PTC124

Biological Activity

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.

Customer Product Validations & Biological Datas

	Source	Mol Pharm (2014). Figure 4. PTC124
	Method	fluorescence microscopy
	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
	Concentrations	12 μM
	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

Protocol

Cell Experiment
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.
Concentrations	0~100μM
Incubation time	24 hr

Animal Experiment
Animal models	Cftr−/− hCFTR-G542X mice
Formulation	saline
Dosages	15, 30 or 60mg/kg initiated 16 days after birth and continued once daily until the animals were killed
Administration	Subcutaneous injections

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

Chemical Information

References

[1] Goldmann et al. Hum Gene Ther. PTC124-mediated translational readthrough of a nonsense mutation causing Usher syndrome type 1C.

[2] Wilschanski et al. Eur Respir J. Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis.

[3] Auld DS, et al. Proc Natl Acad Sci U S A. Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression.

[4] Du M, et al. Proc Natl Acad Sci U S A. PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model.