A novel glucagon analog with an extended half-life, HM15136, normalizes glucose levels in rodent models of congenital hyperinsulinism

Abstract

Congenital hyperinsulinism (CHI) is a rare genetic condition characterized by uncontrolled insulin secretion, resulting in hypoglycemia. Although glucagon has lately been regarded as a therapeutic option for CHI, its use is severely hampered by its poor solubility and stability at physiological pH, as well as its short duration of action. To address these constraints, we developed HM15136, a novel long-acting glucagon analog composed of a glucagon analog conjugated to the Fc fragment of human immunoglobulin G4 via a polyethylene glycol linker. In this study, we established that HM15136 was more soluble than natural glucagon (≥ 150 mg/mL vs 0.03 mg/mL). Next, we confirmed that HM15136 activated glucagon receptor in vitro and induced glycogenolysis and gluconeogenesis in rat primary hepatocytes. Pharmacokinetics (PK)/Pharmacodynamics (PD) analysis of HM15136 shows that HM15136 has a markedly longer half-life (36 h vs. < 5 min) and increased bioavailability (90%) compared to native glucagon in mice. Further, HM15136 could effectively reverse acute hypoglycemia induced by insulin challenge, and multiple doses of HM15136 could sustain increased blood glucose levels in CHI rats. In conclusion, our findings indicate that HM15136 promotes sustained elevation of blood glucose, demonstrating the potential for development as a once-weekly therapy for CHI.

Figure 1

Structure of HM15136, conjugate of glucagon analog and human immunoglobulin G4 Fc. (A) Structure of HM15136. (B) Primary sequence of glucagon analog. (C) Physical stability of HM15136 in phosphate-buffered saline (pH 7.0) at 25 °C. (D) Solubility of HM15136 (pH 7.0) at 25 °C.

Figure 2

HM15136 stimulates GCCR and increases cAMP levels. (A) HM15136 induced a dose-dependent increase in intracellular cAMP levels in hGCGR/CHO cells. Symbols denote native glucagon (open circle) and HM15136 (open square). The concentration of individual test agents ranged from 0.0002 to 194.4 nM (for native glucagon) and from 0.0015 to 1555.2 nM (for HM15136). (B,C) Glucose production in rat primary hepatocyte treated with either HM15136 or native glucagon for 30 min (B, for glycogenolysis) and 6 h (C, for gluconeogenesis). Isolated hepatocytes were incubated with insulin to promote glycogen production and then, treated with HM15136 for 30 min to induce glycogenolysis. For gluconeogenesis, serum-starved hepatocytes were treated with HM15136 for 6 h in the presence of pyruvate and lactate. After treatment, the glucose level in the collected medium was determined via a GOPOD assay. Results represent mean ± S.D. of duplicate assessments. *p < 0.05, **p < 0.01, ***p < 0.001 vs 0 nM. (D) Mechanism of HM15136 for elevation of glucose blood level.

Figure 3

HM15136 interacts with FcRn and does not bind FcγR. The sensorgrams for the FcγRIA (A–C), C1q (D–F) binding of IgG1 (= I.V.-Globulin SN), HMC001, and HM15136. IgG1 control (A,D), HMC001 (B,E) and HM15136 (C,F). The concentration of individual test from top to bottom were 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM for IV-Globulin SN (A) and HMC001 (B) and 486 nM, 243 nM, 121.5 nM, 60.75 nM, and 30.38 nM for HM15136 (C). The concentration of the individual test agents: IV-Globulin SN (D), HMC001 (E), and HM15136 (F) from top to bottom were 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM for C1q.

Figure 4

HM15136 exhibited good bioavailability and had high systemic exposure and long-lasting PK properties in mice, rat, and dog. (A) A single dose of HM15136 was administered via the intravenous and subcutaneous routes in ICR mice (n = 3/time point/group). The error bars indicate standard deviation. The symbols represent the following: (filled circle) HM15136 (i.v.) 260 μg/kg; (open square) HM15136 (s.c.) 260 μg/kg; (open triangle) HM15136 (s.c.) 519 μg/kg; (open reverted triangle) HM15136 (s.c.) 1558 μg/kg; (B) A single dose of HM15136 was administered via the intravenous and subcutaneous routes in SD rats (n = 3/group). The error bars indicate standard deviation. The symbols represent the following: (filled circle) HM15136 (i.v.) 260 μg/kg; (open square) HM15136 (s.c.) 260 μg/kg; (open triangle) HM15136 (s.c.) 519 μg/kg; (open reverted triangle) HM15136 (s.c.) 1558 μg/kg. (C) A single dose of HM15136 was administered via the intravenous and subcutaneous routes in beagle dogs (n = 3/group). The error bars indicate standard deviation. The symbols represent the following: (filled circle) HM15136 (i.v.) 52 μg/kg; (open square) HM15136 (s.c.) 52 μg/kg; (open triangle) HM15136 (s.c.) 104 μg/kg; (open reverted triangle) HM15136 (s.c.) 260 μg/kg.

Figure 5

HM15136 increases blood glucose levels in hyperinsulinemia-induced acute and chronic hypoglycemic rats. (A) Hypoglycemia was induced with 0.65 IU/kg human insulin treatment in male SD rats fasted for 4 h. The indicated doses of HM15136 and native glucagon were administered intravenously once at 45 min (n = 5/group). Blood glucose of all animals were measured at 0, 45, 75, 120, and 180 min with OneTouch® Ultra® (Johnson & Johnson, USA). Each symbol represents the following: vehicle control, s.c. (filled circle), HM15136 268 μg/kg, i.v. (filled square), HM15136 536 μg/kg, i.v. (filled triangle), HM15136 1071 μg/kg, i.v. (filled reverted triangle), and glucagon 209 μg/kg, s.c. (open circle). (B) Experimental scheme for insulin-induced chronic hypoglycemia in SD rats. The indicated doses of HM15136 were subcutaneously administered every 3 days to mimic the human weekly dosing interval (n = 6–15/group). (C) Blood glucose levels with treatment over time. Each symbol represents the following; vehicle control for rats implanted with an insulin pump (filled circle), implanted pump containing vehicle in place of insulin (filled circle), HM15136 at 156 μg/kg (filled square), HM15136 at 234 μg/kg (filled triangle), HM15136 at 312 μg/kg (filled reverted triangle), and HM15136 at 416 μg/kg (filled diamond). (D) Quantification of the area under the curve (AUC) from blood glucose/time graph. Blood glucose after 2-h fasting was measured daily with OneTouch^® Ultra^® (Johnson & Johnson, USA). Each symbol represents the following; implanted pump containing vehicle in place of insulin (open square), vehicle control for rats implanted with an insulin pump (filled square), HM15136 at 156 μg/kg (square with horizontal stripes), HM15136 at 234 μg/kg (square with vertical stripes), HM15136 at 312 μg/kg (square with leftward cross stripes), and HM15136 at 416 μg/kg (square with rightward cross stripes). Values are expressed as mean ± SEM. (*p < 0.05 and ***p < 0.001 vs. Pump vehicle group).

Figure 6

High levels of HM15136 were observed in the liver where gluconeogenesis and glycogenolyis occur.