Chitosans as nasal absorption enhancers of peptides: comparison between free amine chitosans and soluble salts

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Abstract

A total of three free amine chitosans (CS J, CS L and CS H) and two soluble chitosan salts (CS G and CS HCl) were evaluated for their efficacy and safety as nasal absorption enhancers of peptides based on in situ nasal perfusion and subacute histological evaluation in rat. At 0.5% w/v, all chitosans were effective in enhancing the nasal absorption of [d-Arg2]-Kyotorphin, an enzymatically stable opioid dipeptide. The enhancing effect of the free amine chitosans increased as the pH was decreased from 6.0 to 4.0 (P<0.05). However, the pH effect was not significant for the two chitosan salts (P>0.05), suggesting that their adjuvant activity may be less pH-dependent than the free amine form. CS J and CS G were subsequently selected for further studies. At only 0.02% w/v, their enhancing effect was already significant and comparable to that of 5% w/v hydroxypropyl-β-cyclodextrin (HP-β-CD). Both chitosans at 0.1% caused minimal release of total protein and phosphorus from the rat nasal mucosa, with the values similar to that of 5% HP-β-CD. At 0.5% the two chitosans also stimulated smaller release of lactate dehydrogenase, an intracellular enzyme used as marker of nasal membrane damage, than 1.25% dimethyl-β-cyclodextrin. Morphological evaluation of the rat nasal mucosa following 2-week daily administration indicated that the two chitosans (1.0%) produced only mild to moderate irritation. In conclusion, both the free amine and the acid salt forms of chitosans are effective in enhancing the nasal absorption of [d-Arg2]-Kyotorphin and have potential for further studies as a safe and effective nasal absorption enhancer of peptide drugs.

Introduction

Chitosan is a polymer obtained from deacetylation of chitin, a naturally-occurring structural polymer abundant in crab and shrimp shells. It is a cationic polysaccharide with linear chain consisting of β-(1,4)-linked 2-acetamido-2-deoxy-β-d-glucopyranose (GlcNAc) and 2-amino-2-deoxy-β-d-glucopyranose (GlcN) (Mathur and Narang, 1990). The greater the extent of deacetylation, the smaller is the proportion of GlcNAc in the polymer chain.

Recently, chitosan has been shown to enhance nasal and intestinal absorption of hydrophilic drugs like peptide hormones in both the in vitro and in vivo models (Artursson et al., 1994; Illum et al., 1994; Lueβen et al., 1996, Lueβen et al., 1997). According to the study using an in vitro Caco-2 cell model, its absorption enhancing mechanisms were reported to be a combination of mucoadhesion and an effect on the opening of the tight junctions (Artursson et al., 1994). Schipper et al. (1996), using the same in vitro model, reported that the structural properties of chitosans such as degree of acetylation and molecular weight, are very important for its absorption enhancement of hydrophilic drugs. They found that a low degree of acetylation (i.e. high percent deacetylation with greater charge density) and/or a high molecular weight appear to be necessary for chitosans to increase the epithelial permeability. Toxicity of chitosan also depends on its high charge density but appears to be less affected by the molecular weight.

Although this Caco-2 cell model has yielded a good deal of relevant information, the experimental conditions were far from the actual environment of the nasal cavity. In vivo absorption studies in animals have been carried out to evaluate the nasal absorption promoting activity of chitosan using insulin as a model peptide (Illum et al., 1994; Aspden et al., 1996). However, no correlation was found between the molecular weight nor the degree of acetylation of chitosan and its enhancing effect on the in vivo nasal absorption of insulin in rats (Aspden et al., 1996). Therefore, it appears that the results could vary among different experimental models and that more information is needed to further characterize the safety and efficacy of chitosan as a nasal absorption enhancer of peptides.

In addition, most of the studies utilized the salt forms of chitosan as the absorption enhancer, either hydrochloride or glutamate salt. Thus, it would be interesting to study the absorption enhancing effect of some free amine chitosans and compare the results with the soluble salt forms. The in situ nasal perfusion technique was utilized in this study due to its simple experimental set-up and its ability to characterize both the efficacy and safety of several nasal absorption enhancers (Tengamnuay and Mitra, 1990; Shao and Mitra, 1992; Shao et al., 1992). Furthermore, few longer-term histological studies of the effect of chitosans on the nasal mucosa are available. For example, Aspden et al. (1997) studied the histology of human inferior turbinates following daily nasal administration of 0.25% w/v chitosan glutamate to healthy volunteers for 7 days. A study with a longer exposure period and using higher concentration of chitosans would further characterize their safety profiles under a more rigorous condition.

Therefore, the objectives of this study were: (1) to evaluate the efficacy of chitosans as nasal absorption enhancers of peptides using the in situ rat nasal perfusion technique; (2) to study the effects of physicochemical factors such as chemical form (free amine versus salt form), pH, and chitosan concentration on their nasal absorption promoting activity; and (3) to study the possible deleterious effects of chitosans based on the release of various mucosal components and morphological evaluation of the nasal mucosa following 2-week daily nasal administration of chitosan solutions to intact rats.

Section snippets

Chemicals

A total of three free amine chitosans (CS J, CS H and CS L) and two salt forms (CS G and CS HCl) were used. CS J was obtained from Kyowa Technos, Japan, whereas CS H and CS L were donated by Unicord Public, Bangkok, Thailand. Chitosan glutamate (CS G, Seacure© G 210+) and chitosan hydrochloride (CS HCl, Seacure© Cl 210) were gifts from Pronova Biopolymer, Drammen, Norway. The viscosity average molecular weight (Mv) of chitosans was determined from intrinsic viscosity ([η]) measurements using

Effect of chitosan type (free amine versus salt forms), pH and concentration on their nasal absorption enhancing efficacy

In situ perfusion experiments revealed that all chitosans studied were capable of enhancing the nasal absorption of [d-Arg2]-Kyotorphin as compared to controls (P<0.05). The absorption pattern, as seen from the loss of the dipeptide from the perfusate, appeared to follow first order process (r2=0.90–0.99), indicating that the substance was transported across the nasal mucosa by passive diffusion. Fig. 1 shows semilogarithmic plots of percent [d-Arg2]-Kyotorphin remaining in the perfusate versus

Conclusion

According to the in situ rat nasal perfusion, all the chitosans tested are effective in enhancing the nasal absorption of a model dipeptide [d-Arg2]-Kyotorphin. The enhancing effect of the free amine chitosans (CS J, CS H, CS L) is pH-dependent and increases with decreasing pH whereas the salt forms tested (CS G and CS HCl) appear to be less pH-dependent. Based on % dipeptide remaining at 120 min, the ranking of the enhancing effect of five chitosans (all at 0.5% under their respective optimum

Acknowledgements

This project was supported by a grant from the Rachadapisek Research Fund of Chulalongkorn University, Bangkok, Thailand. The authors wish to thank the technical assistance of Prapasri Sinswat and Arisara Muangkum for determination of the average molecular weight and LDH studies of chitosans.

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