CFTR/cystic fibrosis transmembrane conductance regulator
(R)-Olacaftor specifically targets the cystic fibrosis transmembrane conductance regulator (CFTR) protein. As a CFTR modulator, it is designed to bind to the defective CFTR protein and modify its function. In the context of CF research, (R)-Olacaftor is used as a tool to study the correction or potentiation of CFTR activity..

Specific in vitro activity data, such as EC50 or IC50 values, for (R)-Olacaftor is not detailed in the provided search results. However, it is classified as a CFTR modulator with promising potential for CF research, indicating that it is expected to have biological activity on the CFTR protein, likely acting as a corrector to improve its processing and trafficking or as a potentiator to enhance its channel function..

Olacaftor (VX-440) a next generation corrector was assessed in a phase 2 trial, randomized, double blind, placebo, and active-controlled study designed to evaluate the safety and tolerability of VX-440 in triple combination with tezacaftor and ivacaftor in patients with CF who are heterozygous for the F508del mutation and a MF CFTR mutation not likely to respond to tezacaftor and/or ivacaftor therapy (F508del-MF), or who are homozygous for the F508del mutation (ClinicalTrials.gov Identifier: NCT02951195)[https://pmc.ncbi.nlm.nih.gov/articles/PMC7088950/].

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Specific in vivo activity data for (R)-Olacaftor is not detailed in the provided search results. The parent compound Olacaftor (as part of combination therapies) has been evaluated in preclinical studies. Given that (R)-Olacaftor is the R-enantiomer, its in vivo efficacy would be assessed in a similar manner to other CFTR modulators: by measuring improvements in CFTR-dependent chloride transport in animal models of cystic fibrosis (such as the CFTR-knockout mouse expressing the human F508del-CFTR mutation).

There is no specific cell-free enzyme/receptor binding protocol for (R)-Olacaftor detailed in the search results. As a CFTR modulator, its activity is typically assessed using cell-based assays that measure CFTR protein processing and chloride channel function. For an isolated system, researchers could perform a binding assay using a radiolabeled CFTR modulator and purified CFTR protein reconstituted into liposomes, though this is a highly specialized and challenging protocol. Therefore, the standard method for evaluating this compound is through cellular assays.

The standard in vitro cellular assay for evaluating CFTR modulators like (R)-Olacaftor involves the use of Fischer rat thyroid (FRT) cells or primary human bronchial epithelial (HBE) cells that are stably transfected with the human F508del-CFTR mutation (the most common cause of CF). The cells are seeded in 96-well plates and allowed to adhere. Then, they are treated with varying concentrations of the compound (0.001 uM to 10 uM) for 24-48 hours. After incubation, CFTR function is measured using a fluorescence-based plate reader in a halide-sensitive yellow fluorescent protein (HS-YFP) quenching assay. The cells are exposed to an iodide gradient, and the rate of fluorescence decay is recorded. The EC50 value is calculated from the dose-response curve.

An in vivo protocol for (R)-Olacaftor would likely be similar to that of other CFTR modulators. It would involve a relevant animal model, such as the F508del-CFTR rat or a CFTR knockout mouse expressing human F508del-CFTR. The compound would be administered by oral gavage at doses ranging from 1-30 mg/kg, typically once daily for 14-28 days. Efficacy would be assessed by measuring CFTR-dependent chloride secretion in the intestinal tract using Ussing chamber technology (short-circuit current). Secondary endpoints would include improvement in weight gain, survival, and histological analysis of lung and intestinal tissues to assess the reduction of disease pathology.

Detailed pharmacokinetic (PK) data for (R)-Olacaftor is not provided. However, the parent compound Olacaftor is an orally administered small molecule. For research purposes, (R)-Olacaftor (MW 539.66) is solubilized in DMSO for in vitro experiments (100 mg/mL) and can be formulated for in vivo studies using a vehicle such as 10% DMSO/40% PEG300/5% Tween-80/45% saline. It would be expected to be absorbed from the gastrointestinal tract and metabolized by the liver, likely by cytochrome P450 enzymes (e.g., CYP3A4). Its terminal half-life would be determined by the rate of metabolism.

Specific toxicological data for (R)-Olacaftor is not available. Standard safety screening for CFTR modulators includes an in vitro MTT assay on primary human hepatocytes to assess general cytotoxicity and a hERG (human Ether-à-go-go-Related Gene) channel inhibition test to assess the risk for cardiac arrhythmia (QT prolongation). A CYP450 (cytochrome P450) inhibition panel would be performed to evaluate the potential for drug-drug interactions. In vivo, a 14-day repeat-dose oral toxicity study in rats would be required to determine the Maximum Tolerated Dose (MTD) and identify any target organ toxicity.

[1]. Modulators of Cystic Fibrosis Transmembrane Conductance Regulator. Patent US20160095858A1.

Background: Cystic fibrosis (CF) is a common genetic disorder that shortens lifespan and is caused by mutations in the cystic fibrosis transmembrane transport regulator (CFTR) protein. The class II CFTR mutation F508del is the most common pathogenic mutation (found in up to 90% of CF patients). The F508del mutation results in the loss of CFTR protein function—the defective protein is degraded before reaching the cell membrane, the site essential for CFTR proteins to perform transepithelial salt transport. Corrective therapies hold promise for many CF patients. This review evaluated single corrective agents (monotherapy) and any combination of corrective agents (most commonly lumacaftor, tezacaftor, elexacaftor, VX-659, olacaftor (VX-440), or VX-152) with enhancers (e.g., ivacaftor) (dual and triple therapies).
Objective: To evaluate the clinical benefits and harms of CFTR correctors (with or without combination with enhancers) in patients with cystic fibrosis (pwCF) of any age carrying class II CFTR mutations (most commonly F508del).
Search Methods: We searched the Cochrane Cystic Fibrosis Trial Registry (November 28, 2022), reference lists of relevant articles, and online trial registries (December 3, 2022).
Inclusion Criteria: Randomized controlled trials (RCTs) comparing the use of CFTR correctors in patients with cystic fibrosis carrying class II CFTR mutations to control groups (parallel design).
Data Collection and Analysis: Data were independently extracted by two authors, risk of bias was assessed, and the degree of certainty of evidence (GRADE) was determined; we contacted researchers for further data.
Main results: We included 34 RCTs (4781 participants) with durations ranging from 1 day to 48 weeks; two extensions of the lumacaftor-ivacaftor study provided an additional 96 weeks of safety data (1029 participants). We evaluated 8 monotherapy RCTs (344 participants) (4PBA, CPX, lumacaftor, cavsonstat, and FDL169), 16 dual-drug RCTs (2627 participants) (lumacaftor-ivacaftor or tezacaftor-ivacaftor), and 11 triple-drug RCTs (1804 participants) (elexacaftor-tezacaftor-ivacaftor/deutivacaftor; VX-659-tezacaftor-ivacaftor/deutivacaftor; Olaftor (VX-440)-tezacaftor-ivacaftor; VX-152-tezacaftor-ivacaftor). Of the participants in 21 randomized controlled trials (RCTs), 7 had participants with the F508del/F508del genotype, 7 had participants with the F508del/minimum function (MF) genotype, 1 had participants with the F508del/gated genotype, 1 had participants with the F508del/F508del or F508del/residual function genotype, 1 had participants with the F508del/gated genotype or F508del/residual function genotype, and 3 had participants with the F508del/F508del or F508del/MF genotype. Risk of bias assessments varied across comparisons. Due to age limitations (e.g., adults only) or non-standard designs (e.g., switching from monotherapy to combination therapy), results from 16 RCTs may not be applicable to all patients with cystic fibrosis. No deaths or clinically meaningful improvements in quality of life (QoL) were observed in monotherapy studies. There is insufficient evidence to determine its effect on lung function. Randomized controlled trials (RCTs) of monotherapy without placebo control showed differences in mild, moderate, or severe adverse events (AEs); the clinical significance of these events was difficult to assess due to the wide variety of adverse events and the small number of participants (all F508del/F508del mutants). Regarding dual therapy, one death occurred in the tezacaftor-ivacaftor group (considered unrelated to the study drug). At all time points, both the lumacaftor-ivacaftor and tezacaftor-ivacaftor treatment groups showed better quality of life scores (respiratory domain) compared to placebo (moderate certainty evidence). At 6 months, all dual therapy groups showed an improvement in the percentage of predicted forced expiratory volume in one second (FEV1) compared to placebo (high to moderate certainty evidence). More patients with cystic fibrosis reported early transient dyspnea after using lumacaftor-ivacaftor (odds ratio (OR) 2.05, 99% confidence interval (CI) 1.10 to 3.83; I² = 0%; 2 studies, 739 participants; high-quality evidence). Over 120 weeks (initial study period and follow-up period), patients (80 participants) taking 400 mg lumacaftor-ivacaftor twice daily experienced an increase in systolic blood pressure of 5.1 mmHg and an increase in diastolic blood pressure of 4.1 mmHg. These adverse events were not reported in randomized controlled trials of tezacaftor-ivacaftor. Compared with placebo, patients with cystic fibrosis receiving ivacato in combination with other therapies had a lower rate of acute pulmonary exacerbations (all evidence was of moderate certainty): lumamacato 600 mg (hazard ratio (HR) 0.70, 95% confidence interval 0.57 to 0.87; I² = 0%; 2 studies, 739 participants); lumamacato 400 mg (HR 0.61, 95% confidence interval 0.49 to 0.76; I² = 0%; 2 studies, 740 participants); and tezacato (HR 0.64, 95% confidence interval 0.46 to 0.89; 1 study, 506 participants). Triple therapy: No deaths were reported in studies (high certainty evidence). All other evidence was of low to moderate certainty. Patients in the triple therapy group may have improved respiratory domain scores for quality of life at six months compared with the control group (6 studies). Triple therapy may result in greater relative and absolute changes in FEV1 as a percentage of predicted value (four studies for all combinations). For F508del/MF patients receiving elexacaftor-tezacaftor-ivacaftor, the absolute change in FEV1 as a percentage of predicted value was likely greater than in the placebo group (mean difference 14.30, 95% CI 12.76 to 15.84; 1 study, 403 participants; moderately definitive evidence), with similar results observed for other drug combinations and genotypes. There was little difference in the incidence of adverse events between the triple therapy and control groups (10 studies). No studies reported the timing of the next acute pulmonary exacerbation, but in F508del/F508del patients treated with elexacaftor-tezacaftor-ivacaftor, the proportion of acute pulmonary exacerbations at 4 weeks was low (OR 0.17, 99% CI 0.06 to 0.45; 1 study, 175 participants), and the proportion of acute pulmonary exacerbations at 24 weeks was also low (OR 0.29, 95% CI 0.14 to 0.60; 1 study, 405 participants); similar results were observed with other triple therapies and genotype combinations.
Author Conclusion: There is insufficient evidence to suggest that monotherapy with corrective agents is clinically meaningful in patients with F508del/F508del cystic fibrosis. Other data in this review reduce the evidence for the efficacy of dual therapies; these agents are no longer considered standard treatments. Their use may be appropriate in special circumstances (e.g., patients who cannot tolerate triple therapy or due to age). Both dual therapies (lumacaftor-ivacaftor and tezacaftor-ivacaftor) provided similar, slight improvements in quality of life and respiratory function, and reduced the rate of acute pulmonary exacerbations. While the efficacy in quality of life and FEV1 remained superior to dual therapy, it was lower than in our previous studies. Lumacaftor-ivacaftor was associated with early transient dyspnea and long-term hypertension (this was not observed with tezacaftor-ivacaftor). Tezacaftor-ivacaftor had a better safety profile, but data in children under 12 years of age were lacking. In children under 12 years of age, lumacaftor-ivacaftor significantly improved respiratory function without significant short-term safety issues; however, the hypertension and dyspnea observed in long-term adult data should be weighed when considering lumacaftor-ivacaftor. Data from triple therapy trials showed improvements in several key endpoints, including FEV1 and quality of life. For patients aged 12 years and older with cystic fibrosis (pwCF) carrying one or two F508del variants, the incidence of adverse events with triple therapy (elexacaftor-tezacaftor-ivacaftor/deutivacaftor; VX-659-tezacaftor-ivacaftor/deutivacaftor; Olaftor (VX-440)-tezacaftor-ivacaftor; VX-152-tezacaftor-ivacaftor) may differ little or no (moderate certainty evidence). Further randomized controlled trials are needed in children under 12 years of age and in patients with more severe lung disease. Reference: Cochrane Database Syst Rev. 2023 Nov 20;11(11):CD010966. https://pubmed.ncbi.nlm.nih.gov/37983082/

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(R)-Olacaftor ((R)-VX-440) is a research-grade compound and is not approved for clinical use. It is an enantiomer of Olacaftor, which is a CFTR modulator that has been investigated in clinical trials for the treatment of cystic fibrosis. This compound serves as a valuable tool for researchers to study the stereospecific pharmacology of CFTR modulators and to understand the precise molecular interactions between the drug and its target. It is typically stored at -20degC and is soluble in DMSO..

C29H34FN3O4S

539.661369800568

539.225

C, 64.54; H, 6.35; F, 3.52; N, 7.79; O, 11.86; S, 5.94

1899111-41-1

Olacaftor;1897384-89-2

130203218

White to light yellow solid powder

6.3

1

7

8

38

902

1

S(C1C=CC=CC=1)(NC(C1=CC=C(C2C=C(C=C(C=2)OCC(C)C)F)N=C1N1C[C@H](C)CC1(C)C)=O)(=O)=O

NHOUNZMCSIHKHJ-HXUWFJFHSA-N

InChI=1S/C29H34FN3O4S/c1-19(2)18-37-23-14-21(13-22(30)15-23)26-12-11-25(27(31-26)33-17-20(3)16-29(33,4)5)28(34)32-38(35,36)24-9-7-6-8-10-24/h6-15,19-20H,16-18H2,1-5H3,(H,32,34)/t20-/m1/s1

N-(benzenesulfonyl)-6-[3-fluoro-5-(2-methylpropoxy)phenyl]-2-[(4R)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide

(R)-Olacaftor; 1899111-41-1; SCHEMBL19097503; (R)-VX-440;

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: 250 mg/mL (463.25 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.85 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 (3.85 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.)