Variant fatty acid-like molecules Conjugation, novel approaches for extending the stability of therapeutic peptides

Abstract

The multiple physiological properties of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the treatment of type 2 diabetes. However, the in vivo half-life of GLP-1 is short due to rapid degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. The poor stability of GLP-1 has significantly limited its clinical utility; however, many studies are focused on extending its stability. Fatty acid conjugation is a traditional approach for extending the stability of therapeutic peptides because of the high binding affinity of human serum albumin for fatty acids. However, the conjugate requires a complex synthetic approach, usually involving Lys and occasionally involving a linker. In the current study, we conjugated the GLP-1 molecule with fatty acid derivatives to simplify the synthesis steps. Human serum albumin binding assays indicated that the retained carboxyl groups of the fatty acids helped maintain a tight affinity to HSA. The conjugation of fatty acid-like molecules improved the stability and increased the binding affinity of GLP-1 to HSA. The use of fatty acid-like molecules as conjugating components allowed variant conjugation positions and freed carboxyl groups for other potential uses. This may be a novel, long-acting strategy for the development of therapeutic peptides.

Figure 1. Stabilization studies of GLP-1 derivatives against DPP IV proteolysis.

Panel (A) A pharmacokinetic study of GLP-1 (○) and derivatives in DPP IV protease solution. Panel (B) A pharmacokinetic study of GLP-1 (○) and derivatives in DPP IV protease solution in the presence of human serum albumin. Legend: The results indicate that the compound 6, 15, 17, 18, 21, 22, 23 and 24 was still detectable at 24 h, whereas native GLP-1 was rapidly degraded within 5 min, showing that these derivatives have prolonged stabilities upon the binding to human serum albumin. Panel (C) AUC_{GLP-1(7–37)} of the circulating GLP-1 concentration in proteolysis solution. Legend: The calculated AUC_GLP-1(7-37) shows that the amount of compound 21, 22, 23 and 24 was significantly increased compared to native GLP-1, P < 0.01. Conditions: GLP-1 and fifteen GLP-1 derivatives were incubated in DPP IV solution with or without the existence of human serum albumin. A human GLP-1 (7–37) ELISA kit was used to quantitatively determine the half-life of GLP-1 and its derivatives in human serum, according to the manufacturer’s instructions.

Figure 2. Blood retention time measurement of GLP-1 derivatives in Wistar rats.

Panel (A) A pharmacokinetic study of the GLP-1 derivatives in Wistar rats. Panel (B) AUC _{GLP-1 (7-37)} of the circulating GLP-1 concentration in treated Wistar rats. Legend: The results show that the serum level of native GLP-1 (○) was rapidly decreased by proteolysis and that the compound 22 (▲) and compound 24 (●) derivatives extended the stabilities in vivo compared with native GLP-1. In addition, the AUC data indicated that a 2.6- and 2.7-fold increase of circulating amount of compound 22 and 24, respectively. Conditions: Wistar rats were injected with the GLP-1 derivatives (compound 18, 21, 22, 23 and 24) (500 μg/kg body weight, n = 5). Blood samples were taken from the tail vein at various time points, and the peptide levels were measured using a GLP-1 (7–37) ELISA kit.

Figure 3. Effects of GLP-1 derivatives on the insulin stimulation and cAMP accumulation after oral glucose administration.

Panel (A) The stimulation of insulin secretion by native GLP-1 (○) and five derivatives (compound 18, 21, 22, 23 and 24) in Wistar rats after oral glucose administration. Panel (B) The effect of GLP-1 derivatives on cAMP levels in INS-1 cells. Legend: The results indicate that the oral administration of glucose increased insulin levels in the rats. The levels of secreted insulin induced by GLP-1 arrived the peak at 10–20 min and returned to baseline at 40 min. The insulin levels in Wistar rats treated with compound 22 or 24 showed distinct difference in insulin secretion. Compared with GLP-1, these two derivatives stimulated the prolonged secretary response significantly. In similarity, the cellular cAMP level stimulated by GLP-1 peaked to 16.34 ± 1.04 pmol/10⁵ cells in 2 min before declining to baseline at 10 min. However, the cells treated with compound 22 (▲) or 24 (●) exhibited longer response than cells treated with GLP-1 remarkably. Conditions: GLP-1 and derivatives (100 μg GLP-1/kg body weight) were injected into Wistar rats; glucose was administered orally (10 g/kg). The concentration of insulin was measured using a rat insulin detection kit. Total cellular cAMP was measured in INS-1 cells (1.0 × 10⁵) at the indicated times using an HTRF-cAMP kit. The data are presented as the means ± SE, P < 0.01.

Figure 4. Effects of compound 22 and 24 on glucose tolerance after a single-dose injection.

Panel (A) The effect of a single injection of the GLP-1 derivatives and Liraglutide on glucose regulation in Wistar rats. Panel (B) The dosage-efficacy of compound 22 and 24, 24 h after administrations. Legend: The administration of compound 24 maintained the blood glucose concentration at normal levels for 48 hours after a single-dose injection approximately, suggesting improved blood glucose clearance activity than liraglutide in long term manner. Conditions: Fasting Wistar rats were injected with the GLP-1 derivatives or GLP-1. Glucose (2 g/kg body weight) was administered 30 min before each time point (0–120 h).

Figure 5. Effect of multiple doses of compound 24 over 42 days treatment.

Panel (A) The long-lasting glucose regulatory effect of compound 24 (●) in ZDF rats. Panel (B) Body weight changes after the treatment of compound 24 in ZDF rats. Legend: The results indicate that the treated ZDF rats maintained relatively constant and lower glucose levels than the control rats; native GLP-1 failed to produce a similar glucose regulatory effect. Another compound 22 and liraglutide is not capable to provide satisfied blood glucose well-control. Treatment of compound 24 also lowered the body weight increase which is the physiological function of GLP-1. Conditions: Compound 24 (●) (300 μg /kg body weight) was administered every two days during the experimental period, which lasted 42 days. A glucometer was used to measure the glucose levels at various time intervals.