Insulin is a short-lived species in the circulatory system. After binding to its receptor sites and transmission of its biological signals, bound insulin undergoes receptor-mediated endocytosis and consequent degradation. An inactive insulin derivative that is not recognized by the receptor has a longer circulation life, but obviously is biologically impotent. (Fmoc)2 insulin is an insulin derivative purified through high-performance liquid chromatography in which two 9-fluorenylmethoxycarbonyl (Fmoc) moieties are covalently linked to the (alpha-amino group of phenylalanine B1 and the epsilon-amino group of lysine B29. It has 1-2% of the biological potency and receptor binding capacity of the native hormone. After incubation, (Fmoc)2 insulin undergoes a time-dependent spontaneous conversion to fully active insulin in aqueous solution at 37 degrees C and a pH range of 7-8.5. At pH 7.4, the conversion proceeds slowly (t1/2 = 12 +/- 1 days) and biological activity is generated gradually. A single subcutaneous administration of (Fmoc)2 insulin to streptozocin-treated diabetic rats normalized their blood glucose levels and maintained the animals in an anabolic state over 2-3 days. A broad shallow peak of immunoreactive insulin was found to persist in circulation over this period. To confirm further that the long-acting effect of (Fmoc)2 insulin proceeds via slow release in the blood circulation itself, we administered native insulin, NPH insulin, or the (Fmoc)2 derivative intraperitoneally. The rats recovered from hypoglycemia at t1/2 = 8.0 +/- 0.3 and 10 +/- 0.4 h after administration of native and NPH insulin, respectively. In contrast, (Fmoc)2 insulin was active for a significantly longer time, with an extended onset of t1/2 = 26 +/- 1h, and a glucose-lowering effect even 40 h after administration. (Fmoc)2 insulin was also found to be more resistant to proteolysis. Finally, we found that (Fmoc)2 insulin does not induce antigenic effects. In summary, we present here a new concept for prolonging the half-life of insulin in the circulatory system, in which receptor-mediated endocytosis and degradation is delayed and accompanied by a time-dependent generation of basal insulin.

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