Deciphering Transporter‐Mediated Drug Interactions Affecting Marbofloxacin Residues in Sheep Milk: From In Silico Predictions to In Vivo Validation

Veterinary drug residues in milk represent an important food safety concern, particularly under conditions of co-administration where transporter-mediated drug-drug interactions may alter residue profiles. Marbofloxacin, a fluoroquinolone antimicrobial, is actively secreted into milk through the ATP-binding cassette transporter ABCG2 (BCRP), whereas ivermectin has been reported to interact with ABC transporters. The present study integrated in silico and in vivo approaches to investigate whether ivermectin may influence marbofloxacin secretion into sheep milk through transporter-mediated mechanisms. A homology model of ovine ABCG2 was constructed using the human cryo-electron microscopy structure as a template. Molecular docking and molecular dynamics simulations were subsequently performed to evaluate ligand-transporter interactions and complex stability. Docking analysis demonstrated that ivermectin exhibited a broader and more favorable interaction profile within the ABCG2 transmembrane cavity compared with marbofloxacin, with partial overlap at residues associated with substrate recognition, particularly within the Asp129-Asp130 region. Molecular dynamics simulations confirmed stable accommodation of ivermectin within the transporter throughout the simulation period without structural destabilization of the protein. The in vivo component included lactating dairy ewes receiving marbofloxacin alone or in combination with ivermectin under routine field conditions. Milk samples collected at the predicted peak milk concentration were analyzed using a validated HPLC-PDA/MS method. Marbofloxacin was detected in all milk samples with high analytical specificity. Ewes treated with ivermectin exhibited significantly lower marbofloxacin concentrations in milk compared with animals receiving marbofloxacin alone, while significant within-animal variability between udder halves was also observed.