Sufficient quantities of metabolites M1a and M2 were produced for in vitro characterization of their potential involvement in drug-drug interactions involving the drug efflux transporter, P-glycoprotein (P-gp). For evaluation of their interaction with P-gp as substrates, bidirectional permeability assays were performed in Transwell® cultures of MDR1-MDCK cell monolayers, a canine renal epithelial cell line transfected with MDR1, the gene coding for human P-gp.
Substrates of efflux transporters such as P-gp have low apical-to-basolateral (A→B) apparent permeability (Papp) values and higher basolateral-to-apical (B→A) Papp values, resulting in an efflux ratio (B→A Papp/A→B Papp) greater than 1. This is due to the fact that P-gp, which is expressed on the apical surface of many epithelial cells, blocks the entry of its substrates across the apical membrane and facilitates the transport of drugs in the opposite direction.
In this manner, ritonavir, M1a, and to a lesser extent M2 (each at a concentration of 5 µM), were identified as substrates of P-gp.
To evaluate their interactions as inhibitors of P-gp, bidirectional permeability assays were performed in Transwell cultures of Caco-2 monolayers, a human intestinal epithelial cell line that naturally expresses P-gp. Digoxin, known to be effluxed by P-gp, was used as the probe substrate.
Inhibitors of P-gp reduce the efflux ratio of digoxin in this assay. Ritonavir completely inhibited P-gp, M1a inhibited by 55%, and M2 was a weak inhibitor. Each compound was tested at a concentration of 20 µM, approximately the plasma Cmax for ritonavir in humans.
It should not be surprising that drug metabolites, like the parent drugs themselves, could potentially be involved in drug interactions as substrates or inhibitors of macromolecules involved in drug disposition. Just as drug metabolites can be pharmacologically active, they can also be pharmacokinetically active. As illustrated here, the drug interaction potential of metabolites cannot necessarily be inferred based on that of the parent molecule. Biomimetic oxidation, a relatively quick and simple means of generating semi-preparative quantities of metabolites, can make the job easier.