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-acting insulin derivative whose protracted action in vivo is based on a new principle rather than on slower absorption rates of suspended insulin formulations. To this end, we have prepared (9-fluorenylmethoxycarbonyl-SO(3)H)(3)-insulin ((FMS)(3)-insulin), a derivative having three 9-fluorenylmethoxycarbonyl-SO(3)H (FMS) moieties covalently linked to the three amino side chains of 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 biological 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 rats 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 and 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 administered, at high dose, with no concern for hypoglycemia. Because of its decreased receptor-binding affinity, (FMS)(3)-insulin evades receptor-mediated 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, manifesting a prolonged glucose-lowering action in vivo. According to the data presented here, (FMS)(3)-insulin represents a typical prodrug: a compound which by itself shows only marginal activity but over time it is chemically hydrolyzed to the fully active hormone.