Effects of protein binding on the in vitro activity of antitumour acridine derivatives and related anticancer drugs

Purpose: We set out to measure drug binding to serum proteins. These have been shown to reduce the free plasma concentrations of a number of anticancer drugs, particularly of those of complex organic structure, in both experimental studies and clinical trials. Methods: We have used cultures of murine Lewis lung carcinoma cells as sensors of available drug to measure the effects of two drug-binding plasma proteins, alpha-acid glycoprotein (AAG) and human serum albumin (HSA), as well as of bovine serum albumin (BSA) on drug activity. Results: The concentrations required for 50% growth inhibition (IC50 values) of a number of anti-cancer drugs were found to be linear functions of the added proteins. Assuming that cells respond to free drug, the data provide estimates of the product K . n, where K is the binding constant of the protein and n is the number of drug binding sites per protein molecule. Amsacrine, the amsacrine analogue asulacrine, camptothecin, DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide), doxorubicin, etoposide, mitoxantrone, paclitaxel and vincristine were tested. The K . n values for AAG were 30, 2400, 8.7. 340, 29, 290 x 10(3) M-1 and 120 x 10(3) M-1, respectively, and the K . n values for HSA were 16, 580, 530, 10, 6.2, 4.3 x 10(3) M-1 and 0.0 x 10(3) M-1, respectively. The combined data allowed the estimation of free fractions of drug in plasma, assuming that AAG and HSA contributed most to protein binding. The data were in general comparable with that reported using equilibrium dialysis and ultrafiltration. Data for drug binding to BSA were different from those for HSA, in some cases by a large factor with values for HSA generally higher. The applicability of the method to analogue development was illustrated by examining the binding to AAG of a series of DACA analogues, and binding was found to be primarily related to lipophilicity. Conclusion: IC50 determinations provide a rapid means of estimating drug binding to plasma proteins and have utility in the assessment of new anticancer drugs.