Preparation, Characterization And Biological Activity Of Chemically-Modified Insulin

Insulin is the most effective drug to treat diabetes. It can't be administered orally because of its degradability by proteases. Chemically-modified insulin can remain its blood glucose-lowering effect, enhance its stability, extend its action profile in the body, change its lipophilicity, improve its permeation characteristics and absorption in intestines and increase the receptor binding potency. And it may be possible to protect insulin from degradation with proteases. These maybe hold promise of orally insulin delivery. In this paper, some appropriate modifiers were used to modify the (-amino group of LysB29 of insulin selectively without using any specific protecting agents,and several (-NB29-monoacylated insulins were synthesized. The modification products of insulin were characterized, and the biological activity, inhibitory effect against trypsin degradation and receptor binding potency of these modified insulins were investigated. The main results are as follows:1. The modifiers were activated with benzotriazole by means of acylchloride or dicyclohexylcarbodiimide (DCC) coupling method. A series of activated amides such as 1-lipoyl benzotriazole, 1-dehydrocholyl benzotriazole, 1-oleoyl benzotriazole, 1-linoleoyl benzotriazole, 1-stearyl benzotriazole, 1-stearyl-glycinyl benzotriazole, 1-acetylcholyl benzotriazole were synthesized, and their structure were characterized by IR and 1HNMR.2. A series of novel insulin analogues such as lipoyl insulin, dehydrocholyl insulin, linoleoyl insulin and oleoyl insulin were obtained by using a series of activated amides of (-lipoic acid (LA), dehydrocholic acid (DHC), linoleic acid (LOA), oleic acid (OA) and their derivatives without using any specific protecting group, respectively. The modification product was analyzed by native discontinuous polyacrylamide gel electrophoresis (PAGE) and the lipoyl insulin, dehydrocholyl insulin showed a faster migrating band than native insulin, respectively. The separation and purification of the modification products of insulin with LA and DHC were conducted by reversed-phase high performance liquid chromatography (RP-HPLC), and the results showed that the major product was monolipoyl insulin with a yield of 73% and monodehydrocholyl insulin with a yield of 66%, respectively. The monolipoyl insulin and monodehydrocholyl insulin with a purity of above 95% were obtained by semiprep RP-HPLC, respectively. 3. The molecular weight of each component of modified insulin with LA, DHC, LOA and OA was accurately identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The measured molecular weight of each component was coincident with the theoretical values of the corresponding modified insulin and confirmed the modification. The content of monodehydrocholyl insulin or monolipoyl insulin was consistent with the results of RP-HPLC. MALDI-TOF-MS of the raw product of LOA- or OA-modified insulin also showed that monoacylated insulin was the major product, and a little diacylated insulin was observed and no triacylated insulin was detected. Especially, linoleic acid showed a higher selectivity to the modification of insulin. With MALDI-TOF-MS analyses of the substrate of trypsin treatment about lipoyl-insulin and dehydrocholyl-insulin, the main modified site was confirmed to be ε-amino group of LysB29, that is, the major modification product was ε-NB29-lipoyl insulin and ε-NB29-dehydrocholyl insulin, respectively. 4. The in vivo glucose-lowering effect and in vitro inhibitory potency from trypsin about these modified insulins were investigated with animal experiments. The results showed that the modified insulin retained significant biological activity. ε-NB29-lipoyl insulin, ε-NB29-dehydrocholyl insulin and linoleoyl insulin showed a protracted duration of action and a protective effect from trypsinic degradation. Compared with other modified insulin, ε-NB29-lipoyl insulin demonstrated a more strongly inhibitory effect against trypsin. The stabilization may be attributed...