A novel approach for a water soluble long-acting insulin prodrug: Design, preparation, and analysis of [(2-sulfo)-9-fluorenylmethoxycarbonyl](3)-insulin

In this study we designed, prepared, and analyzed a water-soluble, long-act ing insulin derivative whose protracted action in vivo is based on a new pr inciple rather than on slower absorption rates of suspended insulin formula tions. To this end, we have prepared (9-fluorenylmethoxycarbonyl-SO3H)(3)-i nsulin ((FMS)(3)-insulin), a derivative having three 9-fluorenylmethoxycarb onyl-SO3H (FMS) moieties covalently linked to the three amino side chains o f insulin. (FMS)(3)-insulin is soluble in aqueous buffers at neutral pH, at a concentration range of 0.15-0.60 mM, and has about 1% of both the biolog ical potency and the receptor-binding affinity of the native hormone. Upon incubation at pH 7.4 and 37 degrees C, it undergoes a slow hydrolysis with linear regeneration of insulin possessing full biological potency. A single subcutaneous administration of (FMS)(3)-insulin to streptozocin-treated ra ts lowered circulating glucose levels for a prolonged period (t(1/2) = 30 h ). Similarly, intraperitoneal administration of (FMS)(3)-insulin to healthy rats had a prolonged glucose-lowering effect. In this experimental system, recovery from hypoglycemia proceeded with a t(1/2) value of 14 +/- 1 h, as compared with t(1/2) = 8.0 +/- 1 h for native insulin and t(1/2) = 10.0 +/ - 1 h for NPH-insulin. (FMS)(3)-insulin is more resistant to proteolysis an d appears to be nonimmunogenic. On the whole, (FMS)(3)-insulin represents a prototype version of a water-soluble, long-acting preparation of insulin. It is nearly inactive at the time of administration, and therefore can be a dministered, at high dose, with no concern for hypoglycemia. Because of its decreased receptor-binding affinity, (FMS)(3)-insulin evades receptor-medi ated endocytosis and degradation and, hence, persists for a long period in the circulation. The insulin constituent of the (FMS)(3)-insulin conjugate undergoes a slow, spontaneous activation in the circulatory system, manifes ting a prolonged glucose-lowering action in vivo. According to the data pre sented here, (FMS)(3)-insulin represents a typical prodrug: a compound whic h by itself shows only marginal activity but over time it is chemically hyd rolyzed to the fully active hormone.