Springer Online Journal Archives 1860-2000
Summary In studies on (antitumor antibody)-drug conjugates as potential antitumor agents, the amide derivatives of methotrexate (MTX) with cysteine and with 2-mercaptoethylamine (cysteamine) (MTX-Cys and MTX-MEA, respectively) were linked via a disulfide bond with a monoclonal antibody (αMM46) to a mouse mammary tumor MM46 with attached 3-(2-pyridyldithio) propionyl groups to give conjugates of MTX with αMM46 (MTX-Cys-SS-αMM46 and MTX-MEA-SS-αMM46, respectively). These two conjugates are both linked by a disulfide bond and are very similar in structure, but MTX-MEA-SS-αMM46 showed only weak in vitro cytotoxicity against MM46 cells, whereas MTX-Cys-SS-αMM46 had strong cytotoxicity. The cytotoxicity of the latter was comparable to that of the conventional direct MTX-αMM46 conjugate prepared with an MTX-active ester. However, this conjugate had a greater selectivity than that of the direct conjugate, calculated as the IC50 (concentration of a conjugate by MTX equivalence required for suppression of the number of viable MM46 cells to 50% of that of the untreated control) for the corresponding nonspecific conjugate divided by the IC50 for the αMM46 conjugate. The inhibitory activities of MTX-Cys and MTX-MEA on dihydrofolate reductase were similar. The cytotoxicity of MTX-Cys-SS-αMM46 was not affected by thiamine pyrophosphate, an inhibitor of the active transport of MTX across the cell membrane, but was decreased significantly by ammonium chloride, a lysosomotropic amine. However, the cytotoxicity was decreased only to a small extent by leupeptin, an inhibitor of lysosomal cysteine proteases cathepsins B, H, and L. These results suggest that the cytotoxicity is mediated by lysosomes, and may involve lysosomal enzymes other than cathepsins B, H, and L.
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