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| Targets |
Dystrophin (DMD) pre-mRNA. Eteplirsen binds to a specific region within exon 51 of the dystrophin pre-mRNA. By sterically blocking the spliceosome's access to this exon, it causes the splicing machinery to skip over exon 51, joining exon 50 to exon 52. This restores the disrupted open reading frame (ORF) in patients with certain deletions in the DMD gene (e.g., deletion of exon 50 or exon 45-50). The mechanism produces a shortened, internally deleted but functional dystrophin protein, converting a severe Duchenne phenotype to a milder Becker-like phenotype. The restoration of dystrophin synthesis is the therapeutic effect.
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| ln Vitro |
By specifically skipping exon 51 in the faulty gene variant, eteplirsen restores the translational reading frame in Duchenne muscular dystrophy (DMD) and subsequently increases the production of dystrophin [1].
As an antisense oligonucleotide, eteplirsen is not used in cell-free enzyme inhibition assays. Its activity is measured in cell-free systems by determining its ability to hybridize to its complementary RNA target sequence. Melting temperature (Tm) analysis is performed by mixing the PMO with a synthetic RNA oligonucleotide corresponding to the target site in exon 51. The mixture is heated (25-95degC), and the absorbance at 260 nm is monitored to determine the Tm, which is a measure of binding affinity. Eteplirsen forms a stable duplex (Tm > 60degC). RNase H cleavage assays are not applicable, as PMOs do not recruit RNase H; they act by steric blockage. |
| ln Vivo |
In cell-based assays, eteplirsen is tested in DMD patient-derived myoblasts that have a deletion mutation amenable to exon 51 skipping (e.g., deletion of exon 50). Cells are cultured in a differentiation medium to form myotubes. Eteplirsen is added to the medium (free uptake) at concentrations of 0.1-100 uM for 48-96 hours. Exon skipping is measured by RT-PCR using primers in exons 49 and 53, and the product is resolved by capillary electrophoresis. The percentage of exon 51 skipping is quantified. Dystrophin protein restoration is measured by Western blotting using a monoclonal antibody (e.g., DYS-1) and immunofluorescence staining to confirm membrane localization. Eteplirsen restores dystrophin expression to a small percentage of normal levels (typically 0.5-5%). The EC50 for exon 51 skipping is in the low micromolar range.
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| Enzyme Assay |
Eteplirsen's activity can be assessed in a cell-free splicing assay using a minigene construct. A plasmid containing the human DMD gene exons 50-52 with flanking introns is engineered. The minigene is transcribed in vitro to produce pre-mRNA. The pre-mRNA is incubated with a nuclear extract (e.g., from HeLa cells) supplemented with ATP. Eteplirsen (0.1-10 uM) is added to the reaction. After 2-4 hours, RNA is extracted, and RT-PCR is performed using primers spanning exons 50-52. The product is run on a gel to visualize the skipped (exon 51 absent) vs. full-length product. This confirms that the antisense oligonucleotide directly inhibits spliceosome assembly.
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| Cell Assay |
For cellular activity, primary human myoblasts from DMD patients with a mutation amenable to exon 51 skipping (e.g., deletion of exon 50) are used. Cells are cultured in a differentiation medium (DMEM + 2% horse serum) for 5-7 days to form myotubes. Eteplirsen is added at concentrations of 0.1-100 uM during the last 48-72 hours of differentiation. RNA is extracted with Trizol. Exon skipping is evaluated by nested RT-PCR using a primary PCR (exons 49 and 53) and a secondary PCR with fluorescently labeled primers. Products are resolved by capillary electrophoresis. Percent skipping is calculated based on peak area. Dystrophin protein is quantified by Western blot using a DYS2 antibody and normalized to alpha-actinin. Immunofluorescence on myotube cultures visualizes sarcolemmal dystrophin. The limit of detection for protein is typically around 1-2% of normal.
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| Animal Protocol |
In vivo efficacy of eteplirsen is evaluated in the mdx52 mouse model, which lacks exon 52 (some models) or a hDMD transgenic mouse model. Mice are treated with eteplirsen at doses of 10-100 mg/kg via intravenous (IV) or subcutaneous (SC) injection weekly or twice weekly for 4-12 weeks. The primary endpoint is the restoration of dystrophin protein in skeletal muscles (quadriceps, diaphragm, heart), measured by Western blot and immunofluorescence. Functional tests include grip strength and treadmill running tests. Serum creatine kinase (CK) levels are measured as a biomarker of muscle damage. Histological analysis of muscle sections includes H&E staining to assess central nucleation and fibrosis. Eteplirsen has been shown to produce significant dystrophin expression (1-5% of normal) in animal models.
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| ADME/Pharmacokinetics |
Eteplirsen is administered intravenously in humans. In preclinical PK studies in animals, following IV or SC administration, eteplirsen shows a short plasma half-life (hours) but prolonged retention in muscle (days). It is distributed primarily to the kidney, liver, and muscle. The compound is cleared by endocytosis and lysosomal degradation. It is not metabolized by CYP enzymes, so drug-drug interactions are minimal. The major excretion route is urine. In human PK studies, eteplirsen (e.g., 30 mg/kg weekly IV) shows linear PK. The Tmax is at the end of infusion, and the elimination half-life is 2-4 hours for plasma. The muscle half-life is much longer. The high molecular weight and hydrophilicity prevent oral absorption.
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| Toxicity/Toxicokinetics |
In preclinical toxicology studies, eteplirsen was generally well-tolerated at clinically relevant doses. The most common findings in animals were injection site reactions, minimal to moderate glomerulonephritis, and tubular vacuolization in the kidney at high doses, which were reversible. No significant hepatotoxicity, cardiotoxicity, or CNS effects were observed. In human clinical trials (Phase 2 studies), the most common adverse events were vomiting, pyrexia, nasopharyngitis, and cough. Proteinuria (transient) has been observed. No serious drug-related adverse events or deaths were reported. It is not genotoxic. The FDA approved eteplirsen in 2016 under a controversial accelerated approval based on a surrogate endpoint (increased dystrophin production). Long-term safety is monitored.
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| References | |
| Additional Infomation |
Eteplirsen (Exondys 51) was the first FDA-approved drug for the treatment of Duchenne muscular dystrophy (DMD) in the US (approved in 2016). It is developed by Sarepta Therapeutics. The approval is for DMD patients with a confirmed mutation of the dystrophin gene amenable to exon 51 skipping (approximately 13% of DMD patients). The drug is administered as a 30 mg/kg weekly intravenous infusion. The CAS number is 1173755-55-9. The molecular weight is approximately 5,000-6,000 Da. The sequence is: 5'-CCTCCGGTTCTGAAGGTGTTC-3'. For research use, eteplirsen is provided as a lyophilized powder or solution. The compound is not for human use in research. It is a PMO antisense oligonucleotide.
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| Exact Mass |
10300.59
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| CAS # |
1173755-55-9
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~50 mg/mL (~4.85 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 33.33 mg/mL (3.23 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
(Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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