Evaluating rat and canine microbiota models for predicting human colonic prodrug metabolism

Abstract

The rise of microbiome-aware drug development has placed growing emphasis on the need for reliable preclinical tools to evaluate microbiota-mediated drug metabolism. While human faecal models are used, they suffer from practical limitations such as donor recruitment and regulatory constraints. Larger animals like dogs are often assumed to be more translationally relevant yet are resource-intensive and subject to more complex regulatory and logistical requirements. Rats offer a more accessible, cost-effective and scalable alternative. However, it remains unclear whether their faecal material alone accurately reflects colonic metabolism. Specifically, it is unknown whether faecal samples capture the same metabolic activity as more invasive caecal or colonic contents, or how closely they reflect drug degradation in larger animal models or humans. This study aimed to: (i) compare degradation of three prodrugs across Wistar rat faecal, caecal, and colonic compartments; (ii) determine how rat degradation profiles differ from those observed in Labradors; and (iii) evaluate how closely rat and canine data align with published human in vitro results. Degradation kinetics of sulfasalazine, balsalazide, and olsalazine were first assessed. Bioreactors prepared from 10% faecal, caecal, and colonic contents in rats were used. Faecal material showed equivalent metabolic activity to colonic and caecal material across all drugs (two-way ANOVA, p = 0.233), with sulfasalazine degrading most rapidly (t₁/₂ = 29.1 min), followed by balsalazide (t₁/₂ = 47.9 min), and olsalazine (t₁/₂ = 84.1 min). These findings indicate that faecal material can reliably substitute for more invasive gut content sampling, offering practical and procedural advantages. Subsequent interspecies comparisons revealed that rats exhibited significantly higher degradation rates than dogs (P < 0.05), reflecting known differences in gut microbial density and composition. When benchmarked against published human in vitro data, rat degradation rates were closely aligned with human values, particularly for sulfasalazine (rat: K = 0.025 min⁻¹; human: K = 0.021 min⁻¹) and balsalazide (rat: K = 0.015 min⁻¹; human: K = 0.009 min⁻¹). These findings highlight rat faecal material as a practical and translationally relevant model for microbiota-sensitive prodrug metabolism, offering a low-impact alternative to invasive sampling and larger animal studies.