Continued research is proposed toward the development of a chemical mediated artificial pancreas. This system will deliver glycosylated insulin (G-Ins) in response to glucose concentrations, based on the competitive binding between G-Ins and glucose to a saccharide binding substrate. This application will focus on optimizing the components of the system and comprehensive in vitro and in vivo animal tests. The synthesis, characterization, and isolation of glucosyl, as well as fucosyl, mannosyl, and galactosyl saccharride derivatives of insulin will be performed. By expanding the range of insulin derivatives, improved physical-chemical properties (binding kinetics, glucose exchange rates, and permeation) may be achieved. The interaction of G-Ins with surface bound insulin receptors on an insulinoma cell will be evaluated. This in vitro model will assess the conformation or bioactivity associated with G- Ins through changes in cell growth rates and the amount of insulin secreted by the cell. The immunogenicity of the G-Ins will be investigated. Mice will be injected with G-Ins and the antibody titer to G-Ins will be determined. The central component of the self-regulated insulin delivery system is a substrate that binds both G-Ins and glucose. ConA was evaluated as a soluble monomeric protein, enclosed within microcapsules, and formed into microspheres. This proposal focuses on coupling ConA as a soluble high molecular weight oligomer. This new form will maintain the binding characteristics and not leak or permeate through the device membrane. Other saccharride binding substrates will be investigated particularly, hexokinase and glucokinase. These proteins strongly bind sugars and may achieve a greater sensitivity in G-Ins release to glucose levels. A composite membrane 'pouch' will be used to house the G-Ins/binding substrate complex. A heat sealable, 40K MWCO membrane coated with a thin dense biocompatible material will prevent the permeation of immunoglobulins from the peritoneum into the device and maintain suitable glucose and G-Ins diffusion rates. A vascular graft delivery device will also be developed. G-Ins and binding substrate will he housed in a casing surrounding a vascular graft fabricated from the same membrane as the pouch. Glucose will rapidly equilibrate into the casing and release bound G-Ins. In vitro tests of both systems will evaluate the release of G-Ins in response to glucose concentrations, binding capacity, and effective duration of the device. Both the pouch and the vascular graft systems (anastomosed to the common illiac vein) will be implanted in the peritoneum of pancreatectomized dogs for short and long term in vivo evaluations including: G-Ins levels in response to blood glucose, lowering of blood glucose, biocompatibility, in vivo duration, and decreased long term complication (glycosylated hemoglobin) by the self-regulated insulin delivery device. It is anticipated that by the completion of this application, a final prototype device aimed toward human clinical uses will be realized.
| Liu, F; Song, S C; Mix, D et al. (1997) Glucose-induced release of glycosylpoly(ethylene glycol) insulin bound to a soluble conjugate of concanavalin A. Bioconjug Chem 8:664-72 |
| Baudys, M; Uchio, T; Mix, D et al. (1995) Physical stabilization of insulin by glycosylation. J Pharm Sci 84:28-33 |
| Makino, K; Mack, E J; Okano, T et al. (1991) Self-regulated delivery of insulin from microcapsules. Biomater Artif Cells Immobilization Biotechnol 19:219-28 |
