Abstract
Exendin-4 is a 39-amino acid peptide that shares several glucoregulatory activities with
the mammalian incretin hormone, glucagon-like peptide-1 (GLP-1). The synthetic form of
exendin-4, exenatide, was approved as adjunctive therapy by subcutaneous injection for patients
with type 2 diabetes failing to achieve glycemic control with oral antidiabetic agents. Basically,
the oral route is the most convenient way of drug administration for patients because it can avoid
pain and reduce the complex complication. Base on our previous study, we developed
nanoparticles (NPs) composed of chitosan (CS), poly-γ-glutamic acid (γPGA) with Fe ions as the
carrier for exendin-4 delivery via the oral route. The size and zeta potential of the prepared NPs
were ~300nm and ~30mV, respectively. The exendin-4 loaded in NPs could get ~60% loading
efficiency (LE) and ~15% Loading Content (LC). To protect NPs in the acidic stomach
environment, gelatin hard capsules filled with NPs were coated with enteric coating polymer.
The enteric coating polymer, Eudragit L55-100, was dissolved beyond pH 6.0 that mimetic the
pH environment of duodenum in the GI track. In the dissolution study, the test capsule could be
dissolved immediately as it reached the duodenal region and NPs could then release exendin-4
into the systemic circulation via the opened paracellular pathway. For the in vivo study, the
biodistribution of exendin-4 loaded NPs following the oral administration to rats was performed
using the single-photon emission computed tomography (SPECT)/computed tomography (CT).
The results obtained in the SPECT/CT study indicate that the orally administered exendin-4 was
significantly absorbed into the systemic circulation. There was very high circulating exendin-4
(~48%) in the peripheral tissue and plasma (PP) at 2 h post ingestion. In the pharmacodynamic
(PD) and pharmacokinetic (PK) evaluation in a mild diabetic rat model, the orally administered
exendin-4 loaded NPs produced a slower hypoglycemic response for a prolonged period of time.
The relative bioavailability of exendin-4 orally delivered by test NPs in gelatin capsules coated
with 10% Eudragit was found to be 13.7 ± 2.1%. The results suggest the suitability of the NP
system to be used as a non-invasive alternative for the basal exendin-4 therapy.

ii

Contents
Abstract…………………………………………………………………………………………….i
Contents………………………..……………………..……………………..……………………..ii
List of Figures……………………..……………………..……………………..…………………ii
List of Tables……………………..……………………..……………………..………………… v
Chapter 1. Introduction………………………………………………………………………… 1
1.1 Exendin-4…………………………………………………………………………………….. 1
1.2 Strategies for exendin-4 delivery…………………………………………………………….. 2
1.3 Nanoparticles composed of Chitosan and poly(γ-glutamic acid) ……………………………. 4
Chapter 2. Materials and methods……………………………………………………………... 9
2.1 Depolymerization of chitosan by enzyme Hydrolysis……………………………………….. 9
2.2 Preparation of low-MW γPGA……………………………………………………………….. 9
2.3 Preparation and characterization of Exendin-4 loaded CS- γPGA Nanoparticles………….. 10
2.3.1 Preparation of Exendin-4 loaded NPs…………………………………………………. 11
2.3.2 Freeze drying exendin-4 loaded NPs………………………………………………….. 11
2.3.3 Characterization of exendin-4 loaed NPs……………………………………………... 12
2.4 Transepithelial electrical resistance (TEER) measurements………………………………… 12
2.4.1 In vitro growing Caco-2 cell monolayers…………………………………………….. 12
2.4.2 TEER study…………………………………………………………………………… 13
2.5 Dissolution study of enteric-coated capsules……………………………………………….. 13
2.5.1 In vitro dissolution study……………………………………………………………… 13
2.5.2 In vivo dissolution study……………………………………………………………… 14
2.6 In vivo study………………………………………………………………………………… 15
2.6.1 Biodistribution study…………………………………………………………………. 15
iii

2.6.2 Creating the mild diabetic rat models for PD/PK study……………………………… 15
2.6.3 In vivo PD and PK study…………………………………………………………….. 15
Chapter 3. Results and Discussions…………………………………………………………. 18
3.1 Depolymerization of chitosan and γPGA………………………………………………….. 18
3.2 Characterization of exendin-4 loaded NPs…………………………………………………. 19
3.2.1 Optimization the formulation of exendin-4 loaded NPs……………………………… 19
3.2.2 Freeze drying exendin-4 loaded NPs…………………………………………………. 20
3.3 TEER measurements……………………………………………………………………….. 23
3.4 Dissolution study of enteric-coated capsules……………………………………………….. 26
3.4.1 In vitro dissolution study……………………………………………………………… 26
3.4.2 In vivo dissolution study……………………………………………………………… 28
3.5 Bioditribution study…………………………………………………………………………. 31
3.6 In vivo PK and PD study……………………………………………………………………. 33
Chapter 4. Conclusions……………………………………………………………………….. 38
References……………………………………………………………………………………… 39

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