Rapid Measurement of Intracellular Unbound Drug Concentrations

Intracellular
unbound drug concentrations determine affinity to
targets in the cell interior. However, due to difficulties in measuring
them, they are often overlooked in pharmacology. Here we present a
simple experimental technique for the determination of unbound intracellular
drug concentrations in cultured cells that is based on parallel measurements
of cellular drug binding and steady-state intracellular drug concentrations.
Binding in HEK293 cells was highly correlated with binding in liver-derived
systems, whereas binding in plasma did not compare well with cellular
binding. Compound lipophilicity increased drug binding, while negative
charge and aromatic functional groups decreased binding. Intracellular
accumulation of unbound drug was consistent with pH-dependent subcellular
sequestration, as confirmed by modeling and by inhibition of subcellular
pH gradients. The approach developed here can be used to measure intracellular
unbound drug concentrations in more complex systems, for example,
cell lines with controlled expression of transporters and enzymes
or primary cells.