Unique Drug Combination for Reversal of Type 1 Diabetes Sarah A. Tersey, Jeffrey D. Carter, Elizabeth Z. Kropf, Lawrence Rosenberg*, Jerry L. Nadler Department of Medicine, University of Virginia, Charlottesville, VA and * McGill University, Montreal, CANADA Introduction Type 1 diabetes mellitus (T1DM) is an autoimmune disorder characterized by immune damage to pancreatic beta-cells. Lisofylline (LSF) is an immunomodulator that reduces interleukin 12 signaling and reduces the onset of Type 1 diabetes in non-obese diabetic (NOD) mice. Islet neogenesis associated protein (INGAP) is a member of the Reg3 family of pancreatic proteins and can induce new islet formation and restore euglycemia in streptozotocin-induced diabetic mice. We hypothesize that a combination therapy with both LSF and INGAP peptide provides protection from autoimmune destruction via LSF and also stimulates beta-cell regeneration with INGAP peptide. Treatment Protocol NOD/Ltj female mice from Jackson (Bar Harbor, Maine) were placed randomly into a treatment group after 2 consecutive non-fasting blood glucose values over 250mg/ml. LSF was delivered via the Alzet osmotic minipumps (DURECT Co., Cupertino, CA) to provide consistently systemic administration at 27mg/kg/day. INGAP was administered by intraperitoneal injection at 2mg/kg/day once a day for 6 weeks. Each mini-pump was placed subcutaneously on the back of a mouse under a general analgesia. All mice received subcutaneous insulin pellets (~0.1 U/24 hr/pellet; Linshin, Ontario, Canada) to maintain euglycemia during the course of treatment. Insulin pellets were removed after 6 (7 weeks for pretreatment) of treatment and we continued to monitor blood glucose for one week before euthanasia. Results Figure 3. Number of insulin pellets required to maintain euglycemia. All mice received subcutaneous insulin pellets (~0.1 U/24 hr/pellet) to maintain euglycemia during the course of treatment. Mice in group 5 (pretreatment) required an average of only 1.1 pellets compared to 2.5 pellets in the saline control group (p<0.01) during treatment to maintain a blood glucose value below 250mg/dl. Results and Conclusions 1.Pretreatment with LSF followed by 5 additional weeks on both INGAP and LSF resulted in remission for 70% of the animals regardless of starting blood glucose. 2.The mice in the pretreatment group also required fewer insulin pellets over the course of treatment than the other groups. The animals receiving INGAP peptide had higher random fed serum insulin levels. 3.Mice in the pretreatment group also showed more Ki67 staining in positive insulin cells, indicating more active cell proliferation in their islets. 4.In the pretreatment combined group, we found islets ranging from positive staining with insulin and Pdx1 to positive staining with both Pro-insulin and Pdx1 without mature insulin, indicating a potential of newly formed islets. Acknowledgments This study was supported in part by the NIH-NIDDK, the Farish Foundation, the Ella Fitzgerald Foundation, and the Iacocca Foundation. We thank DiaKine Therapeutics for providing the Lisofylline and Kinexum LCC for providing the INGAP peptide. For further information please contact or Saline Control LSF Alone INGAP Alone INGAP/LSF Pretreatment Saline Control LSF Alone INGAP Alone INGAP/LSF Pretreatment X X X X X Insulin LSF INGAP No treatment Figure 1: Treatment Protocol Figure 2: Blood glucose values. A, Starting blood glucose values for all treatment mice. B, Blood glucose values during treatment while being maintained euglycemic via insulin pellets. * A.B. Blue – Hoechst; Green – Insulin; Red – Ki67 A.B.C. D.E. C57bl/6 controlNon-diabetic NODInsulin Alone Pretreatment Figure 7: Insulin and Ki67 co-immunofluorescence. All treatment groups were co-stained for insulin and Ki67. A, C57bl/6 control. B, age matched non-diabetic NOD control. C, representative insulin alone. D, E, representative pretreatment islets. Figure 6: Random Fed Serum Insulin. All mice treated with INGAP and LSF produced higher serum insulin levels (1.01ng/ml) than saline treated controls (0.18ng/ml) and were similar to non- diabetic NOD mice (1.4ng/ml), INGAP alone and Pretreatment groups were statistically greater than saline control (p>0.05). B. A. Figure 4: Remission rates of treatment groups. After treatment, insulin pellets were removed and random blood glucose was monitored. A, average blood glucose value during final week with no insulin treatment. B, rate of remission (<250mg/dl). Mice in the pretreatment group had a 70% remission rate, regardless of blood glucose starting level. Saline Control LSF Alone INGAP Alone INGAP/LSF Pretreatment Figure 5: Successful long term pretreatment mouse. One pretreatment mouse has maintained euglycemia with no insulin for 6 weeks following treatment. Figure 8: Insulin, Pdx1, and pro-insulin immunohistochemistry. All treatment groups were stained for insulin, Pdx1, and pro-insulin. Shown are representative islets (20X) from three groups stained consecutively for each antibody. A, B, C, representative age-matched non-diabetic NOD control. D-F, representative islets from insulin alone treatment. G-L, representative pretreatment islets. Non-Diabetic Saline Control LSF Alone INGAP Alone INGAP/LSF Pretreatment Control non-diabetic NOD A.B.C. Pretreatment InsulinPdx1Pro-Insulin D.E.F. G.H.I. J.K.L. Insulin only In conclusion, we show new data suggesting a potential therapy for type 1 diabetes using a novel combined treatment utilizing Lisofylline and INGAP peptide. Studies are currently ongoing to determine if the pretreatment will be effective for late-onset diabetes as well as more in depth immunohistochemistry and gene expression. * *