Cytotoxicity of phosphoramidate, bis-amidate and cycloSal prodrug metabolites against tumour and normal cells

Abstract

Phosphonate and phosphate prodrugs are integral to enhancing drug permeability, but the potential toxicity of their metabolites requires careful consideration. This study evaluates the impact of widely used phosphoramidate, bis-amidate, and cycloSal phosph(on)ate prodrug metabolites on BxPC3 pancreatic cancer cells, GL261-Luc glioblastoma cells, and primary cultured mouse astrocytes. 1-Naphthol and 2-naphthol demonstrated the greatest toxicity. Notably, 2-naphthol exhibited an ED₅₀ of 21 μM on BxPC3 cells, surpassing 1-naphthol with an ED₅₀ of 82 μM. Real-time xCELLigence experiments revealed notable activity for both metabolites at a low concentration of 16 μM. On primary cultured mouse astrocyte cells, all prodrugs exhibited reduced viability at 128 to 256 μM after only 4 hours of exposure. A cell-type-dependent sensitivity to phosph(on)ate prodrug metabolites was evident, with normal cells showing greater susceptibility than corresponding tumour cells. The results suggest it is essential to consider the potential cytotoxicity of phosph(on)ate prodrugs in the drug design and evaluation process.

Fig. 1. Representation of the prodrug concept. A) Phosph(on)ate is deprotonated at physiological pH resulting in poor membrane permeability. B) Neutral phosph(on)ate prodrug allows for efficient cell membrane permeability before being cleaved intracellularly into the charged active drug. Created with https://Biorender.com

Fig. 2. A) Chemical structure of clinically used anti-virals with pivaloyloxymethyl (POM) and isopropyloxymethyl carbonate (POC) prodrugs. B) Example applications of the phosphoramidate, bis-amidate and cycloSal prodrugs.

Fig. 3. Chemical structure and activation pathways of phosph(on)ate prodrugs and their metabolites explored in this study.

Fig. 4. Evaluation of the viability of BxPC3, GL261-Luc and primary mouse astrocytes treated with prodrug moieties. A) Fluorescence image of BxPC3 cells treated with prodrug moieties for 72 hours and then measured with CyQUANT. The image was produced using the Amersham™ Typhoon 5™. B) and D) cell viability measured 72 hours after treatment using CyQUANT assay. Fluorescence readings were quantified using ImageQuant™ TL and normalised to the control. C) Viability measured by luciferase assay 72 hours after treatment where luminescence was measured by plate reader and normalised to the control. E–G) Time-course of cell index reflecting viability over 72 hours using the xCELLigence system. The cell index measurements were taken every hour and normalised to the control. Statistical significance is shown relative to the control. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data is shown as mean ± S.D of three independent experiments.

Fig. 5. Dose–response curves of 1-naphthol and 2-naphthol on BxPC3 cells over 72-hours. Cell index measurements using the xCELLigence system were taken every hour and normalised to the control. A) Cell index of BxPC3 cells decreased with increasing concentrations of 1-naphthol. B) Dose–response curve of 1-naphthol (ED₅₀ = 82 μM). C) Cell index of BxPC3 cells decreased greatly with increasing concentrations of 2-naphthol. D) Dose–response curve of 2-naphthol (ED₅₀ = 21 μM). Data is shown as mean ± S.D. of three independent experiments.

Fig. 6. Caspase 3/7 activation of BxPC3 and GL261-Luc cells treated with phosph(on)ate prodrug moieties for 72 hours. The fluorescence was normalised to cell number using CellTag 700. Statistical significance is shown relative to the control. * p ≤ 0.05, *** p ≤ 0.001. Data is shown as mean ± S.D. of three independent experiments.