Oral insulin delivery: the potential of thiolated chitosan-insulin tablets on non-diabetic rats

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Abstract

It was the aim of this study to develop a delivery system providing an improved efficacy of orally administered insulin utilizing a thiolated polymer. 2-Iminothiolane was covalently linked to chitosan. The resulting chitosan-TBA (chitosan-4-thiobutylamidine) conjugate exhibited 453.5±64.1 μmol thiol groups per gram polymer. 3.1% of these thiol groups were oxidised. Additionally, the enzyme inhibitors BBI (Bowman-Birk-Inhibitor) and elastatinal were covalently linked to chitosan representing 3.5±0.1% and 0.5±0.03% of the total weight of the resulting polymer conjugate, respectively. Chitosan-TBA conjugate (5 mg), insulin (2.75 mg), the permeation mediator reduced glutathione (0.75 mg) and the two inhibitor conjugates (in each case 0.75 mg) were compressed to so-called chitosan–TBA–insulin tablets. Control tablets consisted of unmodified chitosan (7.25 mg) and insulin (2.75 mg). Chitosan–TBA–insulin tablets showed a controlled release of insulin over 8 h. In vitro mucoadhesion studies showed that the mucoadhesive/cohesive properties of chitosan were at least 60-fold improved by the immobilisation of thiol groups on the polymer. After oral administration of chitosan–TBA–insulin tablets to non-diabetic conscious rats, the blood glucose level decreased significantly for 24 h corresponding to a pharmacological efficacy of 1.69±0.42% (means±S.D.; n=6) versus s.c. injection. In contrast, neither control tablets nor insulin given in solution showed a comparable effect.

According to these results the combination of chitosan-TBA, chitosan-enzyme-inhibitor conjugates and reduced glutathione seems to represent a promising strategy for the oral application of insulin.

Introduction

Since the discovery of insulin diabetics have to inject their insulin doses subcutaneously. Oral insulin would present a more convenient form of application as pain caused by the injection, stress of multiple daily injections and possible infections could be avoided leading to a higher patient compliance. Furthermore insulin would be directly transported to the liver after oral administration preventing the patient from peripheral hyperinsulinemic side effects. The oral administration of insulin, however, is encountered with many difficulties, as insulin has to confront prima facie insuperable barriers in the gut on the way from its delivery system to the blood stream. Different enzymes of the stomach and small intestine make the protein ineffective, and mucus and absorption membrane complicate the diffusion of the protein to its site of action [1]. To overcome these barriers for orally administered insulin, permeation enhancers [2] and/or enzyme inhibitors [3] can be added. However, enzyme inhibitors may cause several side effects such as systemic intoxications and/or hypertrophy and hyperplasia of the pancreas, if they are co-administered to the drug delivery system. By the covalent attachment of enzyme inhibitors to the polymer-matrix, they should remain concentrated at the delivery system and local and systemic toxic side effects should be excluded [4]. Additionally, an intimate contact of the drug delivery system to the mucosa of the gastrointestinal tract can be guaranteed by the use of mucoadhesive polymers. Such an intimate contact of the dosage form to the membrane seems advantageous as an enzymatic degradation of insulin on the way between the delivery system and the absorption membrane can be avoided. Moreover, a steep concentration gradient on the absorption membrane representing the driving force for passive drug uptake can be provided.

Polymers showing strong mucoadhesive properties are so-called thiolated polymers—polymers with thiol groups that are believed to form disulfide bonds with cysteine-rich subdomains of mucus glycoproteins [5]. Among them chitosan-TBA (chitosan-4-thiobutylamidine conjugate) [6] turned out to exhibit the comparatively strongest mucoadhesion measured with the rotating cylinder method [7]. Furthermore thiolated polymers display also permeation enhancing effects, which can be further improved in the presence of reduced glutathione [8].

The aim of this study was to develop an oral insulin delivery system by the combination of the enzyme inhibiting properties of covalently linked enzyme inhibitors and the mucoadhesive and permeation enhancing properties of a thiolated polymer. To achieve this goal insulin loaded chitosan-TBA tablets were generated comprising the enzyme inhibitors Bowman-Birk inhibitor (BBI) and elastatinal covalently linked to chitosan and the permeation mediator reduced glutathione. The formulation was tested in vitro regarding its mucoadhesive properties and drug release. Subsequently, after oral administration of the tablets to non-diabetic rats, the decrease of the blood glucose level as pharmacological response was determined.

Section snippets

Synthesis and purification of the chitosan-TBA conjugate

Initially, 500 mg of chitosan (medium molecular mass: 400 kDa; Fluka, Buchs, Switzerland) were dissolved in 50 ml of 1% acetic acid. After adjusting the pH to 6.5 with 1 M NaOH 400 mg of 2-iminothiolane HCl were added. The coupling reaction was allowed to proceed for 14 h at room temperature under continuous stirring. For purification the resulting polymer conjugate was dialysed (Sigma, St. Louis, MO, cellulose membrane dialysing tubing with molecular weight cutoff of 12 kDa) against 5 mM HCl,

Characterisation of the chitosan-TBA conjugate

TBA was attached to chitosan via an amidine bond between the carboxylic group of the reagent and a free primary amino group of the polymer. The purified chitosan-TBA conjugate exhibited 453.5±64.1 μmol thiol groups per gram polymer (mean±S.D., n=4). Thereof 3.1% were oxidised thiol groups. The obtained polymer was white, odourless and showed a fibrous structure.

Characterization of the chitosan-inhibitor conjugates

The amount of covalently bound BBI on the chitosan backbone was 3.5±0.1% (m/m; n=4), the amount of immobilised elastatinal was

Discussion

Within this study insulin tablets comprising chitosan-TBA as carrier matrix, chitosan-BBI-conjugate and chitosan-elastatinal-conjugate as enzyme inhibitors as well as reduced glutathione as permeation mediator were developed.

Chitosan is often used in order to improve the efficacy of orally given insulin because of its strong mucoadhesive properties. The blood glucose level of non-diabetic rats, for instance, was significantly decreased after oral administration of insulin loaded and chitosan

Conclusion

The chitosan–TBA–insulin formulation used in this study features excellent mucoadhesive properties, combined with the enzyme inhibiting properties of two covalently linked enzyme inhibitors and the permeation mediating properties of reduced glutathione. The blood glucose levels of non-diabetic rats were significantly decreased after oral administration of these tablets compared to unmodified polymer insulin tablets. As no other insulin tablet described in literature showed such a pronounced

Acknowledgements

This work was supported by Grant No. P15373-MOB from the Fonds zur Förderung der wissenschaftlichen Forschung (FWF) to A. Bernkop-Schnürch. The authors wish to thank Mr. Ströbel and co-workers from the slaughterhouse Totzenbach for the supply of porcine intestinal mucosa.

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