New concept for long-acting insulin - Spontaneous conversion of an inactive modified insulin to the active hormone in circulation: 9-fluorenylmethoxycarbonyl derivative of insulin

Insulin is a short-lived species in the circulatory system. After binding t o its receptor sites and transmission of its biological signals, bound insu lin undergoes receptor-mediated endocytosis and consequent degradation. An inactive insulin derivative that is not recognized by the receptor has a lo nger circulation life, but obviously is biologically impotent. (Fmoc)(2) in sulin is an insulin derivative purified through high-performance liquid chr omatography in which two 9-fluorenylmethoxycarbonyl (Fmoc) moieties are cov alently 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 recep tor binding capacity of the native hormone. After incubation, (Fmoc), insul in undergoes a time-dependent spontaneous conversion to fully active insuli n in aqueous solution at 37 degrees C and a pH range of 7-8.5. At pH 7.4, t he conversion proceeds slowly (t(1/2) = 12 +/- 1 days) and biological activ ity is generated gradually. A single subcutaneous administration of (Fmoc)( 2) insulin to streptozocin-treated diabetic rats normalized their blood glu cose 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 circ ulation over this period. To confirm further that the long-acting effect of (Fmoc)(2) insulin proceeds via slow release in the blood circulation itsel f, we administered native insulin, NPH insulin, or the (Fmoc), derivative i ntraperitoneally. The rats recovered from hypoglycemia at t1/2 = 8.0 +/- 0. 3 and 10 +/- 0.4 h after administration of native and NPH insulin, respecti vely. In contrast, (Fmoc)(2) insulin was active for a significantly longer time, with an extended onset of t(1/2) = 26 +/- 1 h, 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 conce pt for prolonging the half-life of insulin in the circulatory system, in wh ich receptor-mediated endocytosis and degradation is delayed and accompanie d by a time-dependent generation of basal insulin.