Design, synthesis and in vitro testing of methotrexate carrier conjugates linked via oligopeptide spacers.

A range of methotrexate (MTX)-spacer-human serum albumin (HSA) conjugates have been prepared. They have been designed to release one of the following materials: MTX, MTX-Lys (alpha-epsilon) or MTX-Lys (gamma-epsilon). These materials have previously been shown to be the low-molecular-weight products released on hydrolysis of a MTX-HSA conjugate by rat liver tritosomes. Preliminary studies were carried out using a lysine residue, linked by the epsilon-amino group to the oligopeptide chain, as a model for the polymer. This use of this model system indicated that an Ala-Leu-Ala-Leu spacer was cleaved by lysosomal enzymes with release of the appropriate derivative. Analysis of the lysosomal digestion products of the MTX-spacer-HSA conjugates showed that the model system appeared to work well. A maximum rate of release of 100% MTX-Lys (alpha-epsilon) from a MTX-spacer-HSA within 48 h was obtained, as compared to 5% in 48 h obtained for MTX-HSA. Determination of the in vitro cytotoxicity of the conjugates showed that release of 'free' MTX was 10-fold more cytotoxic than either of the MTX-Lys derivatives, but the level of release of MTX from MTX-HSA was 10-fold less than that of the MTX-Lys materials.