A novel permeation enhancer: N-succinyl chitosan on the intranasal absorption of isosorbide dinitrate in rats

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Abstract

The purpose of this paper is to study the potential of N-succinyl chitosan as a novel permeation enhancer for the intranasal absorption of isosorbide dinitrate (ISDN). A series of N-succinyl chitosan (NSCS) with different degree of succinylation (DS) and molecular weight were synthesized. An in situ nasal perfusion technique in rats was utilized to investigate the effect of NSCS substitution degree, NSCS molecular weight and concentration on the intranasal absorption of ISDN. The absorption enhancing effect of NSCS was compared with that of chitosan. It was found that all the NSCS investigated improved the intranasal absorption of ISDN remarkably. Better promoting effect was observed for 0.1% NSCS 50 (63) compared with 0.5% chitosan 50. In nasal ciliotoxicity test, both NSCS and chitosan investigated showed good safety profiles. Thereafter, in vivo studies of the selected formulations were carried out in rats and the pharmacokinetic parameters were calculated and compared with that of intravenous injection. Both in situ and in vivo studies demonstrated that NSCS is more effective than chitosan in promoting intranasal absorption of ISDN. Taking both absorption enhancing and safety reason into account, we suggest NSCS is a promising intranasal absorption enhancer.

Graphical abstract

Mean plasma concentration–time profiles after intranasal application of isosorbide dinitrate with 0.5% chitosan 50 and 0.1% N-succinyl chitosan 50 (63) in rats, compared with the control group, intragastric administration and intravenous injection group. Indicated values are the mean of five experiments (n = 5). This study indicated the potential of N-succinyl chitosan as a novel absorption enhancer for mucosal drug delivery.

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Introduction

Recently, intranasal administration attracted more and more attention as a viable option for local or systemic delivery of diverse therapeutic compounds (Malerba et al., 2011, Florence et al., 2011). Intranasal route allows a rapid onset of therapeutic effect, potential for direct-to-central nervous system delivery, avoidance of first-pass metabolism, and it is convenient for drug administration (Henkin, 2011). However, two barriers limit efficient nasal absorption of drugs. One is the low membrane permeability and the other is the rapid clearance of the administered formulation from the nasal cavity (Illum, 2003). So far, various intranasal absorption enhancers have been reported to increase nasal membrane permeability (Chandler et al., 1995, Duan and Mao, 2010). Anyhow, despite of their effectiveness, some of them were limited to use in nasal formulations due to their unacceptable membrane toxicity (Chandler et al., 1995, Duan and Mao, 2010). In view of reports in the literature, only limited permeation enhancers, such as hydropropel-β-cyclodextrin (HP-β-CD), chitosan, and poloxamer 188, alkylsaccharides, low methylated pectin, polyglycol mono-and diesters of 12-hydroxystearate (70%), polyethylene glycol (30%) are regarded as safe (Illum, 2012, Na et al., 2010). Therefore, it is absolutely essential to search for novel safe and effective permeation enhancers applicable for mucosal drug delivery.

Chitosan was evaluated for the enhancing effect on in vivo nasal absorption of salmon calcitonin (sCT) in rats and the results were subsequently compared with beta-cyclodextrins, one of the most commonly studied enhancers. It was shown that the inclusion of 1% CS resulted in twofold increase in the AUC(0–180) of plasma sCT relative to that of the control group. Addition of 5% DM-beta-CD led to 1.56-fold increase in absorption over the control group (Sinswat and Tengamnuay, 2003), implying that chitosan may have a better absorption enhancing effect. Chitosan is a biocompatible, biodegradable and low toxic cationic polysaccharide derived by partial deacetylation of chitin isolated from crustacean shells (Illum, 1998). Every deacetylated subunit of chitosan contains a primary amine group with a pKa value of about 6.5 (Mao et al., 2010). Thus, chitosan is soluble in acidic media with positive charge. However, it becomes insoluble at neutral and alkaline pH values, which limits its application to specified conditions. To improve the solubility of chitosan, a quaternized derivative, N-trimethyl chitosan (TMC) was synthesized, which is freely soluble over a wider pH range and showed absorption-enhancing effects even in neutral and basic pH environments (Van der Merwe et al., 2004). Unfortunately, TMC was shown to be cytotoxic in L929 mouse fibroblast cells as indicated by MTT assay (Mao et al., 2005). Similarly, it was reported that reversibility of transepithelial resistance at 0.5% concentrations of TMC with different degree of quaternization could not be demonstrated at pH 6.2 and 7.4 in Caco-2 cells (Kotze′ et al., 1999) and the cytotoxicity of TMC is molecular weight and quaternization dependent (Jintapattanakit et al., 2008), although some other studies indicated TMC was safe (Thanou et al., 1999, du Plessis et al., 2010). Therefore, it is desirable to search for other possibilities to increase the solubility of chitosan, and in the meantime with good safety profile and permeation enhancing effect.

N-succinyl-chitosan (NSCS), which can be obtained from a simple reaction between chitosan and succinic anhydride, has been found to exhibit several biological properties, such as good solubility at various pH, nontoxicity, biocompatibility and long systemic circulation in mice, and the maximum tolerable dose for the intraperitoneal injection of Suc-Chi to mice was greater than 2 g/kg (Kato et al., 2004). For this reason, NSCS has been applied as drug delivery carriers such as microspheres and hydrogel beads (Kato et al., 2004, Ubaidulla et al., 2007, Dai et al., 2008) and as gene delivery carrier for improved chitosan solubility and gene transfection (Toh et al., 2011). Therefore, we assume NSCS might be a potential mucosal permeation enhancer with acceptable safety. This hypothesis was tested in this study.

Isosorbide dinitrate (ISDN) is commonly used for therapy of stable angina pectoris and traditionally administrated via oral or sublingual routes. However, loss of consciousness appears in patients when angina pectoris breaks out, and thus it is difficult for patients to take medicine by themselves. Additionally, ISDN administrated orally has low bioavailability due to its high first-pass metabolism in the gastrointestinal tract and liver (Zhao et al., 2007). Moreover, the critical point of antianginal therapy depends, to a certain extent, on the ability of the drug to produce an immediate effect. Thus, intranasal (i.n.) delivery may be an appropriate administration route for ISDN.

Therefore, in the present work, first of all, NSCS with different degree of substitution (DS) and molecular weight were synthesized. Thereafter, taking ISDN as a model drug, the feasibility of NSCS as an intranasal absorption enhancer was studied and influence of succinylation degree of NSCS, NSCS molecular weight and concentration on the absorption permeation effect was investigated using in situ test. Safety properties of different NSCSs were further studied and compared with chitosan of the same molecular weight by using in situ toad palate model. Moreover, based on the in situ results, the selected intranasal formulations were evaluated in vivo by measuring drug blood concentration after intranasal administration in rats. To the best of our knowledge, this is the first time that NSCS was used as a permeation enhancer for mucosal drug delivery.

Section snippets

Materials

ISDN was purchased from Shandong Keyuan Inc. Paracetamol (APAP, 99.6% purity, internal standard) was obtained from Shandong Xinhua Pharmaceutical Company, Ltd. (Jinan, China). The standard of isosorbide minitrate (ISMN) was purchased from National Institute for the Control of Pharmaceutical and Biological Products. Chitosan 400 kDa (CS 400) with a degree of deacetylation of 85% was purchased from Weifang Kehai Chitin Co., Ltd. Chitosan 20 kDa (CS 20), chitosan 50 kDa (CS 50) and chitosan 100 kDa

Synthesis and characterization of NSCS

N-Succinyl-chitosans (NSCSs) of different succinylation degree and molecular weight were synthesized by the introduction of succinyl groups into chitosan at the N-position of the glucosamine units. The degree of succinylation could be easily modified by changing reaction time. Furthermore, the molecular weight of NSCS was easily controlled by using chitosans of different molecular weight as the raw material. Significant difference in DS was found between 2 h and 6 h samples (P < 0.1), but the

Conclusion

In this paper, NSCSs with different DS and molecular weight were successfully prepared. The absorption enhancing effect of NSCS increased with the increase of DS in the range studied. No statistical difference in absorption was found between NSCS 50 and NSCS 100 at comparable DS, both of them are much higher than that of NSCS 20. 0.1% NSCS is sufficient to achieve good permeation enhancing effect. Ciliotoxicity study indicated that NSCS was even safer than chitosan. The high effectiveness of

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