Enhancing effect of chitosan on nasal absorption of salmon calcitonin in rats: comparison with hydroxypropyl- and dimethyl-β-cyclodextrins

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Abstract

Two types of chitosan, i.e. the free amine (CS J) and the glutamate salt (CS G), were evaluated for their enhancing effect on in vivo nasal absorption of salmon calcitonin (sCT) in rats. The results were subsequently compared with β-cyclodextrins, one of the most commonly studied enhancers. Solutions containing sCT and chitosan (0–1.25% w/v) in isotonic phosphate buffers (IPB; pH 3.0–6.0) were nasally administered at the dose of 10 IU/kg. The plasma calcium lowering effect in each sCT-treated rat was determined by calculating the total percent decrease in plasma calcium (%D). CS J showed an increase in %D as the solution pH was decreased in accordance with the increased ionization and hydration of the free amine chitosan at the more acidic pH. However, CS G showed an increase in %D with increasing pH, with maximum hypocalcemic effect observed at pH 6.0. At their optimal pH (4.0 for CS J and 6.0 for CS G), the absorption enhancing effect of both chitosans was concentration dependent from 0.25 to 1.0% and leveled off at 1.25%. Using specific RIA, the absolute bioavailability of sCT after comparison with i.v. administration was determined to be 2.45, 1.91, and 1.22% for 1% CS J, 5% dimethyl-β-cyclodextrin (DM-β-CD) and control group (intranasal (in) sCT alone), respectively. Although the absolute nasal bioavailability seemed to be low when compared to the i.v. administration, the inclusion of 1% CS J resulted in two-fold increase in the AUC0–180 of plasma sCT relative to that of the control group. Addition of 5% DM-β-CD also led to 1.56-fold increase in absorption over the control group. All the enhancers showed significant absorption enhancement (P<0.05) with the highest effect observed with CS J. In conclusion, cationic polymer chitosan may have promising potential as a safe and effective nasal absorption enhancer of sCT.

Introduction

Salmon calcitonin (sCT) is an endogenous polypeptide hormone consisting of 32 amino acids which plays a vital role in both calcium homeostasis and bone remodeling. Nasal administration of sCT has been used therapeutically for the treatment of hypercalcemia, Paget’s disease, and osteoporosis (Stevenson and Evans, 1981, Jacobs, 1985). However, the relative bioavailability of sCT was reported to be poor, only 1.6% that of intramuscular preparation (Kagatani et al., 1996). To enhance the absorption efficacy of sCT, various enhancers have been investigated including bile salts, lauroylcarnitine chloride, and sodium tauro-24,25-dihydrofusidate (Kagatani et al., 1991, Kagatani et al., 1996, Lee et al., 1994). However, the use of these enhancers was often associated with some types of nasal membrane damages. For example, bile salts can induce significant release of several membrane components following nasal exposure in rats (Shao and Mitra, 1992). Other compounds reported to enhance the nasal absorption of peptides include laureth-9, lysophosphatidylcholine, and dimethyl-β-cyclodextrin (DM-β-CD; Gill et al., 1994, Shao et al., 1992, Merkus et al., 1991). However, all of these enhancers have been reported their membrane-irritating effect according to histology, ciliotoxicity, and membrane component release studies (Merkus et al., 1993, Marttin et al., 1995).

Chitosan is a cationic polysaccharide obtained from deacetylation of chitin, a structural polymer abundant in cretaceous animals like crabs and shrimps. Due to its biocompatibilty, biodegradability, and low toxicity, chitosan represents an attractive biopolymer for a variety of pharmaceutical applications (Illum, 1998, Paul and Sharma, 2000, Takahashi et al., 1990, Meshali and Gabr, 1993). Chitosans are potent absorption enhancers for poorly absorbed hydrophilic drugs. They improve, for example, the uptake of polypeptides such as atenolol, insulin, and buserelin across nasal and intestinal epithelia (Schipper et al., 1997, Illum et al., 1994, Luessen et al., 1996). The mechanism of action was suggested to be a combination of mucoadhesion (Henriksen et al., 1996, Witschi and Mrsny, 1999) an effect on the gating properties of the tight junction (Artursson et al., 1994, Illum et al., 1994).

In our previous work, we compared the absorption enhancing activity and safety of different types of chitosan with that of DM-β-CD and hydroxypropyl-β-cyclodextrin (HP-β-CD) using in situ rat nasal perfusion techniques (Tengamnuay et al., 2000). We found that both the free amine and soluble salt forms of chitosans were effective nasal absorption enhancers of l-Tyr-d-Arg, a model opioid dipeptide. The more soluble salt form appeared to be less dependent on pH whereas the enhancing activity of the free amine chitosan increased as the pH was lowered from 6 to 4. We also found that both chitosans had a relatively mild effect on the rat nasal membrane. The extent of total protein, phosphorus, and lactate dehydrogenase (LDH) release after nasal perfusion with chitosans (0.1 and 0.5%) was low and much less than that induced by 1.25 and 5% DM-β-CD, an effective enhancer reported to have marked membrane-irritating effects (Muangkum and Tengamnuay, 1999, Krishnamoorthy et al., 1995, Marttin et al., 1995, Yoshida et al., 1988). Despite the promising potential of chitosan as a safe and effective nasal absorption enhancer, studies to confirm its in vivo efficacy are limited. Also, its in vivo enhancing activity relative to other commonly studied enhancers has not been extensively investigated.

Thus, the primary purpose of this study was to evaluate the in vivo efficacy of chitosan as a nasal absorption enhancer of sCT in rat. Changes in plasma calcium (hypocalcemic effect) and sCT levels were used as indicators of sCT absorption. The effects of chitosan type, pH, and concentration were investigated and results were subsequently compared with that of DM-β-CD and HP-β-CD.

Section snippets

Chemicals

Chitosan free amine (CS J) with viscosity average molecular weight (MWv) of 1860 kDa and 80% deacetylation was purchased from Kyowa Technos Co. (Japan). Chitosan glutamate (CS G) (Seacure G 210+, MWv 800 kDa and >70% deacetylated) was donated by Pronova Biopolymer (Norway). The actual molecular weight of the chitosan content in CS G approximately 480 kDa taken into account of the 35–45% presence of glutamic acid in the CS G. Salmon calcitonin, synthetic (sCT) was purchased from Sigma Chemicals Co.

Effect of pH of chitosan on in vivo nasal absorption enhancing efficacy

The enhancing activity of both chitosans appear to depend on pH. The enhancing effect of CS J was observed to increase as the pH is decreased (Fig. 2a). This could be due to the ability of the free amine form which require an acidic condition for ionization and hydration. Chitosan is a basic polymer with an intrinsic pKa value of about 6.5 (Schipper et al., 1996). As the pH is lowered below its pKa, the fraction of the ionized groups in the chitosan molecules increases. However, too acidic pH

Conclusion

The in vivo absorption data indicated the potential of CS J and CS G as effective nasal absorption enhancers of peptides like sCT. Their enhancing effects were equivalent to β-cyclodextrins, particularly 1% CS J which exhibited strong activity comparable to 5% DM-β-CD under their corresponding optimum pH. However, our previous study found that DM-β-CD was more irritating to the rat nasal mucosa than CS J and CS G (Tengamnuay et al., 2000). Perfusion of the rat nasal cavity with 1.25% DM-β-CD

Acknowledgements

This project was supported by a grant from the Rachadapisek Research Fund of Chulalongkorn University, Bangkok, Thailand.

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