Synthesis and evaluation of insulin-human serum albumin conjugates.

A series of human insulin maleimido derivatives with short and long linkers was synthesized by exploiting the variations in the pK(a) values and environment of the three amino groups present in the protein. The syntheses were accomplished in organic solvent because of maleimide's instability in basic aqueous media. The derivatives thus obtained were conjugated to the free thiol on Cys34 of human serum albumin (HSA) and purified. A structure-activity relationship based on in vitro receptor binding and activation results for this series of insulin-HSA conjugates showed that the best compounds were attached at the B1 position of insulin with either short or long linkers. Two conjugates were administered subcutaneously to streptozotocin-induced diabetic rats and found to possess blood glucose normalizing activity up to 8 h post-administration. The return to diabetic plasma glucose levels was not observed within the time frame of the experiment (48 h). In comparison, the insulin-treated group's normalization activity lasted 2 h and returned to a diabetic level at 8 h. The onset of the conjugate activities were delayed by 1 h when compared to the activity of human insulin. The study results led to the identification of CJC-1575 as a potent and long lasting human insulin analogue.