PTC124 (Ataluren) is a novel small molecular CFTR-G542X nonsense allele inhibitor. In safety pharmacology studies in rats and dogs, oral administration of PTC124 (Ataluren) induces no adverse neurological, pulmonary, or cardiovascular effects at doses through 1500 mg/kg. In toxicology studies in rats and dogs at oral doses through 1500 mg/kg for 28 days, PTC124 (Ataluren) has shown good tolerability. Both PTC124 doses were similarly active, improving total chloride transport with a combined mean change of -5.4 mV (p<0.001), and on-treatment responses (at least -5 mV improvement) and hyperpolarisations (values more electrically negative than -5 mV) in 61% (p<0.001) and 56% (p = 0.002) of patients. CFTR function was greater with time and was accompanied by trends toward improvements in pulmonary function and CF-related coughing. The high readthrough efficiency of PTC124 in combination with excellent biocompatibility makes it a promising therapeutic agent for PTCs in USH1C.
|Cell lines||Grip-Tite 293 cells transfected with the pFLuc190UGA construct|
|Preparation method||Luciferase Reporter Assays. All cell-based assays were performed in 1,536-well plates with an assay volume of 4.5 μL per well. Comparative cell-based experiments were performed with cells from the same batch of transfected cells to control for transfection efficiency. Cells were incubated with compound or antibiotic for 16–72 h. In experiments with Grip-Tite cells, cells were washed with PBS before the addition of detection reagent containing the FLuc or RLuc substrate. Luminescence from luciferase activity was detected by using ViewLux (PerkinElmer). Experimental plates contained the transcriptional activator MS-275 as a positive control and DMSO-treated cells or mock-transfected cells as a negative control. Percentage activity was defined as the percentage signal above DMSO-treated cells.|
|Incubation time||24 hr|
|Animal models||Cftr−/− hCFTR-G542X mice|
|Dosages||15, 30 or 60mg/kg initiated 16 days after birth and continued once daily until the animals were killed|
|Body Surface Area (m2)||0.007||0.025||0.15||0.05||0.02||0.5|
|Animal A (mg/kg) = Animal B (mg/kg) multiplied by||Animal B Km|
|Animal A Km|
For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.
|Solubility||DMSO ≥55 mg/mL|
|Source||Mol Pharm (2014). Figure 4. PTC124|
|Cell Lines||HeLa FLuc-opal cell|
|Concentrations||6, 9, and 12 μM|
|Incubation Time||72 h|
|Results||After exposition of HeLa cells to 6 μM, 9 μM, and 12 μM PTC124 we observed the presence of green cells indicating that PTC124 induced the production of a full length functional H2B-GFP protein, despite the presence of a premature stop codon in its coding sequence, as evidenced by the clearly dose-dependent increase in the number of green cells.|
|Source||Mol Pharm (2014). Figure 3. PTC124|
|Method||luciferase activity assay|
|Cell Lines||HeLa FLuc-opal cell|
|Incubation Time||24 h|
|Results||Following PTC124 exposition pFLuc190UGA transfected HeLa cells showed an increase of luciferase activity, this result is consistent with what previously published|
PTC124-mediated translational readthrough of a nonsense mutation causing Usher syndrome type 1C.
Goldmann et al. Hum Gene Ther. 2011 May;22(5):537-47. PMID: 21235327.
Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis.
Wilschanski et al. Eur Respir J. 2011 Jul;38(1):59-69. PMID: 21233271.
Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression.
Auld DS, et al. Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3585-90. PMID: 19208811.
PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model.
Du M, et al. Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):2064-9. PMID: 18272502.
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