“All-inclusive” evaluation of the efficacy and safety of methotrexate in a murine breast cancer model integrating the 3Rs to enhance preclinical assessment

Abstract

Safety pharmacology evaluation plays a crucial role in the preclinical assessment of anti-cancer drugs, ensuring their tolerability and minimizing potential adverse effects before clinical translation. While there is broad consensus around the importance of safety assessment in cancer drug evaluation at the clinical stage, this is poorly investigated at the preclinical level. This study aims to comprehensively evaluate the safety pharmacological properties of Methotrexate, a folate antagonist, in a preclinically relevant murine model of breast cancer and emphasizing the interest of such approach for 3Rs (Replacement, Reduction, and Refinement) in animal research. Female BALB/c mice were orthotopically implanted with 4 T1 mouse mammary carcinoma cells to establish breast cancer tumors. The mice were randomized into treatment or control groups. Methotrexate was injected at 25 and 1000 mg/kg (slow i.v. once a week for 3 weeks). Tumor growth kinetics, tumor volume, metastatic potential, hematological profile, and overall survival were assessed. Additionally, respiratory (whole body plethysmography) and behavioral (Irwin) functions were investigated longitudinally over four different timepoints to monitor the adverse effects associated with Methotrexate treatment. Interestingly, this approach aligns with the 3Rs by using an “all-inclusive” model that reduces the number of animals needed through the longitudinal assessment of multiple efficacy and safety parameters within the same study. This global approach minimizes potential risks prior to clinical development and provides valuable insights into the pharmacological properties of drugs for cancer therapy while adhering to ethical standards in animal research.