CFTR

In vitro activity: VX-661, known as a CTFR corrector, allows F508del mutant channels to escape degradation and transit to the cell membrane. Kinase Assay: In vitro, a combination of VX-661 and ivacaftor results in greater CFTR activity compared with VX-661 alone. 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

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.

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.

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 used in combination with ivacaftor, the Cmax, Tmax, and AUC of tezacaftor were 5.95 mcg/ml, 2–6 h, and 84.5 mcg·h/ml, respectively. Co-administration with a high-fat meal increased tezacaftor/ivacaftor exposure by 3-fold. 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 in urine as the metabolite M2. Notably, less than 1% of the administered dose is excreted unchanged in urine; therefore, renal excretion is not the primary route of clearance. In one study, the apparent volume of distribution of tezacaftor was 271 L in postprandial patients receiving 100 mg tezacaftor every 12 hours. In one clinical trial, the apparent clearance of tezacaftor in postprandial patients was 1.31 L/h.

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Metabolism/Metabolites
Tezacotto 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.

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

Description 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 different 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. Therefore, 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
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 position 508 is deleted, thus impairing CFTR channel function 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 reposition the CFTR protein on the cell surface to the correct location, thereby ensuring normal ion channel formation and increasing the transport of water and salt ions across the cell membrane. The simultaneous use of tezacaftor aims to maintain channel openness, increase chloride ion transport, and reduce mucus production.

C26H27F3N2O6

520.497597932816

520.182

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

1226709-85-8

Tezacaftor;1152311-62-0;Tezacaftor-d4;1961280-24-9

46199801

Typically exists as solid at room temperature

2.9

4

9

8

37

858

0

FC1=CC2=C(C=C(C(C)(C)CO)N2CC(CO)O)C=C1NC(C1(C2=CC=C3C(=C2)OC(O3)(F)F)CC1)=O

MJUVRTYWUMPBTR-UHFFFAOYSA-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)

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

(Rac)-Tezacaftor; 1226709-85-8; 1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-Methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxaMide; Tezacaftor (Racemate); SCHEMBL322363; MJUVRTYWUMPBTR-UHFFFAOYSA-N; HMS3652D20; HMS3748O05;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)