Feasibility of localized immunosuppression: 1. Exploratory studies with glucocorticoids in a biohybrid device designed for cell transplantation.

Emerging biotechnologies, such as the use of biohybrid devices for cellular therapies, are showing increasing therapeutic promise for the treatment of various diseases, including type 1 diabetes mellitus. The functionality of such devices could be greatly enhanced if successful localized immunosuppression regimens could be established, since they would eliminate the many otherwise unavoidable side effects of currently used systemic immunosuppressive therapies. The existence of local immune privilege at some specialized tissues, such as the eye, CNS, or pregnant uterus, supports the feasibility of localized immunomodulation, and such an approach is particularly well-suited for cell transplant therapies where all transplanted tissue is localized within a device. Following the success of syngeneic transplantation in a subcutaneous prevascularized device as a bioartificial pancreas in a rodent model, we now report the first results of exploratory in vivo islet allograft studies in rats using locally delivered glucocorticoids (dexamethasone phosphate and the soft steroid loteprednol etabonate). Following in vitro assessments, in silico drug distribution models were used to establish tentative therapeutic dose ranges. Sustained local delivery was achieved via implantable osmotic mini-pumps through a central sprinkler, as well as with a sustained-delivery formulation for loteprednol etabonate using poly(D,L-lactic) acid (PLA) microspheres. Doses delivered locally were approximately hundred-fold smaller than those typically used in systemic treatments. While several solubility, stability, and implantation problems still remain to be addressed, both compounds showed promise in their ability to prolong graft survival after tapering of systemic immunosuppression, compared to control groups.

Effects of systemic immunosuppression on islet engraftment and function into a subcutaneous biocompatible device.

The aim of this study was to explore the effect of sirolimus (Sir) and tacrolimus (Tac) on islets implanted into a subcutaneous (SC), prevascularized device in syngeneic rats. Animals received a 40-day treatment with Tac and Sir (alone or in combination) starting either on day 0 or 40 days after islet transplantation. Controls received no treatment. A 40-day washout period was performed after immunosuppression (IS). Glycemia and intravenous glucose tolerance tests (IVGTT) were assessed at follow-up. In the control group, 75% of recipients achieved stable normoglycemia after islet transplantation, while none reversed diabetes with any IS regimen started on day 0. Graft dysfunction was irreversible after IS withdrawal. Glucose clearance (IVGTT) was significantly impaired among Tac-treated compared with control groups (P < .05 with IS; P < .01 after washout). Among animals with established grafts, islet dysfunction which occurred under IS treatment persisted after washout in animals treated with Tac and Sir plus Tac. When compared with controls, glucose clearance was significantly impaired in the Tac and Tac plus Sir groups before and after IS (P < .01, Tac; P < 0.01, Tac plus Sir). Sir and Tac showed profound deleterious effects on islet cell engraftment and function, which may hinder the success of implantation into biohybrid devices. Nondiabetogenic IS protocols must be developed for clinical application of islet transplantation into biohybrid devices.

Rapamycin impairs beta-cell proliferation in vivo.

During pregnancy a high rate of beta-cell proliferation occurs, making of this a useful model for the study of islet cell expansion in vivo. We used the murine pregnancy model to assess the effect of Rapamycin treatment on islet cell proliferation in vivo. Rapamycin is routinely used for the prevention of graft rejection in transplanted patients, including islet transplant recipients. As expected, pregnancy led to increased beta-cell proliferation, islet yield and skewing in size distribution after isolation and pancreatic insulin content, when compared to non-pregnant females. Rapamycin treatment resulted in reduced beta cell proliferation in pregnant mice, while minimal effects of Rapamycin treatment were observed on islet function both in vivo and in vitro. Rapamycin treatment of islets resulted in reduced phosphorylation of p70s6k, a downstream effector molecule of mTOR and increased ERK1/2 phosphorylation. In conclusion, beta-cell replication is reduced under Rapamycin treatment in vivo, suggesting that this mechanism may be operational and impair beta-cell renewal in transplanted patients.

Soft corticosteroids for local immunosuppression: exploring the possibility for the use of loteprednol etabonate for islet transplantation.

Transplantation of pancreatic islets into subcutaneous, neovascularized devices is one of the possibilities explored as part of our search for a cure of diabetes. We have recently reported that syngeneic transplantation in a subcutaneous prevascularized device can restore euglycemia and sustain long-term function in rats and that explanted grafts showed preserved islets and intense vascular networks. Because all of the transplanted tissue is localized within the device, if such a bioartificial pancreas approach is used, localized immunosuppression might provide sufficient protection against rejection to achieve long-term function, while also avoiding the serious systemic side effects and the susceptibility for opportunistic infections that are commonly associated with systemic immunosuppressive therapies as only much smaller and localized doses are needed. Soft steroids are obvious candidates because soft drugs are specifically designed to produce targeted local activity, but no systemic side effects due to prompt metabolic (preferably extrahepatic, e.g., hydrolytic) inactivation. However, local concentrations that are effective for immunosuppression, but non-toxic to insulin-producing beta-cells have to be found, and nontrivial difficulties related to long-term local deliverability have to be addressed. Here, we report preliminary results obtained using in vitro studies with human islets used to establish a tentative therapeutic concentration range together with fully scaled three-dimensional finite element method (FEM)-based Comsol multiphysics computational models that were used to explore various possibilities to achieve and maintain these concentration levels within the device.