Proinsulin-Transferrin Fusion Protein as a Novel Long-acting Insulin Analogue for the Inhibition of Hepatic Glucose Production Running title: ProINS-Tf as a long-acting inhibitor of HGP

Proinsulin-transferrin (ProINS-Tf) fusion protein was evaluated for its in vivo pharmacokinetics, efficacy and mechanism. Our previous studies have shown that ProINS-Tf was converted to active insulin (INS)-Tf via the Tf-Tf receptor mediated pathway in hepatoma cells. We hypothesized that this fusion protein can be administered as a prodrug, and be converted to a biologically active protein with specificity for the liver versus other INS-sensitive tissues (muscle and adipose). Administration as an inactive prodrug with liver-specific action compared to other INS-sensitive tissues conceivably reduces negative side-effects seen with other INS analogs. In this report, the data show that ProINS-Tf exhibited a slow, but sustained, in vivo hypoglycemic efficacy and long plasma half-life. The fusion protein showed activity in the liver, as evidenced by decreased expression of two key hepatic glucose production (HGP) enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, and by increased glycogen levels under feeding conditions. Furthermore, the INS-receptor (IR) phosphorylation (activation) in liver and muscle tissues was compared post-injection of INS or ProINS-Tf. While INS activated IR in both the liver and muscle, ProINS-Tf only showed activation in the liver. Thus, ProINS-Tf fusion protein can potentially be administered as a prodrug with sustained Tfmediated activation and selectivity in inhibiting HGP. Page 2 of 42 Diabetes