We investigated whether human serum albumin (HSA) in suspended human hepatocytes would affect the uptake clearance of anionic drugs with high binding to HSA and improve the extrapolation of in vivo hepatic clearance from in vitro uptake clearance by the hepatocytes via the "albumin-mediated" hepatic uptake mechanism. The uptake clearances for total forms (PS inf) and for unbound forms (PS u,inf) of 11 anionic drugs [all of which were organic aniontransporting polypeptide (OATP) substrates] were determined with suspended human hepatocytes in varying concentrations of HSA. The fraction of unbound drugs (f u) was determined using an equilibrium dialysis at the various HSA concentrations. The PS inf values decreased with increasing concentrations of HSA, whereas the unbound uptake clearances (PS u,inf (+) = PS inf / f u) in the presence of HSA increased substantially, thus demonstrating the "albuminmediated" hepatic uptake mechanism. The relationships between PS inf and HSA concentration were well described by the previously proposed facilitated-dissociation model, in which the drug-albumin complex interacts with the cell surface, enhancing the dissociation of the complex and providing unbound drug for hepatic uptake. Furthermore, the PS u,inf (+) values in in vivo conditions (at 5% HSA) were predicted from those obtained in isolated hepatocytes on the basis of the facilitated-dissociation model, revealing compatibility with the overall hepatic intrinsic clearance in vivo. We conclude that the "facilitated-dissociation" model is useful for describing the "albumin-mediated" hepatic uptake phenomenon of OATP drugs and to predict hepatic uptake clearance in vivo.

A macrocyclic peptide scaffold with well-established structure−property relationship is desirable for tackling undruggable targets. Here, we adopted a natural macrocycle, cyclosporin O (CsO) and its derivatives (CP1−3), and evaluated the impact of conformation on membrane permeability, cyclophilin A (CypA) binding, and the pharmacokinetic (PK) profile. In nonpolar media, CsO showed a similar conformation to cyclosporin A (CsA), a well-known chameleonic macrocycle, but less chameleonic behavior in a polar environment. The weak chameleonicity of CsO resulted in decreased membrane permeability; however, the more rigid conformation of CsO was not detrimental to its PK profile. CsO exhibited a higher plasma concentration than CsA, which resulted from minimal CypA binding and lower accumulation in red blood cells and moderate oral bioavailability (F = 12%). Our study aids understanding of CsO, a macrocyclic peptide that is less explored than CsA but with greater potential for diversity generation and rational design.

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