Comparison of Human and Canine P‐Glycoprotein Substrates at R, P, and H Binding Sites

P‐glycoprotein (P‐gp) greatly impacts substrate drug disposition, so much so that regulatory agencies recommend ascertaining the P‐gp status of active pharmaceutical ingredients (APIs) intended for human use. Arguably, the P‐gp status of drugs intended for canine patients is equally, if not more, important. Our research objectives were to assess whether human P‐gp substrate data can predict canine P‐gp substrate status and to explore the three previously reported binding sites within the P‐gp binding pocket, the H‐, R‐, and P‐sites. Competitive efflux assays employing cell lines expressing canine or human P‐gp were used to compare the degree of overlap or independence of the three binding sites in canine versus human P‐gp using site‐specific fluorescent P‐gp substrates rhodamine 123, calcein AM and Hoechst 33342. Because calcein AM can also be transported by multidrug resistance protein 1 (MRP1), experiments were performed to assess its potential influence on calcein AM efflux studies. Results indicate that: (i) MRP1 is either a non‐factor or negligible factor for cells expressing canine or human P‐gp respectively; (ii) determining an API's P‐gp binding site may provide clinically relevant information; and (iii) use of human P‐gp substrate data as a proxy for canine P‐gp substrate data will often prove inaccurate.