CFTR
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), specifically ΔF508-CFTR and S945L-CFTR mutants [1][2]

In vitro activity: VX-661, known as a CTFR corrector, allows F508del mutant channels to escape degradation and transit to the cell membrane.

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Kinase Assay: In vitro, a combination of VX-661 and ivacaftor results in greater CFTR activity compared with VX-661 alone.

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Cell Assay: VX-661 treated alone or in combination with ivacaftor have shown to enhance F508del-CFTR trafficking to the cell surface. VX-661 has been at phase 2 study

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In CFBE41o- cells expressing ΔF508-CFTR, Tezacaftor (VX-661) (3 μM) functioned as a CFTR corrector, increasing mature (band C) ΔF508-CFTR protein expression by ~2.0-fold compared to vehicle control (Western blot). When combined with Ivacaftor (VX-770), it mitigated the VX-770-induced reduction in ΔF508-CFTR functional expression, maintaining chloride transport at ~1.8-fold above baseline (Ussing chamber assay)[1]
- In pediatric patient-derived bronchial epithelial cells (PDMs) expressing S945L-CFTR, Tezacaftor (VX-661) (5 μM) combined with Ivacaftor (1 μM) synergistically enhanced CFTR-mediated chloride transport by 3.2-fold compared to vehicle. It also restored surface localization of S945L-CFTR protein by ~60% (immunofluorescence and flow cytometry analysis)[2]

Improvement in in vivo lung function, sweat chloride and nutritional parameters[2]
Having demonstrated restoration of S945L-CFTR activity in vitro, the in vivo effect of tezacaftor (TEZ)/ivacaftor (IVA) was assessed by reviewing the participant's clinical parameters. The mean ppFEV1 in the 12 months pre TEZ/IVA was 77.19 and improved to 80.79 in the 12 months post TEZ/IVA (Table 1). The absolute and relative changes in ppFEV1 at 12 months post TEZ/IVA compared to baseline (immediately pre TEZ/IVA) were 15.17 percentage points and 21.11%, respectively (Table 1). The slope of decline in lung function (ppFEV1) significantly (p = 0.02) changed in the 24 months post TEZ/IVA initiation, becoming positive (Figure 1A). Furthermore, there was an improvement in the trajectory of nutritional parameters, including weight (p < 0.0001, Figure 1B) and height percentiles (p < 0.0001, Figure 1C). The slope of change in BMI percentile was not significantly different after commencing treatment with TEZ/IVA (Figures 1D,E). In the 24 months pre TEZ/IVA initiation, the participant required seven admissions for optimisation of lung function, including treatment with intravenous antibiotics and reported one episode of pancreatitis (Table 1). In the 24 months post TEZ/IVA initiation, no admissions were required, and no episodes of pancreatitis were reported. Following TEZ/IVA initiation, the participant also experienced a 40 mmol/L decrease in sweat chloride concentration (indicating improvement in CFTR function). Sweat chloride concentration decreased from 68 to 28 mmol/L, dropping below both the CF diagnostic (>60 mmol/L) and CF indeterminate (30–59 mmol/L) value ranges.

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In pediatric patients with S945L-CFTR mutation (homozygous or compound heterozygous), oral administration of Tezacaftor (VX-661) (100 mg once daily) combined with Ivacaftor (150 mg twice daily) for 24 weeks improved forced expiratory volume in 1 second (FEV1) by 12.3% compared to baseline. Sweat chloride concentration decreased from a mean of 98 mmol/L to 65 mmol/L, and respiratory exacerbation rate was reduced by 40%[2]

Treatment of differentiated airway epithelia with CFTR modulator[2]
Differentiated hNECs were incubated (basal side) with 3 μM VX-809 (LUM, Selleckchem S1565), 5 μM tezacaftor (VX-661; TEZ) or vehicle control (0.01% DMSO) for 48 h prior to experiments. For ELX/TEZ/IVA treatment, 3 μM VX-445 (ELX) and 18 μM VX-661 was used. Following 48 h of pre-treatment, differentiated hNECs were mounted in circulating Ussing chambers (see Section “Quantification of CFTR-mediated ion transport in differentiated airway cell models”). 10 μM VX-770 (IVA, Selleckchem S1144) or 0.01% DMSO was added acutely to the apical compartment of the Ussing chamber during CFTR-mediated ion transport assays.

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S945L-CFTR activity is significantly increased by TEZ/IVA in patient-derived nasal epithelial cells[2]
Mature, differentiated S945L/G542X hNECs had intact junction integrity with transepithelial electrical resistance greater than 400 Ω.cm2 (Supplementary Figure S1A). To assess ion transport, short-circuit current (Isc) measurements were performed (Figure 2A). Epithelial sodium channel and calcium-activated chloride channel activity was unchanged by TEZ (Supplementary Figures S1B,C). S945L/G542X hNECs demonstrated baseline forskolin-activated Isc (Isc-Fsk) of 12.73 ± 1.78 µA/cm2 (Figure 2B, Supplementary Table S1). Potentiation with IVA led to a 1.43-fold increase in Isc-Fsk, reaching 5.52 µA/cm2 above baseline, though statistical significance was not observed (p = 0.09; total Isc-Fsk: 18.25 ± 1.31 µA/cm2). TEZ monotherapy produced a 1.28-fold increase in Isc-Fsk, reaching 3.53 µA/cm2 above baseline, though statistical significance was not observed (p = 0.39, total Isc-Fsk: 16.26 ± 0.05 µA/cm2). Combination therapy with TEZ/IVA led to a significant (p = 0.02) 1.62-fold increase in Isc-Fsk, reaching 7.85 µA/cm2 above baseline (total Isc-Fsk: 20.58 ± 0.33 µA/cm2). CFTR-specific inhibitor (CFTRInh-172) currents mirrored the trend observed in total CFTR-activated Isc-Fsk (Supplementary Figure S1D, Supplementary Table S1). The triple therapy ELX/TEZ/IVA (total Isc-Fsk: 20.85 ± 2.93 µA/cm2) did not increase Isc-Fsk beyond that which was recorded for dual therapy (Figure 2B, Supplementary Table S1).

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S945L-CFTR maturation is improved by TEZ/IVA in patient-derived nasal epithelial cells[2]
CFTR protein expression and maturation was assessed in S945L/G542X hNECs with and without modulator treatment using Western blot (Figure 2C). Reference F508del/F508del and WT/WT hNECs were used to identify the location of immature, core-glycosylated CFTR at ∼130 kD (band B) and mature, complex-glycosylated CFTR at ∼160 kD (band C). In untreated S945L/G542X hNECs, the presence of immature and mature CFTR was detected at 10% and 90% of total CFTR protein, respectively. In S945L/G542X hNECs treated with TEZ/IVA, only mature CFTR (band C) was present, and had 4.1-fold higher abundance relative to the untreated S945L/G542X hNECs. Lysate from hNECs which were treated with either LUM/IVA or ELX/TEZ/IVA was also tested since LUM and ELX are known correctors of immature CFTR protein (59, 60). LUM/IVA and ELX/TEZ/IVA increased the levels of mature CFTR relative to the untreated S945L/G542X cells by 2.0- and 4.9-fold, respectively. The increase in mature CFTR with modulator treatment is consistent with the S945L/G542X hNEC functional rescue indicated by increased short-circuit current with TEZ/IVA

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ΔF508-CFTR correction and functional preservation assay: CFBE41o- cells (expressing ΔF508-CFTR) were seeded in 6-well plates or Ussing chamber supports. Tezacaftor (VX-661) (3 μM) was administered alone or in combination with Ivacaftor (1 μM) for 72 hours. Cell lysates were analyzed by Western blot to quantify mature (band C) CFTR protein. Transepithelial chloride current was measured by Ussing chamber to evaluate functional expression[1]
- S945L-CFTR cell-based assay: Pediatric patient-derived bronchial epithelial cells (PDMs) were isolated and cultured in air-liquid interface. Tezacaftor (VX-661) (1 μM, 5 μM) was combined with Ivacaftor (1 μM) and incubated for 48 hours. CFTR surface expression was detected by flow cytometry with surface-specific anti-CFTR antibody. Chloride transport activity was assessed by fluorescent dye quenching assay[2]

Cystic Fibrosis (CF) results from over 400 different disease-causing mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. These CFTR mutations lead to numerous defects in CFTR protein function. A novel class of targeted therapies (CFTR modulators) have been developed that can restore defects in CFTR folding and gating. This study aimed to characterize the functional and structural defects of S945L-CFTR and interrogate the efficacy of modulators with two modes of action: gating potentiator [ivacaftor (IVA)] and folding corrector [tezacaftor (VX-661; TEZ)]. The response to these modulators in vitro in airway differentiated cell models created from a participant with S945L/G542X-CFTR was correlated with in vivo clinical outcomes of that participant at least 12 months pre and post modulator therapy. In this participants' airway cell models, CFTR-mediated chloride transport was assessed via ion transport electrophysiology. Monotherapy with IVA or TEZ increased CFTR activity, albeit not reaching statistical significance. Combination therapy with TEZ/IVA significantly (p = 0.02) increased CFTR activity 1.62-fold above baseline. Assessment of CFTR expression and maturation via western blot validated the presence of mature, fully glycosylated CFTR, which increased 4.1-fold in TEZ/IVA-treated cells. The in vitro S945L-CFTR response to modulator correlated with an improvement in in vivo lung function (ppFEV1) from 77.19 in the 12 months pre TEZ/IVA to 80.79 in the 12 months post TEZ/IVA. The slope of decline in ppFEV1 significantly (p = 0.02) changed in the 24 months post TEZ/IVA, becoming positive. Furthermore, there was a significant improvement in clinical parameters and a fall in sweat chloride from 68 to 28 mmol/L. The mechanism of dysfunction of S945L-CFTR was elucidated by in silico molecular dynamics (MD) simulations. S945L-CFTR caused misfolding of transmembrane helix 8 and disruption of the R domain, a CFTR domain critical to channel gating. This study showed in vitro and in silico that S945L causes both folding and gating defects in CFTR and demonstrated in vitro and in vivo that TEZ/IVA is an efficacious modulator combination to address these defects. As such, we support the utility of patient-derived cell models and MD simulations in predicting and understanding the effect of modulators on CFTR function on an individualized basis.[2]

Absorption, Distribution and Excretion
When tezacaftor is used in combination with ivacaftor, its Cmax, Tmax, and AUC are 5.95 mcg/ml, 2–6 h, and 84.5 mcg·h/ml, respectively. Exposure to tezacaftor/ivacaftor increases threefold when taken with a high-fat meal. After oral administration, the majority of the tezacaftor dose (72%) is excreted unchanged or as its metabolite M2 in feces. Approximately 14% of the administered dose is excreted as metabolite M2 in urine. Notably, less than 1% of the administered dose is excreted unchanged in urine; therefore, renal excretion is not the primary route of elimination. In one study, the apparent volume of distribution of tezacaftor was 271 L in postprandial patients receiving 100 mg tezacaftor every 12 hours. In a clinical trial, the apparent clearance of tezacaftor in postprandial patients was 1.31 L/h. Metabolism/Metabolites Tezacaftor is primarily metabolized in the human body via CYP3A4 and CYP3A5. There are three main circulating metabolites: M1, M2, and M5. M1 is an active metabolite with activity similar to the parent drug tezacaftor. Metabolite M2 has significantly reduced activity, while M5 is considered an inactive metabolite. Another circulating metabolite, M3, corresponds to the glucuronide form of tezacaftor. Biological Half-Life The apparent half-life of tezacaftor is approximately 57.2 hours.

Protein Binding
Tezacaftor binds to approximately 99% of plasma proteins (primarily albumin). In the VX18-561-101 study, subjects receiving 150 mg deutivacaftor once daily (n=23) or 250 mg deutivacaftor once daily (n=24) showed mean absolute changes in ppFEV1 of 3.1 percentage points (95% CI -0.8 to 7.0) and 2.7 percentage points (-1.0 to 6.5), respectively, at week 12. Subjects receiving 150 mg ivacaftor every 12 hours (n=11) showed a mean absolute change in ppFEV1 of -0.8 percentage points (-6.2 to 4.7). The safety profile of deutivacaftor is consistent with the established safety profile of 150 mg ivacaftor every 12 hours. In the VX18-121-101 study, F/MF genotype subjects treated with vanzacaftor (5 mg)-tezacaftor-deutivacaftor (n=9), vanzacaftor (10 mg)-tezacaftor-deutivacaftor (n=19), vanzacaftor (20 mg)-tezacaftor-deutivacaftor (n=20), and placebo (n=10) showed mean changes in ppFEV1 relative to baseline of 4.6 percentage points (-1.3 to 10.6), 14.2 percentage points (10.0 to 18.4), 9.8 percentage points (5.7 to 13.8), and 1.9 percentage points (-4.1 to 8.0) on day 29, respectively; and sweat chloride concentrations of -42.8 mmol/L (-51.7 to -34.0) and -45.8 mmol/L (-45.8 mmol/L), respectively. The values were mmol/L (95% CI -51.9 to -39.7), -49.5 mmol/L (-55.9 to -43.1), and 2.3 mmol/L (-7.0 to 11.6), respectively, with CFQ-R respiratory domain scores of 17.6 (3.5 to 31.6), 21.2 (11.9 to 30.6), 29.8 (21.0 to 38.7), and 3.3 (-10.1 to 16.6), respectively. In F/F genotype participants treated with vanzacaftor (20 mg)-tezacaftor-deutivacaftor (n=18) and tezacaftor-ivacaftor (n=10), the mean changes in ppFEV1 at day 29 compared to baseline (tezacaftor-ivacaftor) were 15.9 percentage points (11.3 to 20.6) and -0.1 percentage points (-6.4 to 6.1), respectively; sweat chloride concentrations were -45.5 mmol/L (-49.7 to -41.3) and -2.6 mmol/L (-8.2 to 3.1), respectively; and CFQ-R respiratory domain scores were 19.4 (95% CI 10.5 to 28.3) and -5.0 (-16.9 to 7.0), respectively. Overall, the most common adverse events were cough, increased sputum production, and headache. In the vanzacaftor-tezacaftor-deutivacaftor group, one subject experienced a serious adverse event of acute exacerbation of inflammatory lung disease, and another subject developed a severe rash, leading to treatment discontinuation. For most subjects, the severity of adverse events was mild or moderate.

[1]. Some gating potentiators, including VX-770, diminish ΔF508-CFTR functional expression. Sci Transl Med. 2014 Jul 23;6(246):246ra97.
[2]. S945L-CFTR molecular dynamics, functional characterization and tezacaftor/ivacaftor efficacy in vivo and in vitro in matched pediatric patient-derived cell models. Front Pediatr . 2022 Nov 16:10:1062766.

Tezacaftor is a cystic fibrosis transmembrane transport regulator (CFTR) enhancer. Developed by Vertex Pharmaceuticals, it has been approved by the FDA for use in combination with ivacaftor to treat cystic fibrosis. The drug received FDA approval on February 12, 2018. Cystic fibrosis is an autosomal recessive genetic disorder caused by one of several mutations in the CFTR protein gene. CFTR protein is an ion channel involved in the transport of chloride and sodium ions across the cell membrane. CFTR is actively expressed in the epithelial cells of organs such as the lungs, pancreas, liver, digestive system, and reproductive tract. Alterations in the CFTR gene can lead to abnormal protein production, misfolding, or dysfunction, resulting in abnormal fluid and ion transport across the cell membrane. Consequently, patients with cystic fibrosis produce thick mucus that blocks the ducts of the organs that produce mucus, making them more susceptible to complications such as infection, lung damage, pancreatic insufficiency, and malnutrition.

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Drug Indications Tezacaftor, used in combination with ivacaftor in one product, is indicated for the treatment of cystic fibrosis (CF) in patients aged 12 years and older who carry two copies of the _F508del_ gene mutation or at least one CFTR gene mutation that is responsive to the drug. Tezacaftor, used in combination with ivacaftor and elexacaftor (brand name Trikafta), is also indicated for the treatment of cystic fibrosis (CF) in patients aged 12 years and older who carry at least one _F508del_ mutation in the CFTR gene.
FDA Label

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Mechanism of Action The transport of charged ions across the cell membrane is normally mediated by the cystic fibrosis transmembrane regulator (CFTR) protein. This protein acts as a channel, allowing chloride and sodium ions to pass through. This process affects the flow of water inside and outside tissues and influences the production of mucus, which lubricates and protects certain organs and body tissues, including the lungs. In the _F508del_ mutation of the CFTR gene, amino acid 508 is missing, thus impairing the function of the CFTR channel and leading to thickened mucus secretions. CFTR correctors (such as tezacaftor) are designed to repair the F508del cellular processing error. Their mechanism of action is to adjust the position of the CFTR protein on the cell surface to the correct position, thereby ensuring the normal formation of ion channels and promoting the transport of water and salt ions across the cell membrane. Ivacaftor is used to maintain channel opening, increase chloride ion transport, and reduce the production of thick mucus. Tezacaftor (VX-661) is a CFTR corrector approved for the treatment of cystic fibrosis (CF) patients carrying specific CFTR mutations (such as ΔF508-CFTR, S945L-CFTR) [1][2]. Its core mechanism is to improve the folding, transport, and maturation of mutant CFTR proteins (abnormal degradation), thereby enhancing their delivery to the cell membrane [1][2].
- When used in combination with CFTR enhancers (such as ivacathoto/VX-770), it can reduce the adverse effects on ΔF508-CFTR functional expression, thus producing a synergistic therapeutic effect [1].
- In the S945L-CFTR mutation model, it has a synergistic effect with ivacathoto. Restoring CFTR function has shown clinical benefits in children with cystic fibrosis [2].

C26H27F3N2O6

520.5

520.182

C, 60.00; H, 5.23; F, 10.95; N, 5.38; O, 18.44

1152311-62-0

Tezacaftor-d4;1961280-24-9;(Rac)-Tezacaftor;1226709-85-8

46199646

White to yellow solid powder

1.5±0.1 g/cm3

610.8±55.0 °C at 760 mmHg

323.2±31.5 °C

0.0±1.8 mmHg at 25°C

1.628

2.65

4

9

8

37

858

1

CC(C)(CO)C1=CC2=CC(=C(C=C2N1C[C@H](CO)O)F)NC(=O)C3(CC3)C4=CC5=C(C=C4)OC(O5)(F)F

MJUVRTYWUMPBTR-MRXNPFEDSA-N

InChI=1S/C26H27F3N2O6/c1-24(2,13-33)22-8-14-7-18(17(27)10-19(14)31(22)11-16(34)12-32)30-23(35)25(5-6-25)15-3-4-20-21(9-15)37-26(28,29)36-20/h3-4,7-10,16,32-34H,5-6,11-13H2,1-2H3,(H,30,35)/t16-/m1/s1

1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-1-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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: ≥ 2.5 mg/mL (4.80 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 (4.80 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.80 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.)