Preclinical characterization of recombinant human tissue kallikrein-1 as a novel treatment for type 2 diabetes mellitus

Abstract

Modulation of the kallikrein-kinin system (KKS) has been shown to have beneficial effects on glucose homeostasis and several other physiological responses relevant to the progression of type 2 diabetes mellitus (T2D). The importance of bradykinin and its receptors in mediating these responses is well documented, but the role of tissue kallikrein-1, the protease that generates bradykinin in situ, is much less understood. We developed and tested DM199, recombinant human tissue kallikrein-1 protein (rhKLK-1), as a potential novel therapeutic for T2D. Hyperinsulinemic-euglycemic clamp studies suggest that DM199 increases whole body glucose disposal in non-diabetic rats. Single-dose administration of DM199 in obese db/db mice and ZDF rats, showed an acute, dose-dependent improvement in whole-body glucose utilization. Sub-acute dosing for a week in ZDF rats improved glucose utilization, with a concomitant rise in fasting insulin levels and HOMA1-%B scores. After cessation of sub-acute dosing, fasting blood glucose levels were significantly lower in ZDF rats during a drug wash-out period. Our studies show for the first time that DM199 administration results in acute anti-hyperglycemic effects in several preclinical models, and demonstrate the potential for further development of DM199 as a novel therapeutic for T2D.

Figure 1. Purified DM199.

1, 2 and 4 µg of purified DM199 were either non-reduced (lanes 2, 3 & 4) or reduced (lanes 6, 7 & 8) with NuPAGE Sample Reducing Agent (Life Technologies, Carlsbad, CA) and loaded on to a NuPage Novex 4–12% BIS-TRIS pre-cast polyacrylamide gel (Life Technologies). The gel was stained with Colloidal Blue Staining (Life Technologies, Carlsbad, CA). Lanes 1, 5 and 9: molecular weight marker (SeeBlue Plus2 Standard, Life Technologies, Carlsbad CA).

Figure 2. The effect of DM199 on glucose infusion rate during a hyperinsulinemic-euglycemic clamp.

Catheterized Sprague-Dawley rats (n = 4; age 11–12 weeks) were injected s.c. with either PBS (Control), or DM199 (7 and 35 µg/kg) 30 minutes prior to commencement of a 120-minute hyperinsulinemic-euglycemic clamp (HEC). (A) Profile of glucose infusion rates during HEC. (B) Glucose infusion rate total AUC. Data are presented as the mean ± SEM, **p<0.01 vs. control.

Figure 3. The effect of DM199 on fasting blood glucose and OGTT in db/db mice.

(A) Blood glucose levels were measured in overnight-fasted female db/db mice (n = 9–10 per group) just prior to (t = 0 min) s.c. administration of PBS (Control), Insulin (5 U/kg) or DM199 (7, 144 or 360 µg/kg), and subsequently every 30 min over a 240 minute period. (B) After 7 days, the same cohorts of mice were fasted overnight, injected with test substances as in (A), and after 30 minutes, orally gavaged with 2 g/kg of glucose solution. Blood glucose was measured just prior to s.c. administration and oral gavage, and subsequently at 30 min intervals over a 240 minute period. (C) Mean glucose total AUC was computed from data in (B). Data are presented as the mean ± SEM. *p<0.05 vs. control.

Figure 4. Effects of acute DM199 dosing in ZDF rats.

11-week old male ZDF rats (n = 8) were fasted for 12 hrs, fasting blood glucose was measured, and rats were injected s.c. with either PBS (control) or DM199 (25, 100 or 400 µg/kg). After 120 min rats were gavaged with 2 g/kg glucose solution (t = 0), and blood samples were drawn at 30-min intervals over a 120-minute period. (A) Average blood glucose levels before and during OGTT. (B) Average plasma insulin levels during OGTT. (C) Average plasma glucagon levels during OGTT. Data are presented as the mean ± SEM. *p<0.05; **p<0.01 vs. control.

Figure 5. Effects of sub-acute DM199 dosing in ZDF rats.

At 72-hour intervals (Day 1, 4 and 7), 11-week old male ZDF rats (n = 8) were fasted overnight (12 hrs.) and injected s.c. with either PBS (Control) or DM199 (25, 100 or 400 µg/kg). (A) After 120 min, rats were gavaged with 2 g/kg glucose solution, blood was sampled just prior to oral gavage (t = 0), and subsequently at 30 min intervals over a 120 minute period. For each dosing, glucose iAUC was calculated using t = 0 glucose value as background. (B) Average plasma insulin levels from fasted rats (t = 0). *p<0.05; **p<0.01 vs. control.

Figure 6. Effects of sub-acute DM199 dosing and drug wash-out.

11-week old male ZDF rats (n = 8) were fasted overnight (12 hrs.) and injected s.c. with either PBS (Control) or DM199 (25, 100 or 400 µg/kg) on days 1, 4 and 7 (arrows), with no subsequent treatments for a 7 day period. Fasting blood glucose levels were measured daily just prior to treatment. *p<0.05; **p<0.01 with respect to control treatment.