A depot-forming glucagon-like peptide-1 fusion protein reduces blood glucose for five days with a single injection

Abstract

Peptide drugs are an exciting class of pharmaceuticals for the treatment of a variety of diseases; however, their short half-life dictates multiple and frequent injections causing undesirable side effects. Herein, we describe a novel peptide delivery system that seeks to combine the attractive features of prolonged circulation time with a prolonged release formulation. This system consists of glucagon-like peptide-1, a type-2 diabetes drug fused to a thermally responsive, elastin-like-polypeptide (ELP) that undergoes a soluble-insoluble phase transition between room temperature and body temperature, thereby forming an injectable depot. We synthesized a set of GLP-1-ELP fusions and verified their proteolytic stability and potency in vitro. Significantly, a single injection of depot forming GLP-1-ELP fusions reduced blood glucose levels in mice for up to 5 days, 120 times longer than an injection of the native peptide. These findings demonstrate the unique advantages of using ELPs to release peptide-ELP fusions from a depot combined with enhanced systemic circulation to create a tunable peptide delivery system.

Keywords: Drug delivery; Elastin-like polypeptide; Glucagon-like peptide-1; Peptide; Subcutaneous depot.

Figure 1

Design and characterization of (GLP-1)-ELP depots. (A) Modified amino acid sequence of GLP-1: Amino acids highlighted in red are essential for GLP-1 binding; modified amino acids are highlighted in blue (modified after [33]), arrow marks site of activation by DPPIV. (B) ITC purification of (GLP-1)-ELP_Depot: lanes (1) sonicated cell lysate, (2) soluble cell lysate, (3) re-suspended cold spin pellet, (4) purified (GLP-1)-ELP_Depot construct after 2 ITC cycles. (C) Transition temperature of (GLP-1)-ELP_Depot and (GLP-1)-ELP_Sol. (D) CD spectra of monomer native GLP-1, ELP_Depot and (GLP-1)-ELP_Depot (all at 7.5 μM).

Figure 2

(A) Degradation of (GLP-1)-ELP_Depot or GLP-1 by NEP after 18 h incubation (lane 1: (GLP-1)-ELP_Depot lane 2: (GLP-1)-ELP_Depot+NEP, lane 3: GLP-1, lane 4: GLP-1+NEP. NEP may be seen as a faint band at ~85kDa. (B) cAMP response of native GLP vs. (GLP-1)-ELP_Depot in BHK cells expressing the GLP-1R. (C-E) NIR tomography images after a single s.c. injection of (C) (GLP-1)-ELP_Sol, 175 nmol/kg (D) (GLP-1)-ELP_Depot, 175 nmol/kg and (E) (GLP-1)-ELP_Depot, 700 nmol/kg immediately following injection and 24, 72 and 120 h after s.c. injection.

Figure 3

Fed glucose reduction following a single injection of (GLP-1)-ELPs. Daily fed blood glucose levels normalized by baseline glucose levels after a single s.c. injection of PBS control or (A) (GLP-1)-ELP_Depot (175 nmol/kg, ANOVA p<0.001), (B) (GLP-1)-ELP_Depot (350 nmol/kg, ANOVA p<0.0001) (C) (GLP-1)-ELP_Depot (700 nmol/kg, ANOVA p<0.0001) with inset showing the effect of a single injection of GLP-1 (50 nmol/kg) (D) a separate experiment comparing the effects of (GLP-1)-ELP_Sol (175 nmol/kg) with (GLP-1)-ELP_Depot (175 and 700 nmol/kg) (ANOVA p<0.0001). Injection was administered at the 0 h time point. (*** p<0.05, ** p<0.01, * p<0.001). (E-F) IPGTT following a single injection of (GLP-1)-ELP_Depot. Blood glucose levels in 7 week old mice during IPGTT performed (E) 52 h and (F) 102 h after a single injection of (GLP-1)-ELP_Depot (700 nmol/kg, *** p<0.001).