Bioavailability enhancement of glucosamine hydrochloride by chitosan

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Abstract

Glucosamine, as a dietary supplement for management of osteoarthritis, has a low and erratic oral bioavailability due to its transport-mediated absorption and presystemic loss in liver and GI tract. The present study described an effective approach to improve glucosamine intestinal absorption and hence its bioavailability using chitosan. Effects of chitosan on intestinal permeability and pharmacokinetics of glucosamine were evaluated in Caco-2 cell monolayer and rats, respectively. In addition, randomized crossover pharmacokinetic studies in beagle dogs were performed to evaluate the oral bioavailabilities of the developed glucosamine oral formulations containing chitosan (QD-Glu solution and QD-Glu tablet) in comparison to its commercial products. Caco-2 permeability studies demonstrated that chitosan could enhance the absorptive transport of glucosamine by 1.9–4.0-fold via the reversible opening of the cell tight junction. After oral administration of glucosamine solutions containing chitosan in rats, it was found that 0.5% (w/v) chitosan exhibited the highest enhancement in Cmax (2.8-fold) and AUC0−∞ (2.5-fold) of glucosamine. Further pharmacokinetic studies in beagle dogs demonstrated that QD-Glu solution and QD-Glu tablet showed much higher relative bioavailabilities of 313% and 186%, when comparing with Wellesse™ solution and Voltaflex™ tablet, respectively. In conclusion, chitosan could serve as a promising oral absorption enhancer for glucosamine.

Introduction

Osteoarthritis is a degenerative disease leading to chronic joint pain and limitation of movement. Typical features of osteoarthritis are the degeneration or progressive loss of the structure and functionality of articular cartilage (Lorenz and Richter, 2006). Cartilage, a flexible connective tissue, is composed of chondroblasts that produce a large amount of extracellular matrix composed of glycosaminoglycans and proteoglycans (Murray and Keeley, 2009). Glucosamine, also known as 2-amino-2-deoxyglucose (CAS 3416-24-8) (Fig. 1), is an amino sugar biosynthesized endogenously in animals and human. Glucosamine functions as a precursor for glycosaminoglycans and is structurally incorporated in mucopolysaccharides, glycoproteins and proteoglycans. As a food supplement, glucosamine may benefit the generation of cartilage structure, and has been applied to facilitate the alleviation of arthritis (Matheson and Perry, 2003, Rovati et al., 2012, Towheed et al., 2005). Although glucosamine has not been approved by US FDA as a drug, it is regarded as a prescription drug for osteoarthritis in Europe (Russell et al., 2002). However, the oral bioavailability of glucosamine is relatively poor in the vertebrate (6.1–26% in human (Meulyzer et al., 2008, Setnikar et al., 1993), ∼19% in rats (Aghazadeh-Habashi et al., 2002) and ∼12% in dogs (Adebowale et al., 2002)), which may be due to its transport-facilitated limited absorption and presystemic loss in gut and liver (Ibrahim et al., 2012, Setnikar et al., 1993). As a dietary supplement, glucosamine is commercially available in hydrochloride and sulphate salt forms with high water solubility, which demonstrate bioequivalence in vivo (Aghazadeh-Habashi and Jamali, 2011) and linear pharmacokinetics in the dose range of 750–1500 mg in human (Persiani et al., 2005). However, following a conventional dose (1500 mg/day), the plasma concentration achieved (2.7–17.4 μM) is far below the required level for response (Ibrahim et al., 2012). The low and variable bioavailability of glucosamine could have led to inconsistency of clinical trial outcomes, which further casts doubt on its use for clinical treatment of osteoarthritis (Aghazadeh-Habashi and Jamali, 2011, Towheed and Anastassiades, 2007). Therefore, improvement in the oral bioavailability of glucosamine is highly desirable.

Glucosamine is a small hydrophilic molecule like glucose, which cannot easily cross the biological membrane via transcellular pathway. It has reported to be a substrate of glucose facilitative transporters (especially GLUT2) (Uldry et al., 2002), which could be inhibited by flavonoid quercetin (Ibrahim et al., 2012) and the transport process can be saturable (Klip and Paquet, 1990, Persiani et al., 2005). Paracellular transport can also play an important role for the transport of small hydrophilic drugs (especially at the upper small intestine with leaky barriers) (Lafforgue et al., 2008, Sugano et al., 2010). Increasing the paracellular transport of glucosamine is expected to lead to an enhancement in its absorption.

The primary aim of our study was to find an approach to improve the oral bioavailability of glucosamine. Although several classes of compounds, including surfactants, fatty acids, cyclodextrins, cellulose ethers, chelators, and positive charged polymers (Junginger, 2007), are known to be able to enhance absorption of low permeable hydrophilic drugs by acting on the mucous layer, the membrane components or the tight junctions of the intestinal epithelium, it is uncertain whether any of them is suitable for glucosamine absorption enhancement or not. In fact, during our previous formulation development of glucosamine, it was found that: (1) addition of cellulose ethers (including CMC-Na and HPMC) even lead to a lower absorption of glucosamine in rats (data not shown); (2) surfactants like SLS (high toxicity to the intestinal membrane (Uchiyama et al., 1999)) and Tween 80 (odoriferous smell and bitter taste) have safety or taste concerns. In our preliminary study in rats, addition of 1% chitosan (positive charged polymer) exhibited a significant enhancement of glucosamine oral bioavailability, which prompted us to investigate further on the mechanism of such absorption enhancement as well as to optimize this chitosan containing formulation.

Chitosan is a natural and cationic polysaccharide obtained by deacetylation of chitin, a polymer which is abundant in the outer skeleton of crustaceous shells such as crabs and shrimps. Chitosan has been approved as food supplement in many countries and is commonly regarded as non-toxic, biological compatible and biodegradable substance (Kean and Thanou, 2010). With a pKa value of ∼6.5, chitosan exhibits a pH-dependent solubility with high solubility at low pH but insoluble at higher pH ranges. It is soluble in dilute HCl, HNO3, and HClO4. However, chitosan is sparing soluble in H2SO4 at room temperature and incompatible with drugs in sulphate form (Tu et al., 2010). An important property is its ability to reversibly interact with components at the epithelial tight junction when chitosan is protonated in its uncoiled configuration at pH below 6.5, leading to widening of the paracellular route temporarily and increasing the paracellular permeability of drugs across mucosal epithelia (Thanou et al., 2001). A number of studies have confirmed the improvement of permeability of poorly absorbed hydrophilic compounds such as atenolol (Schipper et al., 1999), mannitol (Artursson et al., 1994), dextran (Vllasaliu et al., 2012), insulin (Fernandez-Urrusuno et al., 1999) and salmon calcitonin (Sinswat and Tengamnuay, 2003) across the intestinal or nasal epithelia.

In the present study, we first test its ability by a well-established in vitro model, Caco-2 cell model (Artursson et al., 2001, Fong et al., 2012, Zhang et al., 2006, Zhang et al., 2007), followed by animal studies (rats and beagle dogs) to verify the absorption enhancement.

Section snippets

Materials

Considering the compatibility with chitosan, glucosamine hydrochloride has been chosen for the current study. d-(+)-glucosamine hydrochloride (referred as “glucosamine” in this paper) was purchased from Qingdao Highsun Biochemical Products Co., Ltd., China. Chitosan hydrochloride (referred as “chitosan”) with average molecular weight of ∼200 kDa and deacetylation degree of 83% was purchased from Zhejiang Golden-Shell Biomedical Co., Ltd., China. 9-Fluorenylmethyl chloroformate (FMOC-Cl), sodium

Cytotoxicity of glucosamine and chitosan to Caco-2 cells

The cytotoxicity profile of glucosamine and chitosan on Caco-2 cells was shown in Fig. 2. For glucosamine (Fig. 2A), the results showed that the relative viability of Caco-2 cells maintained to be greater than 80% after 4 h incubation with glucosamine concentrations up to 3 mg/ml in PBS+ 6.0 and 1 mg/ml in PBS+ 7.4.

For chitosan (Fig. 2B), the cell viabilities did not remarkably change over 1–100 μg/ml in PBS+ 7.0 and 1–50 μg/ml in PBS+ 7.4, whereas a concentration-dependent cytotoxicity behaviors

Discussion

The present in vitro and in vivo studies showed a significant enhancement of oral absorption of glucosamine by chitosan. Our results suggest that a glucosamine formulation with chitosan could potentially provide enhanced therapeutic effect at conventional dose. Further clinical studies, however, are needed to confirm such results.

In general, chitosans with high degree of deacetylation (DDA > 80%) and high molecular weight were preferred for absorption enhancement of hydrophilic compounds due to

Conclusion

Our study showed that chitosan could significantly enhance the intestinal permeability of glucosamine by reversibly opening the tight junction of intestinal epithelium cells. Further in vivo studies in rats and beagle dogs demonstrated that the presence of chitosan could increase the plasma concentration and bioavailability of glucosamine without altering its elimination. Chitosan can serve as an effective absorption enhancer for glucosamine.

Acknowledgments

Financial support from General Research Fund CUHK 480809 and Comprehensive Drug Enterprises Limited; Technical support from Dr. Zhao, Lizi, Mr. Zeng, Guixiong in Sun Yat-sen University.

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