Effect of pH and human serum albumin on the cytotoxicity of a glucuronide prodrug of 9-aminocamptothecin

Purpose 9-aminocamptothecin glucuronide (9ACG) is a prodrug of 9-aminocamptothecin (9AC) that displays potent antitumor activity against human tumor xenografts in nude mice. Camptothecins exist in a pH dependent equilibrium between active lactone and inactive carboxy forms that can be altered by binding to human serum albumin (HSA). Here we investigated the influence of pH and HSA on the lactone-carboxy equilibrium, HSA binding, and cytotoxicity of 9ACG. Methods Microfiltration and HPLC were used to measure the influence of pH on lactone to carboxy conversion and HSA binding of 9ACG as compared to other camptothecins. In vitro cytotoxicity of drugs was determined against EJ human bladder carcinoma cells and CL1-5 human lung cancer cells. Results The rate of lactone to carboxy conversion was similar for 9ACG and 9AC. Decreasing the pH from 7.6 to 6.0 increased the equilibrium levels of the lactone forms of the drugs from 20 to almost 95% of total drug. HSA moderately diminished the amount of free 9ACG lactone but did not change the ratio of 9ACG lactone to 9ACG carboxy. Consistent with the effect of pH on lactone levels, lowering the pH of EJ human bladder carcinoma cells from 7.6 to 6.8 decreased the IC₅₀ of 9ACG from 480 to 98 nM and 9AC from 33 to 12 nM. Activation of 9ACG by human β-glucuronidase anchored on the surface of EJ cells further decreased its IC₅₀ value to 26 nM. Although HSA significantly decreased the cytotoxicity of 9AC and 9ACG, activation of 9ACG at cancer cells with an antibody-β-glucuronidase immunoconjugate produced greater cytotoxicity than 9AC. Conclusions Acidification and targeted delivery of β-glucuronidase can enhance 9ACG cytotoxicity even in the presence of HSA.