CFTR

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.

---

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).

---

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

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
The Cmax, Tmax and AUC of tezacaftor, when administered with ivacaftor, are 5.95 mcg/ml, 2-6 h, and 84.5 mcg.h/ml respectively. Exposure of tezacaftor/ivacaftor increases 3-fold when it is administered with a high-fat meal.
After oral administration, the majority of tezacaftor dose (72%) is found excreted in the feces either unchanged or as its metabolite, M2. About 14% of the administered dose is found excreted in the urine as the metabolite, M2. It was noted that less than 1% of the administered dose is excreted unchanged in the urine and thus, renal excretion is not the major elimination pathway.
The apparent volume of distribution of tezacaftor was 271 L in a study of patients in the fed state who received 100 mg of tezacaftor every 12 hours.
The apparent clearance of tezacaftor has been measured at 1.31 L/h for patients in the fed state during a clinical trial.

---

Metabolism / Metabolites
Tezacaftor is metabolized extensively in humans by the action of CYP3A4 and CYP3A5. There are three main circulating metabolites; M1, M2, and M5. The M1 is an active metabolite with similar activity to the parent drug, tezacaftor. The M2 metabolite is significantly less active and M5 is considered an inactive metabolite. An additional circulating metabolite, M3, corresponding to the glucuronide form of tezacaftor.

---

Biological Half-Life
The apparent half-life of tezacaftor is approximately 57.2 hours.

Protein Binding
Tezacaftor is approximately 99% bound to plasma proteins, mainly albumin.

[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 drug of the cystic fibrosis transmembrane conductance regulator (CFTR) potentiator class. It was developed by Vertex Pharmaceuticals and FDA approved in combination with [ivacaftor] to manage cystic fibrosis. This drug was approved by the FDA on February 12, 2018. Cystic Fibrosis is an autosomal recessive disorder caused by one of several different mutations in the gene for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, an ion channel involved in the transport of chloride and sodium ions across cell membranes. CFTR is active in epithelial cells of organs such as of the lungs, pancreas, liver, digestive system, and reproductive tract. Alterations in the CFTR gene result in altered production, misfolding, or function of the protein and consequently abnormal fluid and ion transport across cell membranes. As a result, CF patients produce thick, sticky mucus that clogs the ducts of organs where it is produced making patients more susceptible to complications such as infections, lung damage, pancreatic insufficiency, and malnutrition.

---

Drug Indication
Tezacaftor is combined with ivacaftor in one product for the treatment of cystic fibrosis (CF) in patients aged 12 years or older with two copies of the _F508del_ gene mutation or at least one mutation in the CFTR gene that is responsive to this drug. Tezacaftor, when used in combination with ivacaftor and [elexacaftor] in the product Trikafta, is also indicated for the treatment of CF in patients 12 years of age and older that have at least one _F508del_ mutation in the CFTR gene.
FDA Label

---

Mechanism of Action
The transport of charged ions across cell membranes is normally achieved through the actions of the cystic fibrosis transmembrane regulator (CFTR) protein. This protein acts as a channel and allows for the passage of chloride and sodium. This process affects the movement of water in and out of the tissues and impacts the production of mucus that lubricates and protects certain organs and body tissues, including the lungs. In the _F508del_ mutation of the CFTR gene, one amino acid is deleted at the position 508, therefore, the CFTR channel function is compromised, resulting in thickened mucus secretions. CFTR correctors such as tezacaftor aim to repair F508del cellular misprocessing. This is done by modulating the position of the CFTR protein on the cell surface to the correct position, allowing for adequate ion channel formation and increased in water and salt movement through the cell membrane. The concomitant use of ivacaftor is intended to maintain an open channel, increasing the transport of chloride, reducing thick mucus production.

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.

---

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.

---

View More

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.

---

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