Oral delivery of an anti-diabetic peptide drug via conjugation and complexation with low molecular weight chitosan

Abstract

Despite the therapeutic potential of exendin-4 as a glucagon-like peptide-1 (GLP-1) mimetic for the treatment of type 2 diabetes, its utility has so far been limited because of the low level of patient compliance due to the requirement for frequent injections. In this study, an orally available exendin-4 was produced by conjugating it to low molecular weight chitosan (LMWC). Conjugation between the LMWC and cysteinylated exendin-4 was carried out using a cleavable linker system in order to maximize the availability of the active peptide. The LMWC-exendin-4 conjugate formed a nanoparticle structure with a mean particle size of 101 ± 41 nm through complexation between the positively charged LMWC backbone and the negatively charged exendin-4 of individual conjugate molecules. The biological activity of the LMWC-exendin-4 conjugate was evaluated in an INS-1 cell line. The LMWC-exendin-4 conjugate stimulated insulin secretion in a dose dependent manner as similar as that of native exendin-4. From the pharmacokinetic study after oral administration of the conjugate, a C_max value of 344 pg/mL and a T_max of 6 h were observed, and the bioavailability, relative to the subcutaneous counterpart, was found to be 6.4%. Furthermore, the absorbed exendin-4 demonstrated a significantly enhanced hypoglycemic effect. These results suggest that the LMWC-exendin-4 conjugate could be used as a potential oral anti-diabetic agent for the treatment of type 2 diabetes.

Introduction

Patients with type 2 diabetes have greatly impaired incretin-mediated insulin secretion, mainly owing to decreased secretion of glucagon-like peptide-1 (GLP-1) [1]. Exendin-4 is a GLP-1 mimetic peptide comprising 39 amino acids that has been used in the treatment of type 2 diabetes as it has a significantly enhanced half-life in vivo compared to endogenous GLP-1, and similar gluco-regulatory activity [2], [3]. Even though exendin-4 has been widely used for the treatment of type 2 diabetes, patients are required to undergo frequent subcutaneous injections, resulting in poor patient compliance in addition to side effects such as infection at the sites of injection [4], [5]. Since oral delivery is expected to result in drastically enhanced patient compliance, many attempts have been made to develop oral delivery systems for exendin-4 [6], [7], [8]. However, orally administered peptides encounter formidable barriers to absorption into the blood stream. These typically include physical barriers, such as viscous mucous layers and tight junctions of aligned enterocytes in the gastrointestinal (GI) track; chemical barriers, such as low stomach pH; and biological barriers, such as enzymatic degradation. Overcoming these problems is essential for improving the level of absorption of orally administered peptide-based drugs [9], [10]. There have been a limited number of successful studies into the development of systems for oral exendin-4 delivery. It has been shown that site-specific covalent attachment of biotin to exendin-4 facilitated receptor-mediated intestinal absorption of the peptide, resulting in approximately 3.95% oral bioavailability (BA) [7]. Very recently, a relatively high oral BA of ~ 14.0% was attained using a nanoparticulate system formed by electrostatic complexation between negatively charged exendin-4 and positively charged chitosan, and subsequent coating with anionic poly(γ-glutamic acid) [8]. Unlike previous approaches, we have sought to overcome such oral delivery barriers using chemical conjugation between the therapeutic agent of interest and a mucoadhesive polymer. In the last few years, a large number of mucoadhesive materials have been investigated for oral delivery of peptides and proteins, including chitosan, methacrylate, and alginate, based polymers [11], [12], [13], [14], [15], [16]. Rekha and Sharma reported that anionic/hydrophobic modification of chitosan (LSC) enhanced mucoadhesivity of the nanoparticles and orally administered FITC-insulin loaded LSC particles to diabetic rats showed enhanced insulin absorption and transported insulin across the enterocytes efficiently. We have shown that intestinal absorption of the therapeutic agent could be significantly enhanced by conjugating it to low molecular weight chitosan (LMWC) [17], [18], [19]. Such high absorption is attributed to unique features of LMWC, including its high mucoadhesiveness and its ability to open tight cell-cell junctions, facilitating paracellular transport of drugs [20], [21], [22], [23], [24]. Our previous results suggest that the conjugation of chemical or peptide-based drugs to LMWC may be an efficient strategy for enabling a high level of absorption into the blood stream [18].

In this regard, here we report the development of an oral delivery method for exendin-4 by conjugating it to LMWC. We describe the synthesis and characterization of the conjugate, its activity in the induction of in vitro cellular insulin secretion, and the pharmacokinetic parameters and glucoregulatory effect after oral administration in a diabetic mouse model.