Nasal insulin delivery in the chitosan solution: in vitro and in vivo studies

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Abstract

The effects of chitosan concentrations, osmolarity, medium and absorption enhancers in the chitosan solution on nasal insulin delivery were studied in vitro and in vivo. The penetration of insulin through the mucosa of rabbit nasal septum was investigated by measuring the transmucosal flux in vitro, while the nasal absorption of insulin in vivo was assessed by the efficiency in lowering the blood glucose levels in normal rats. It was demonstrated that increasing concentrations of chitosan up to 1.5% (w/v) caused an increase in the permeability of insulin across the nasal mucosa. Insulin given intranasally in hypo- or hyperosmotic formulation showed a higher hypoglycemic effect than insulin delivered in isoosmotic formulation. Insulin formulation in chitosan solution prepared with deionized water brought to a higher relative pharmacological bioavailability (Fr) value than that prepared with 50 mM pH 7.4 phosphate buffer. A formulation containing both 1% chitosan and 0.1% ethylenediaminetetraacetic acid (EDTA), 5% polysorbate 80 (Tween 80) or 1.2% β-cyclodextrin (β-CD) did not lead to a higher Fr than insulin formulated with 1% chitosan alone. The formulation containing both 5% hydroxypropyl-β-cyclodextrin (HP-β-CD) and 1% chitosan was more effective at reducing blood glucose levels than the formulation containing 5% HP-β-CD or 1% chitosan alone. The studies indicated that chitosan concentrations, osmolarity, medium and absorption enhancers in chitosan solution have significant effect on the insulin nasal delivery. The results of in vitro experiments were in good agreement with that of in vivo studies.

Introduction

Chitosan [2-amino-2-deoxy-(1 → 4)-β-d-glucopyranan] is a mucopolysaccharide obtained by the deacetylation of chitin in crustaceans such as crabs and shrimps. Chitosan is soluble in organic acid (acetic acid) or inorganic acid (hydrochloric acid) and positively charged. The chemical properties of the polymer are determined by the degree of deacetylation, molecular weight and viscosity. Studies (Paul and Garside, 2000) showed that chitosan is non-toxic and its LD50 in mice exceeds 16 g/kg. Because of its biodegradability and biocompatibility, chitosan has been applied as a pharmaceutical excipient in oral, ocular, nasal, implant and transdermal drug delivery (Dodane and Vilivalam, 1998, Illum, 1998). Chitosan has been shown to have mucoadhesive properties because of its viscosity and interaction of the positively charged amino group with the negatively charged sites on the mucosa surface (Artursson et al., 1994, Luessen et al., 1996).

Recent studies indicated that chitosan could enhance absorption of poorly absorbable drugs such as peptides and proteins (Luessen et al., 1996, Illum et al., 2000). The nasal delivery of chitosan was demonstrated to greatly enhance the absorption of insulin across the nasal mucosa of rats and sheep (Illum et al., 1994). In vivo evaluation in rabbits has proved that chitosan nanoparticles were able to improve the nasal absorption to a great extent than chitosan solution probably due to intensified contact of the nanoparticle with the nasal mucosa as compared to chitosan solutions (Fernandez-Urrusuno et al., 1999a, Fernandez-Urrusuno et al., 1999b). Later studies showed that chitosan nanoparticles were not as efficient as chitosan solution nor chitosan powder in terms of their nasal absorption promoting ability in rats and sheep (Dyer et al., 2002). Investigations have suggested that there are two effects of chitosan delivery systems on nasal mucosa. The mucoadhesive properties of the polymer can reduce the clearance rate of drugs from nasal cavity, thereby prolonging the contact time of chitosan delivery system with nasal epithelium. In addition, it has been shown that the interaction of the positively charged amino group of chitosan with the negatively charged sialic acid residues in mucus causes the transient opening the tight junctions and allows large hydrophilic compounds to be transported across the epithelium. The opening mechanism of the tight junctions has been demonstrated by a decrease in ZO-I proteins and the change in the cytoskeletal protein F-actin from a filamentous to a globular structure (Artursson et al., 1994, Schipper et al., 1997.

Most of studies utilized chitosan alone as absorption enhancer. Currently, it is not known if the combination of chitosan and other absorption enhancers, as well as some other factors could exhibits a synergistic effect in the nasal absorption of insulin. It is thought that EDTA affects the tight junctions interconnecting membrane cells by the removal of calcium and consequently increases paracellular transport (Cassidy and Tidball, 1967), and chitosan could also open the tight junctions. So it is interesting to study whether a nasal formulation containing both of these kinds of absorption enhancers could exhibit an additive or synergistic increase of the insulin absorption. Tweens with the ethylene oxide and a long hydrocarbon chain have been used to enhance the absorption of drugs in transdermal delivery systems (Breuer, 1979, Walters et al., 1987), and this kind of nonionic surfactants may penetrate into the intercellular matrix, increase the fluidity, and extract lipid components from biomembrane (Breuer, 1979, Walters et al., 1987). Cyclodextrins (CDs) could also extract the phospholipids and proteins from membrane (Shao et al., 1992). When chitosan interacts with the epithelial membrane, the tight junctions are opened, then Tweens or CDs could penetrate into the opened gaps between cells and extract the phospholipids in biomembrane. Thus, the tight junction proteins such as occludin (Furuse et al., 1993), claudin-1 and -2 (Furuse et al., 1998) are naked and may collapse after the removal of surrounding phospholipids, resulting in these fusion points untied. So the opening of the tight junctions may be strengthened by co-administration of chitosan and Tweens or CDs.

The purpose of this paper was to evaluate the effects of chitosan concentrations, osmolarity, medium and some absorption enhancers in chitosan solution on the insulin permeation across the rabbit nasal mucosa in vitro and the serum glucose concentrations after nasal administration of insulin to normal rats. Moreover, the correlation between the in vitro and in vivo studies was also investigated.

Section snippets

Materials

Crystalline porcine zinc insulin (27.5 IU/mg) was purchased from Xuzhou Biochemical Company (People’s Republic of China), and 125I-insulin was obtained from the China Institute of Atomic Energy. EDTA, Tween 80, β-CD and HP-β-CD were supplied by Sigma (St. Louis, MO, USA). Chitosan (non-salt form) was purchased from Qingdao Haihui Company (People’s Republic of China). The molecular weight of chitosan is about 100,000 Da and the deacetylation degree is 85%. The blood glucose assay kit was a product

Results

The effects of chitosan concentrations, osmolarity of the solution, medium as well as some absorption enhancers on the insulin permeation across the rabbit nasal mucosa in vitro are shown in Fig. 1, Fig. 2, respectively. The permeation coefficients of insulin across the rabbit nasal epithelium in vitro under different conditions are listed in Table 2.

When the concentrations of the chitosan increased from 0 to 1.5%, the amount of insulin across the mucosa increased consequently. There were

Discussion

The influence of chitosan concentrations on the effect of insulin both in vitro and in vivo in our studies indicated that chitosan concentration is one of the impacting factors influencing the enhancement of drugs to pass through the membrane, probably due to the mucoadhesive properties and high viscosity produced by the chitosan solutions, which make the drugs stay in the nasal cavity for a long time and be cleared slowly by mucocilia from nasal mucosa. But in this rat model, the mucociliary

Conclusions

The results in this study indicated that the chitosan concentrations, osmolarity, medium and absorption enhancers in chitosan solution have significant effect on nasal insulin delivery. The maximum hypoglycemic effect was achieved when insulin was administered in a formulation containing both HP-β-CD and chitosan.

Acknowledgements

The authors are very grateful to Mr. W.B. Hu and Miss H.M. Yang for their assistance in the animal experiments.

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