Rybalka, Emma ORCID: https://orcid.org/0000-0002-4854-0036 and Timpani, Cara ORCID: https://orcid.org/0000-0003-4567-4319 (2025) A 28 day repeat dose toxicology study of subcutaneously administered ASA-001 in female C57/Bl6J mice (24-001-01). Research Report. Victoria University, Melbourne, Victoria.

Abstract

The objective of this study was to assess toxicity of ASA-001 administered once daily via sub-cutaneous injection at 800 mg/kg to female C57BL/6J mice for 28 days. Ten female mice were divided into two groups with 5 animals/group. Animals in Group 1 (Animals 1-5) were administered sterile water vehicle (VEH) at a volume of 0.01ml/g body weight by sub-cutaneous injection to the neck scruff daily. Animals in Group 2 (Animals 6-10) were administered ASA-001 dissolved in sterile water VEH neutralised to pH 7.2 with NaOH at a dose of 800mg/kg in a total volume of 0.01ml/ g body weight by the same route. Mice were weighed before the first dose and pre-treatment observations were made (on d-1). Mice were observed for ~5 minutes after dosing and returned to cages thereafter. Food and water consumption, muscle strength and function and in life observations were assessed weekly. Food was removed from cages ~12 hours after the last dose and ~12 hours before the experimental endpoint. Mice were sent in the fasted state to Cerberus Sciences for processing. Mice were euthanised by asphyxiation and blood was drawn by cardiac bleed. Haematological and biochemical variables were assessed at a commercial veterinary pathology laboratory (Gribbles). Gross pathology and tissue level histopathology were evaluated by a certified veterinary pathologist. No mortality was observed in the study. No adverse clinical findings were observed in the study. No gross or tissue level toxicities were observed. Neutrophil concentration was the only hematologic variable that skewed from the normal reference range (in both VEH and ASA-001 treated mice) and was statistically (p<0.05 when compared to vehicle) increased by ASA-001 treatment. However overall White Blood Cell count was within the normal reference range for all ASA-001 treated mice and there was no evidence of tissue injury or abnormality on histological inspection, suggesting higher neutrophil concentration was unrelated to tissue injury. In-life observations of the drug injection site showed skin thickening during the final days of the protocol in all ASA-001 treated mice, and one ASA-001 treated mouse (Mouse 8) showed scar tissue deposition at the injection site. The higher neutrophil content in ASA-001 treated mice may reflect injection injury mediated inflammation of local integument. Since the intended target tissue of ASA-001 is striated muscle, strength and endurance muscle function tests were incorporated into the study protocol. ASA-001 treatment increased body grip strength but not performance endurance tests, likely because of naïve animals used in the study, unlike disease models. To further support this study, the sponsor conducted a bridging non-GLP dose-matched PK study in mice (800 mg/kg), in addition to a dose of 600 mg/kg in rats, representing 50% of the dose utilized in the non-GLP PK study in rats which explored oral (1500 mg/kg), intravenous (100 mg/kg) and subcutaneous (1200 mg/kg) dosing, to enable predictive dose exposure modelling to humans. The data are appended as part of this report as a stand-alone report was not considered necessary. In summary, ASA-001 was well tolerated in mice with no adverse findings, when administered once daily via sub-cutaneous injection at 800mg/kg to female C57Bl/6J mice for 28 days.

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