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.