ELX-02 Generates Protein via Premature Stop Codon Read-Through without Inducing Native Stop Codon Read-Through Proteins.

ELX-02 is a clinical stage, small-molecule eukaryotic ribosomal selective glycoside acting to induce read-through of premature stop codons (PSCs) that results in translation of full-length protein. However, improved read-through at PSCs has raised the question of whether native stop codon (NSC) fidelity would be impacted. Here, we compare read-through by ELX-02 in PSC and NSC contexts. DMS-114 cells containing a PSC in the TP53 gene were treated with ELX-02 and tested for increased nuclear p53 protein expression while also monitoring two other proteins for NSC read-through. Additionally, blood samples were taken from healthy subjects pre- and post-treatment with ELX-02 (0.3-7.5 mg/kg). These samples were processed to collect white blood cells and then analyzed by western blot to identify native and potentially elongated proteins from NSC read-through. In a separate experiment, lymphocytes cultivated with vehicle or ELX-02 (20 and 100 µg/ml) were subjected to proteomic analysis. We found that ELX-02 produced significant read-through of the PSC found in TP53 mRNA in DMS-114 cells, resulting in increased p53 protein expression and consistent with decreased nonsense-mediated mRNA degradation. NSC read-through protein products were not observed in either DMS-114 cells or in clinical samples from subjects dosed with ELX-02. The number of read-through proteins identified by using proteomic analysis was lower than estimated, and none of the NSC read-through products identified with >2 peptides showed dose-dependent responses to ELX-02. Our results demonstrate significant PSC read-through by ELX-02 with maintained NSC fidelity, thus supporting the therapeutic utility of ELX-02 in diseases resulting from nonsense alleles. SIGNIFICANCE STATEMENT: ELX-02 produces significant read-through of premature stop codons leading to full-length functional protein, demonstrated here by using the R213X mutation in the TP53 gene of DMS-114 cells. In addition, three complementary techniques suggest that ELX-02 does not promote read-through of native stop codons at concentrations that lead to premature stop codon read-through. Thus, ELX-02 may be a potential therapeutic option for nonsense mutation-mediated genetic diseases.

Safety, Tolerability, and Pharmacokinetics of Single Ascending Doses of ELX-02, a Potential Treatment for Genetic Disorders Caused by Nonsense Mutations, in Healthy Volunteers.

ELX-02 is an investigational synthetic eukaryotic ribosome-selective glycoside optimized as a translational read-through molecule that induces read through of nonsense mutations, resulting in normally localized full-length functional proteins. ELX-02 is being developed as a therapy for genetic diseases caused by nonsense mutations. Two phase 1a, randomized, double-blind placebo-controlled, single-ascending-dose studies were conducted in healthy human subjects to evaluate the safety and pharmacokinetics of ELX-02. Single subcutaneously injected doses of ELX-02 between 0.3 mg/kg and 7.5 mg/kg showed an acceptable safety profile without severe or serious drug-related adverse events, including a lack of renal and ototoxicity events. Injection of ELX-02 resulted in a rapid time to peak concentration and elimination phase, with complete elimination from the vascular compartment within 10 hours. ELX-02 area under the concentration-time curve to infinity showed dose-exposure linearity (24-fold increase for a 25-fold dose increase), and the maximum concentration showed dose proportionality (17-fold increase for a 25-fold increase). The mean apparent volume of distribution was dose dependent, suggesting an increased distribution and tissue uptake of ELX-02 at higher doses. The primary route of excretion was in the urine, with the majority of the compound excreted unchanged. These results support the evaluation of the safety, pharmacokinetics, and efficacy of repeat dosing in future studies.

Impurities in Oligonucleotide Drug Substances and Drug Products.

This white paper, which is the 10th in a series intended to address issues associated with the development of therapeutic oligonucleotides, examines the subject of product-related impurities. The authors consider chemistry and safety aspects and advance arguments in favor of platform approaches to impurity identification and qualification. Reporting, identification, and qualification thresholds suitable for product-related impurities of therapeutic oligonucleotides are proposed.

Chronic effects of flutamide in male beagle dogs.

Flutamide, a potent nonsteroidal antiandrogen, was administered orally to male beagle dogs for 2, 3, or 4 years at doses of 10, 20, or 40 mg/kg/day. At each study interval, the results of clinical pathology examinations, organ weight determinations, necropsy, and histopathologic examinations generally were similar and included atrophy of the prostate gland, testicular interstitial cell hyperplasia, and seminiferous tubular atrophy and degeneration. After 3 years of drug exposure, there were 3 dogs with testicular interstitial cell adenomas and a few dogs with 1 or more enlarged mammary gland nipples. Based upon the pharmacologic activity of flutamide, these findings were expected and considered the consequence of long-term blocking of testosterone receptors and an exaggerated compensatory response to increased secretion of luteinizing hormone. The findings of this study were consistent with other examples of dysregulated hormone stimulation of target tissues noted during the nonclinical safety assessment of flutamide. In consideration of the clinical indication of flutamide for advanced prostatic carcinoma and based upon reports of minimal flutamide-related adverse clinical responses, the findings of this study pose no concern for human risk assessment.