different mechanism of action, and ivacaftor, a CFTR enhancer that enhances the function of CFTR proteins on the cell surface—this three-drug combination therapy produces a synergistic effect far exceeding that of typical corrector/enhancer dual therapy.

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.)