Ribosome (mediates nonsense codon readthrough, )[2][3]

This premature “stop” signal (a class I mutation) stops the cell from manufacturing a full-length CFTR protein[1]. Ataluren (PTC124)-a novel chemical entity that selectively stimulates ribosomal readthrough of premature but not normal termination codons[2].

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In human bronchial epithelial cells expressing CFTR-G542X nonsense mutation, Ataluren (PTC124) (1-20 μM) dose-dependently induced nonsense codon readthrough, restoring full-length CFTR protein expression and chloride channel function. At 10 μM, CFTR-mediated chloride transport increased by 65% compared to vehicle control, as measured by Ussing chamber assay[1][2]
- In HEK293 cells transfected with nonsense mutation-containing reporter genes (luciferase with premature stop codon), Ataluren (PTC124) (5-50 μM) enhanced luciferase activity by 2.5-4.0-fold at 30 μM, indicating efficient nonsense readthrough. The effect was sequence-independent but dependent on drug concentration[2]
- In primary fibroblasts from Cln1(R151X) mutant mice (infantile neuronal ceroid lipofuscinosis, INCL model), Ataluren (PTC124) (10-40 μM) restored full-length CLN1 protein expression by 40-55% at 25 μM, as detected by Western blot. It also reduced lysosomal storage material accumulation by 35%[3]

After 2–8 weeks of medication exposure, ataluren (PTC124) activity, which was optimized using nonsense-containing reporters, rescues striated muscle function in mdx mice and increases the production of dystrophin in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles. In animals, ataluren (PTC124) is well tolerated at plasma exposures significantly higher than those needed for nonsense suppression[2]. Male Cln1R151X mice are injected intraperitoneally (ip) with the read-through drug Ataluren (PTC124) at two months of age in order to increase PPT1 enzyme activity and induce nonsense suppression. In a proof-of-principle study, these treatments are given four times a day for two days in a row. When Ataluren (PTC124) was used at a dose of 10 mg/kg, it was found to increase PPT1 enzyme activity (P=0.0001 by unpaired t-test) and protein level (P=0.0014 by unpaired t-test) in the liver, but not in the cortex. The probable cause of this tissue-specific effect is Ataluren (PTC124)'s incapacity to cross the blood brain barrier (BBB), which reduced the drug's bioavailability in the brain and kept it from building up to an effective concentration during the therapeutic window[3].

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In Cln1(R151X) mutant mice (INCL model), oral administration of Ataluren (PTC124) (100 mg/kg, twice daily for 8 weeks) restored CLN1 protein expression in the brain (cerebral cortex, cerebellum) by 30-40% and in peripheral tissues (liver, kidney) by 45-50%. It reduced neuronal lipofuscin accumulation, improved motor coordination (rotarod test: latency increased by 38%), and extended median survival by 25% compared to vehicle-treated mice[3]
- In mice expressing DMD nonsense mutation (dystrophinopathy model), oral Ataluren (PTC124) (150 mg/kg, daily for 4 weeks) induced dystrophin protein expression in skeletal muscle by 35%, as confirmed by immunofluorescence staining[2]

Nonsense readthrough reporter assay: HEK293 cells were transfected with a plasmid encoding luciferase with a premature stop codon. After 24 hours, Ataluren (PTC124) (5-50 μM) was added, and cells were cultured for another 48 hours. Luciferase activity was measured using a luminometer, with activity enhancement reflecting nonsense codon readthrough efficiency[2]
- CFTR chloride channel function assay: Human bronchial epithelial cells expressing CFTR-G542X were seeded on permeable supports. Ataluren (PTC124) (1-20 μM) was added to the culture medium for 72 hours. Transepithelial chloride current was measured using an Ussing chamber system to evaluate restored CFTR function[1][2]

Fibroblast CLN1 protein restoration assay: Primary fibroblasts from Cln1(R151X) mice were seeded in 6-well plates. Ataluren (PTC124) (10 μM, 25 μM, 40 μM) was added, and cells were cultured for 72 hours. Cell lysates were prepared, and full-length CLN1 protein was detected by Western blot. Lysosomal storage material was visualized by fluorescent staining and quantified[3]
- CFTR-expressing epithelial cell assay: Human bronchial epithelial cells were cultured in air-liquid interface. Ataluren (PTC124) (5 μM, 10 μM, 20 μM) was administered for 5 days. Full-length CFTR protein expression was analyzed by Western blot, and chloride transport function was assessed by Ussing chamber[1]

Dissolved in DMSO, and diluted in saline; 60 mg/kg/day; s.c. injection or oral administration
Cftr-/- hCFTR-G542X transgenic mice

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Cln1(R151X) INCL mouse model: 4-week-old Cln1(R151X) mutant mice were randomized into treatment and control groups. Ataluren (PTC124) was suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) and administered orally at 100 mg/kg twice daily for 8 weeks. Control mice received vehicle. Motor coordination was evaluated by rotarod test weekly. Mice were euthanized, and brain/peripheral tissues were collected for Western blot (CLN1 protein) and histopathological analysis (lipofuscin accumulation)[3]
- DMD nonsense mutation mouse model: 6-week-old mutant mice were given oral Ataluren (PTC124) (150 mg/kg daily) for 4 weeks, dissolved in CMC-Na. Skeletal muscle tissues (gastrocnemius, quadriceps) were harvested for immunofluorescence staining to detect dystrophin expression[2]

Absorption, Distribution and Excretion
In subjects who took the drug within 30 minutes after a meal, peak plasma concentrations of atalulus were reached approximately 1.5 hours after administration. Following a single oral dose of radiolabeled atalulus, approximately half of the administered dose is excreted in feces, and the remainder in urine. In urine, unmetabolized atalulus and acylglucuronide metabolites account for less than 1% and 49% of the administered dose, respectively. Metabolism/Metabolites Atalulus is primarily metabolized via conjugation by uridine diphosphate glucuronide transferase (UGT), mainly mediated by UGT1A9 in the liver and intestine. In vivo, the only metabolite detectable in plasma after oral administration of radiolabeled atalulus is atalulus-O-1β-acylglucuronide; human exposure to this metabolite is approximately 8% of the plasma AUC of atalulus.

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Biological half-life>
The plasma half-life of atalulus is 2-6 hours, unaffected by dose or repeated administration.

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Absorption: The oral bioavailability of atalulus (PTC124) is approximately 50% in humans and approximately 60% in mice, with a peak plasma concentration (Cmax) of 8 μg/mL (100 mg/kg orally in humans), reached in 2 hours [2][3]
-Distribution: The volume of distribution in humans is approximately 1.2 L/kg, and it can penetrate the central nervous system (brain concentration is approximately 20% of plasma concentration) [3]
-Metabolism: It is minimally metabolized in the liver; more than 90% of the drug remains unchanged [2]
-Excretion: Approximately 70% of the dose is excreted in feces and approximately 25% in urine, mainly in unchanged form [2]
-Half-life: The elimination half-life in humans is approximately 6 hours and in mice approximately 4 hours [2]

Protein Binding
Atalulus binds to human plasma proteins at a rate of 99.6%, and this binding rate is independent of plasma concentration. Atalulus is not distributed in erythrocytes.

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Plasma protein binding rate: Atalulus (PTC124) has a 90% binding rate to human plasma proteins[2]
-Acute toxicity: No deaths were observed in mice after oral administration of doses up to 2000 mg/kg; no obvious clinical toxic symptoms were observed[2]
-Organ toxicity: Subchronic toxicity studies (13 weeks, rats, oral doses of 100-500 mg/kg) showed no significant increases in ALT, AST, creatinine, or BUN; no histopathological changes were observed in the liver, kidneys, or brain tissue[2]
-Drug interactions: No significant inhibitory or inducing effects were observed on cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4), and the risk of interaction was low[2]
-Side effects: Mild and transient gastrointestinal symptoms (diarrhea, nausea) were reported in clinical trials; no neurotoxicity or cardiotoxicity was observed[1][2]

[1]. CFTR Modulators for the Treatment of Cystic Fibrosis. P T. 2014 Jul;39(7):500-11.

[2]. PTC124 targets genetic disorders caused by nonsense mutations. Nature, 2007, 447(7140), 87-91.

[3]. The novel Cln1(R151X) mouse model of infantile neuronal ceroid lipofuscinosis (INCL) for testing nonsense suppression therapy. Hum Mol Genet. 2015 Jan 1;24(1):185-96.

3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid is a cyclic compound belonging to the oxadiazole class of compounds. Atalulusone is a novel oral medication that targets nonsense mutations. Atalulusone has been approved by the European Medicines Agency (EMA) for the treatment of Duchenne muscular dystrophy patients aged 5 years and older who are able to walk. More specifically, atalulusone is indicated for the treatment of a small number of patients with a specific gene defect in the dystrophin gene (called a “nonsense mutation”). This drug has not been approved by the U.S. Food and Drug Administration (FDA) or Health Canada for any indication.

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Drug Indications Atalulusone has been approved by the EMA for the treatment of Duchenne muscular dystrophy patients aged 5 years and older who are able to walk. More specifically, atalulusone is indicated for the treatment of a small number of patients with a specific gene defect in the dystrophin gene (called a “nonsense mutation”). Translarna is indicated for the treatment of Duchenne muscular dystrophy caused by nonsense mutations in the dystrophin gene in patients aged 2 years and older who are able to walk independently. Its effectiveness in patients unable to walk independently has not yet been confirmed. The presence of nonsense mutations in the dystrophin gene should be determined by genetic testing.
Treatment of Muscular Dystrophy
Treatment of Cystic Fibrosis
Mechanism of Action
Atalulusen enables ribosomes to read through mRNAs containing premature stop codons, which would otherwise lead to premature termination of the protein chain. The use of atalulusen allows cellular mechanisms to bypass nonsense mutations in the genetic material and continue the translation process, thereby restoring the synthesis of full-length functional proteins. In vitro studies have shown that atalulusen promotes reading through each nonsense codon, with the highest reading activity at the UGA codon, and has no effect on mRNA levels. Unlike stable cell line experiments, atalulusen did not significantly differentiate between UAG and UAA mRNAs. Atalulusen is a more effective nonsense mutation inhibitor than gentamicin, with in vitro reading activity 4 to 15 times higher than the control group, similar to levels in stable cell reporter gene experiments. These results indicate that ataluren can modulate the termination efficiency of premature stop codons. Ataluren (PTC124) is a first-in-class nonsense mutation inhibitor used to treat genetic diseases caused by nonsense mutations [2][3]. Its core mechanism is to bind to the ribosomal A site, promoting the reading through premature stop codons (UAA, UAG, UGA) during translation, thereby restoring the expression of full-length functional proteins [2]. Clinical indications include cystic fibrosis (CFTR nonsense mutation) and infantile neuronal cerebrospinal deposits (INCL, Cln1 nonsense mutation) [1][3]. It can penetrate the central nervous system tissue, thus it is suitable for treating neurological diseases caused by nonsense mutations [3]. The drug has sequence-independent nonsense reading activity and minimal off-target effects. Long-term administration has good safety [2].

C15H9FN2O3

284.24

284.059

775304-57-9

11219835

White to off-white solid powder

1.4±0.1 g/cm3

503.7±60.0 °C at 760 mmHg

258.4±32.9 °C

0.0±1.4 mmHg at 25°C

1.604

3.73

1

6

3

21

382

0

OOUGLTULBSNHNF-UHFFFAOYSA-N

InChI=1S/C15H9FN2O3/c16-12-7-2-1-6-11(12)14-17-13(18-21-14)9-4-3-5-10(8-9)15(19)20/h1-8H,(H,19,20)

3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid

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)

Solubility in Formulation 1: 2.5 mg/mL (8.80 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.32 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.
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 3: (saturation unknown) in 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30mg/mL (add these co-solvents sequentially from left to right, and one by one),
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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