Novel coumarin modified GLP-1 derivatives with enhanced plasma stability and prolonged in vivo glucose-lowering ability

Abstract

Background and purpose: The short biological half-life limits the therapeutic use of glucagon-like peptide-1 (GLP-1) and chemical modification to improve the interaction of peptides with serum albumin represents an effective strategy to develop long-acting peptide analogues. Coumarin, a natural product, is known to bind tightly to plasma proteins and possesses many biological activities. Therefore, we designed and synthesized a series of coumarin-modified GLP-1 derivatives, hypothesizing that conjugation with coumarin would retain the therapeutic effects and prolong the biological half-life of the conjugates.

Experimental approach: Four cysteine-modified GLP-1 analogues (1-4) were prepared using Gly8 -GLP-1(7-36)-NH2 peptide as a starting point. These analogues were conjugated with two coumarin maleimides to yield eight compounds (conjugates 6-13) for testing. Activation of human GLP-1 receptors, stability to enzymic inactivation in plasma and binding to human albumin were assessed in vitro. In vivo, effects on oral glucose tolerance tests (OGTT) in rats and on blood glucose levels in db/db mice were studied.

Key results: Most conjugates showed well preserved receptor activation efficacy, enhanced albumin-binding properties and improved in vitro plasma stability and conjugate 7 was selected to undergo further assessment. In rats, conjugate 7 had a longer circulating t1/2 than exendin-4 or liraglutide. A prolonged antidiabetic effect of conjugate 7 was observed after OGTT in rats and a prolonged hypoglycaemic effect in db/db mice.

Conclusions and implications: Cysteine-specific coumarin conjugation with GLP-1 offers a useful approach to the development of long-acting incretin-based antidiabetic agents. Conjugate 7 is a promising long-lasting GLP-1 derivative deserving further investigation.

Scheme 1

General synthetic route of coumarin GLP-1 conjugates.

Figure 1

Structures of the eight coumarin-modified GLP-1 derivatives investigated here.

Figure 2

Degradation of the coumarin-modified GLP-1 derivatives 6–13, Gly₈-GLP-1(7–36)-NH₂, exendin-4 and liraglutide by rat plasma. A total of 1000 ng mL⁻¹ Gly₈-GLP-1(7–36)-NH₂, exendin-4, liraglutide and conjugates 6–13 were incubated with rat plasma at 37°C and 100 μL samples were taken from the incubation solution at 0, 1, 2, 4, 6, 8, 12, 24, 36, 48 and 72 h time points. Means ± SD, n = 3.

Figure 3

The plasma stability and albumin-binding test of the coumarin-modified GLP-1 derivatives 6–13, Gly₈-GLP-1(7–36)-NH₂, exendin-4 and liraglutide. Means ± SD, n = 3. *P < 0.05, ****P < 0.0001, compared with liraglutide.

Figure 4

In vivo biological activity tests of 7. Exendin-4, liraglutide and conjugate 7 (25 nmol·kg⁻¹) were injected i.p. into Sprague Dawley rats; glucose was given orally (10 g·kg⁻¹). Plasma insulin levels were measured by a rat insulin detection kit. (A) Insulinotropic activities of exendin-4, liraglutide and conjugate 7 (25 nmol·kg⁻¹) in Sprague Dawley rats. Means ± SD, n = 3. (B) AUC_insulin after oral glucose administration, ****P <0.0001.

Figure 5

Effects of conjugate 7 on plasma insulin levels in db/db mice. Vehicle, exendin-4 (25 nmol·kg⁻¹), liraglutide (25 nmol·kg⁻¹) and conjugate 7 (2.5 nmol·kg⁻¹, 25 nmol·kg⁻¹, 250 nmol·kg⁻¹) were injected i.p. in db/db mice; glucose was given orally (2 g·kg⁻¹). Blood insulin levels were measured by a mouse insulin detection kit. Means ± SD, n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

Pharmacokinetic profiles of exendin-4, liraglutide and 7 after s.c. administration (15 nmol per rat), over 48 h (A). In (B), the same data is shown over 16h, to clarify the early results. Each animal was given a compound by s.c. injection and serial blood samples were collected in EDTA-containing microcentrifuge tubes. Collections were made before dosing and at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 and 48 h after treatment. Means ± SD, n = 3.

Figure 7

Long-term hypoglycaemic effects of conjugate 7 was studied using multiple OGTT in db/db mice. Plasma glucose levels are shown in panels A and B. Exendin-4, liraglutide and conjugate 7 (25 nmol·kg⁻¹) and control (vehicle) were given 0.5 h before the first glucose load, and the glucose loads were administered orally at time points 0, 6 and 12 h. Blood was collected at 0, 0.25, 0.5, 1 and 3 h after the glucose load and the plasma glucose was measured using a blood glucose monitor. The time intervals between collections in each OGTT were the same. The time of the glucose load is indicated by arrows. Means ± SD, n = 6. *P < 0.05, **P < 0.01, compared with exendin-4.

Figure 8

Glucose-lowering and stabilizing effect of exendin-4, liraglutide and conjugate 7 as shown by the duration of hypoglycaemia in non-fasted db/db mice. (A) Time-course average blood glucose levels of db/db mice after an i.p. injection of exendin-4 (25 nmol·kg⁻¹), liraglutide (25 nmol·kg⁻¹) or 7 (25/250 nmol·kg⁻¹). Times (with arrow) represent the duration of blood glucose levels below 8.35 mmol·L⁻¹. (B) Hypoglycaemic effects of exendin-4, liraglutide and 7 based on the calculated glucose AUC_0–48 h values. Means ± SD, n = 6. ***P < 0.001, ****P < 0.0001.