Validation of a flow cytometric competitive efflux assay for assessing clinically important drugs as canine P-glycoprotein substrates.

P-gp-deficient dogs may face increased adverse drug reactions from apomorphine, capromorelin, enrofloxacin, and moxidectin.

This study validated a flow cytometric competitive efflux assay to identify clinically relevant drugs as canine P-glycoprotein (P-gp) substrates, with particular relevance to dogs carrying the MDR1 (ABCB1) mutation, which confers P-gp deficiency. Using a cell line expressing canine P-gp, the assay measured intracellular fluorescence following co-incubation of test drugs with two fluorescent P-gp substrates (rhodamine 123 and calcein AM) that bind distinct sites within the P-gp binding pocket. Mean fluorescence intensity (MFI) ratios were used to classify drugs: ratios below 2 indicated non-substrates (cisplatin, ropinirole, doxycycline, trilostane); ratios of 2–5 indicated weak substrates (apomorphine, capromorelin, enrofloxacin); ratios of 5–10 indicated moderate substrates (trazodone); and ratios above 10 indicated strong substrates (loperamide, moxidectin). Assay specificity, precision, and linearity met established acceptance criteria, supporting its validity as a screening tool. The findings have direct clinical implications: dogs with P-gp deficiency (e.g., Collies, Australian Shepherds, and other herding breeds with MDR1 mutations) may be at heightened risk for adverse drug reactions when treated with apomorphine, capromorelin, enrofloxacin, or moxidectin. Dose-dependent risk was acknowledged for weaker substrates. This validated assay provides a practical and reproducible method for pre-clinical screening of veterinary drugs as canine P-gp substrates, enabling better safety assessments in genetically susceptible dog populations.

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