Tezacaftor Research Articles

Triplet analysis: Quantifying ivacaftor, tezacaftor, and elexacaftor in plasma with mass spectrometry

A novel and highly precise method were developed and rigorously validated for quantifying ivacaftor (IVA), tezacaftor (TEZ), and elexacaftor (ELX) in human plasma. This method leverages multiple reaction-monitoring mass spectrometry for its analytical approach. During the validation process, the method demonstrated its robustness across a wide concentration range: 0.151 to 40.382 ng/ml for ELX, 0.101 to 30.010 ng/ml for IVA, and 20.187 to 6,026.032 ng/ml for TEZ in human plasma. Importantly, this method exhibited exceptional accuracy and reproducibility even at lower concentrations. The mobile phase employed in this analysis consisted of methanol and 0.1% formic acid at a ratio of 85:15 (v/v), with a flow rate set at 1.0 ml/minute. Additionally, linear correlations were established within the human plasma for ELX (R2=0.9983), IVA (R2=0.9992), and TEZ (R2=0.9989). In conclusion, this newly developed method is dependable and precise for the accurate quantification of IVA, TEZ, and ELX, especially when dealing with lower concentrations in human plasma analysis.

Using generic drug elexacaftor/tezacaftor/ivacaftor+ ivacaftor in patients with cystic fidosis in routine clinical practice

Cystic fibrosis (CF) is a multisystem disease of exocrine glands with a progressive course. In recent years, targeted (pathogenetic) treatment aimed at correcting the function of the chloride channel has come to the fore in addition to the symptomatic therapy. The greatest effectiveness was demonstrated with the use of the triple combination of the drug elexacaftor (ELX) / tezacaftor (TEZ) / ivacaftor (IVA) + IVA – Trikafta® (Vertex Pharmaceuticals, USA) that has become the “gold standard” of targeted therapy. Currently, there is virtually no information about the effectiveness and safety of the generic products. The aim of the study was to evaluate the efficacy and safety of the generic drug ELX/TEZ/IVA+IVA (Trilexa®) (Tutor S.A.S.I.F.I.A., Buenos Aires, Argentina) in adult patients with CF in real clinical practice.Methods. The 6-month study included patients (n = 11) aged 18 to 46 years with a diagnosis of CF who were prescribed pathogenetic treatment with ELX/TEZ/IVA+IVA. Their external respiration function (forced vital capacity, forced expiratory volume in 1 second), conductivity of sweat fluid electrolytes (sweat test), anthropometric and other clinical and functional data were analyzed. Results. This study demonstrated pronounced positive effects in relation to indicators of respiratory function, sweat test, and nutritional status. The ELX/TEZ/IVA+IVA treatment was well tolerated, with clinical improvement in the form of a decrease in cough intensity, sputum volume, improvement in daily exercise tolerance, height, and body weight. No serious adverse events were recorded and none of the patients discontinued treatment due to adverse reactions.Conclusion. Obvious clinical and functional positive dynamics and safety over 6 months were demonstrated with ELX/TEZ/IVA+IVA combination.

Post-approval studies with the CFTR modulators Elexacaftor-Tezacaftor-Ivacaftor.

Triple combination therapy with the CFTR modulators elexacaftor (ELX), tezacaftor (TEZ) and ivacaftor (IVA) has been qualified as a game changer in cystic fibrosis (CF). We provide an overview of the body of literature on ELX/TEZ/IVA published between November 2019 and February 2023 after approval by the regulators. Recombinant ELX/TEZ/IVA-bound Phe508del CFTR exhibits a wild type conformation in vitro, but in patient's tissue a CFTR glyoisoform is synthesized that is distinct from the wild type and Phe508del isoforms. ELX/TEZ/IVA therapy improved the quality of life of people with CF in the real-life setting irrespective of their anthropometry and lung function at baseline. ELX/TEZ/IVA improved sinonasal and abdominal disease, lung function and morphology, airway microbiology and the basic defect of impaired epithelial chloride and bicarbonate transport. Pregnancy rates were increasing in women with CF. Side effects of mental status changes deserve particular attention in the future.

Simultaneous Quantification of Ivacaftor, Tezacaftor, and Elexacaftor in Cystic Fibrosis Patients’ Plasma by a Novel LC–MS/MS Method

The new breakthrough cystic fibrosis (CF) drug combination of ivacaftor (IVA), tezacaftor (TEZ), and elexacaftor (ELX), namely "caftor" drugs, directly modulates the activity and trafficking of the defective CF transmembrane conductance regulator protein (CFTR) underlying the CF disease. The role of therapeutic drug monitoring (TDM) of caftor drugs in clinical settings has recently been established. The availability of reliable and robust analytical methods for the quantification of IVA, TEZ, and ELX is essential to support dose-concentration-effect studies. We have developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the rapid and simultaneous quantification of IVA, TEZ, and ELX from the plasma of CF patients. The method was based on a rapid extraction protocol from 50 μL human plasma and separation on a reversed-phase C-18 HPLC column after the addition of deuterated internal standards. Accurate analyte quantification using multiple reaction monitoring (MRM) detection was then obtained using a Thermofisher Quantiva triple-quadrupole MS coupled to an Ultimate 3000 UHPLC. The method has been validated following international (EMA) guidelines for bioanalytical method validation and has been tested on plasma samples from 62 CF patients treated with the three-drug combination IVA/TEZ/ELX, marketed as Kaftrio® or Trikafta®, in steady-state condition. The assay was linear over wide concentration ranges (0.008-12 mg/L) in plasma for IVA, TEZ, and ELX, suitable for a broad range of plasma concentrations, and accurate and reproducible in the absence of matrix effects. The stability of analytes for at least 30 days at room temperature could allow for cost-effective shipment and storage. On the same day of sample collection, a sweat test was evaluated for 26 associated patients' samples, FEV1 (%) for 58, and BMI was calculated for 62. However, Spearman correlation showed no correlation between Cthrough plasma concentrations of analytes (IVA, TEZ, ELX) and sweat test, FEV1 (%), or BMI. Our method proved to be suitable for TDM and could be helpful in assessing dose-concentration-response correlations in larger studies.

S945L-CFTR molecular dynamics, functional characterization and tezacaftor/ivacaftor efficacy in vivo and in vitro in matched pediatric patient-derived cell models.

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 (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.

Quantitation of cystic fibrosis triple combination therapy, elexacaftor/tezacaftor/ivacaftor, in human plasma and cellular lysate

The triple combination modulator therapy (ETI, elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA)) is a recent advancement for the care of patients with cystic fibrosis. To aid in the development of clinical pharmacokinetics studies of this treatment, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantifying the component compounds in human plasma and cell lysate. This assay was optimized for small volumes (10 µL), uses stably labeled isotopes of the ETI compounds as internal standards, and employs a simple methanol protein precipitation method. Chromatography was performed on an ACE Excel C18, 2.1 × 50 mm, reversed phase analytical column, using a step or bump isocratic method, with mobile phases consisting of 0.1% formic acid in water for A, and 0.1% formic acid in acetonitrile for B. Analyte and internal standard detection was conducted with ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 598.0/422.0 for ELX, 521.0/449.0 for TEZ, 393.0/172.0 for IVA, 601.0/422.0 for IS-ELX, 525.0/453.0 for IS-TEZ, and 399.0/178.0 for IS-IVA, respectively. The assay has a dynamic range of 10 to 10,000 ng/mL, with a mean coefficient of determination (r2, mean ± SD) of 0.9970 ± 0.0027 (ELX), 0.9989 ± 0.0004 (TEZ), 0.9981 ± 0.0003 (IVA), regardless of specimen matrix. The mean precision values for all calibration standards ranged from 0.0 to 10.8% (ELX), 0.0 to 6.7% (TEZ), and 0.2 to 5.6% (IVA), while the accuracy for calibration standards was within the range of −5.7 to 3.5% (ELX), −3.2 to 6.0% (TEZ), and −3.8 to 5.2% (IVA). Validation results demonstrated high accuracy (≤7.3, ≤9.8, ≤10.6% deviation) and high precision (≤11.5, ≤6.3, ≤11.0% CV) for the respective ETI quality control samples. This method provides a fully validated assay for ETI quantitation for use in clinical research.

Modulator Therapy in Cystic Fibrosis Patients with cis Variants in F508del Complex Allele: A Short-Term Observational Case Series.

Previous studies reported the influence of cis variants in F508del cystic fibrosis (CF) patients in their responses to CFTR modulators. The current study is a prospective, observational study involving three patients with CF and pancreatic insufficiency, carrying a complex allele including F508del with A238V, I1027T, or L467F. We report clinical data before and after 4 weeks of treatment with tezacaftor (TEZ)/ivacaftor (IVA), elexacaftor (ELX)/TEZ/IVA, and lumacaftor (LUM)/IVA for patients with complex alleles A238V, I1027T, and L467F, respectively. The 50-year-old patient bearing F508del;A238V/D1152H showed a normal sweat test (13 mEq/L) and improvements in forced expiratory volume in the first second (FEV1) (+7 points), body mass index (BMI) (+0.85), and respiratory CF Questionnaire-Revised (CFQ-R) domain (+22.2 points). The 12-year-old patient bearing F508del;I1027T/R709X showed an improvement in a sweat test (−40 mEq/l), FEV1 (+9 points) and the respiratory CFQ-R domain (+16.7 points). No changes in outcomes were observed for the 6-year-old patient F508del;L467F/F508del. Our data highlight that the reported variants do not modify the phenotypic expression of F508del. Searching L467F is crucial in CF patients with F508del nonresponsive to ELX/TEZ/IVA. Further data are needed to evaluate the clinical effect of these variants after a longer follow up.

Theratyping of the Rare CFTR Variants E193K and R334W in Rectal Organoid-Derived Epithelial Monolayers.

Background: The effect of presently available CFTR modulator combinations, such as elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA), on rare CFTR alleles is often unknown. Several assays have been developed, such as forskolin-induced swelling (FIS), to evaluate the rescue of such uncommon CFTR alleles both by established and novel modulators in patient-derived primary cell cultures (organoids). Presently, we assessed the CFTR-mediated electrical current across rectal organoid-derived epithelial monolayers. This technique, which allows separate measurement of CFTR-dependent chloride or bicarbonate transport, was used to assess the effect of ELX/TEZ/IVA on two rare CFTR variants. Methods: Intestinal organoid cultures were established from rectal biopsies of CF patients carrying the rare missense mutations E193K or R334W paired with F508del. The effect of the CFTR modulator combination ELX/TEZ/IVA on CFTR-mediated Cl− and HCO3− secretion was assessed in organoid-derived intestinal epithelial monolayers. Non-CF organoids were used for comparison. Clinical biomarkers (sweat chloride, FEV1) were monitored in patients receiving modulator therapy. Results: ELX/TEZ/IVA markedly enhanced CFTR-mediated bicarbonate and chloride transport across intestinal epithelium of both patients. Consistent with the rescue of CFTR function in cultured intestinal cells, ELX/TEZ/IVA therapy improved biomarkers of CFTR function in the R334W/F508del patient. Conclusions: Current measurements in organoid-derived intestinal monolayers can readily be used to monitor CFTR-dependent epithelial Cl− and HCO3− transport. This technique can be explored to assess the functional consequences of rare CFTR mutations and the efficacy of CFTR modulators. We propose that this functional CFTR assay may guide personalized medicine in patients with CF-like clinical manifestations as well as in those carrying rare CFTR mutations.

Rescue of chloride and bicarbonate transport by elexacaftor-ivacaftor-tezacaftor in organoid-derived CF intestinal and cholangiocyte monolayers

BackgroundIn cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion precipitates the accumulation of viscous mucus in the lumen of respiratory and gastrointestinal epithelial tissues. We investigated whether the combination of elexacaftor (ELX), ivacaftor (IVA) and tezacaftor (TEZ), apart from its well-documented effect on chloride transport, also restores Phe508del-CFTR-mediated bicarbonate transport. MethodsEpithelial monolayers were cultured from intestinal and biliary (cholangiocyte) organoids of homozygous Phe508del-CFTR patients and controls. Transcriptome sequencing was performed, and bicarbonate and chloride transport were assessed in the presence or absence of ELX/IVA/TEZ, using the intestinal current measurement technique. ResultsELX/IVA/TEZ markedly enhanced bicarbonate and chloride transport across intestinal epithelium. In biliary epithelium, it failed to enhance CFTR-mediated bicarbonate transport but effectively rescued CFTR-mediated chloride transport, known to be requisite for bicarbonate secretion through the chloride-bicarbonate exchanger AE2 (SLC4A2), which was highly expressed by cholangiocytes. Biliary but not intestinal epithelial cells expressed an alternative anion channel, anoctamin-1/TMEM16A (ANO1), and secreted bicarbonate and chloride upon purinergic receptor stimulation. ConclusionsELX/IVA/TEZ has the potential to restore both chloride and bicarbonate secretion across CF intestinal and biliary epithelia and may counter luminal hyper-acidification in these tissues.

A Review of Trikafta: Triple Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulator Therapy.

Cystic fibrosis (CF) is a potentially fatal genetic disease that causes serious lung damage. With time, researchers have a more complete understanding of the molecular-biological defects that underlie CF. This knowledge is leading to alternative approaches regarding the treatment of this condition. Trikafta is the third FDA-approved drug that targets the F508del mutation of the CFTR gene. The drug is a combination of three individual drugs which are elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA). This trio increases the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and reduces the mortality and morbidity rates in CF patients. The effectiveness of Trikafta, seen in clinical trials, outperforms currently available therapies in terms of lung function, quality of life, sweat chloride reduction, and pulmonary exacerbation reduction. The safety and efficacy of CFTR modulators in children with CF have also been studied. Continued evaluation of patient data is needed to confirm its long-term safety and efficacy. In this study, we will focus on reviewing data from clinical trials regarding the benefits of CFTR modulator therapy. We address the impact of Trikafta on lung function, pulmonary exacerbations, and quality of life. Adverse events of the different CFTR modulators are discussed.

A rapid RP-HPLC method for the simultaneous estimation of Ivacaftor and Tezacaftor and in silico study of their metabolitic products

BackgroundThis study was designed to develop a reliable method for estimation of Ivacaftor and Tezacaftor in pure and its pharmaceutical dosage form by RP-HPLC in human plasma. Molecular docking studies were carried out and the results were visualized using PyMol and Discovery studio visualizer (Discovery studio visualizer ver. 2.5). The pharmacokinetic properties such as Swiss ADME and pKCSM of the Ivacaftor and its metabolites Ivacaftor M1, M6 and Tezacaftor and metabolites Tezacaftor M1, M2 were predicted. In admetSAR, web-based query tools incorporating a molecular built-in interface enable the database to be queried by SMILES.ResultsA simple, linear, precise, and accurate RP-HPLC method was developed and validated for the determination of Ivacaftor (IVA) and Tezacaftor (TEZ) in human plasma. Chromatographic separation was achieved isocratically on Inspire C18, (4.6 × 250 mm, 5 μm) column at 30 °C. Mobile phase consisting of methanol and 0.05% formic acid in ratio of 95:5 with flow rate of 1 mL/min with injection volume 20 μl detector used is PDA at 235 nm. The developed method was validated according to ICH guidelines and found to be linearity range was found to be for TEZ (10–50 μg/mL) and IVA (15–75 μg/mL). IVA and TEZ drugs and its metabolites were retrieved from the PubChem database and the 2D chemical structures were generated from SMILES notation by using the Chemsketch Software. The structure was viewed using Swiss-PDB Viewer to form a better understanding of the molecule for toxicity and biological activity prediction.ConclusionThe results obtained by the proposed method from validation parameters and from assay confirmed that the determination of Tezacaftor (TEZ) and Ivacaftor (IVA) in their combined dosage form in human plasma was sensitive and selective method. In silico study has revealed that IVA and its metabolites IVA M1, IVA M6 are according to Lipinski rule. The oral bioactivity of IVA was found to be more when compared to its metabolites (Molinspiration) and TEZ and its metabolites TEZ M1, TEZ M2 even though they have the molecular weight > 500, but all other parameters from Molinspiration revealed better oral bioactivity of TEZ M2. Validation of the developed isocratic RP-HPLC procedure revealed that, regardless of how the sample was purified, the method was characterized by good linearity, sensitivity, reproducibility, specificity, and low values of LOD (0.090 μg/mL) and LOQ (0.275 μg/mL). From the in silico docking results, it is quite evident that metabolites of TEZ and IVA have the great potential against cystic fibrosis.

Stability Indicating RP-HPLC Method Development and Validation for the Simultaneous Estimation of Tezacaftor and Ivacaftor in Bulk and Pharmaceutical Dosage Form

Stability Indicating RP-HPLC Method Development and Validation for the Simultaneous Estimation of Tezacaftor and Ivacaftor in Bulk and Pharmaceutical Dosage Form

ICER posts scoping document on tezacaftor and lumacaftor

ICER posts scoping document on tezacaftor and lumacaftor