International Journal of Pharmaceutics 466 (2014) 156–162
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Absorption enhancing effects of chitosan oligomers on the intestinal absorption of low molecular weight heparin in rats Hailong Zhang a , Jie Mi a , Yayu Huo a , Xiaoyan Huang a , Jianfeng Xing a , Akira Yamamoto b , Yang Gao a, * a b
School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi'an 710061, China Department of Pharmaceutics, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 December 2013 Received in revised form 13 February 2014 Accepted 2 March 2014 Available online 11 March 2014
Absorption enhancing effects of chitosan oligomers with different type and varying concentration on the intestinal absorption of low molecular weight heparin (LMWH) were examined by an in situ closed loop method in different intestinal sections of rats. Chitosan hexamer with the optimal concentration of 0.5% (w/v) showed the highest absorption enhancing ability both in the small intestine and large intestine. The membrane toxicities of chitosan oligomers were evaluated by morphological observation and determining the biological markers including amount of protein and activity of lactate dehydrogenase (LDH) released from intestinal epithelium cells. There was no obvious change both in levels of protein and LDH and morphology in the intestinal membrane between control and various chitosan oligomers groups, suggesting that chitosan oligomers did not induce any significant membrane damage to the intestinal epithelium. In addition, zeta potentials became less negative and amount of free LMWH gradually decreased when various chitosan oligomers were added to LMWH solution, revealing that electrostatic interaction between positively charged chitosan oligomers and negative LMWH was included in the absorption enhancing mechanism of chitosan oligomers. In conclusion, chitosan oligomers, especially chitosan hexamer, are safe and efficient absorption enhancers and can be used promisingly to improve oral absorption of LMWH. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Absorption enhancer Intestinal absorption Chitosan oligomers Low molecular weight heparin (LMWH) Membrane toxicity Electrostatic interaction
1. Introduction Low molecular weight heparin (LMWH), an anionic polysaccharide, is preferred clinically as an improved anticoagulant compared to un-fractionated heparin for the prevention and treatment of deep vein thrombosis (DVT) and pulmonary embolism through catalyzing the inactivation of factors Xa and IIa by binding to anti-thrombin, which ultimately leads to the inhibition of the clotting cascade (Hirsh et al., 1998; Paliwal et al., 2011a; Weltermann et al., 2003). However, it has been recognized that the clinic application of LMWH is limited because of the poor oral bioavailability and consequently the requirement for daily subcutaneous injection although it provides a predictable anticoagulant response. Drug delivery via oral route remains the most preferred administration mode due to several advantages including convenient application, patient compliance and low cost. However, the
* Corresponding author at: Xi'an Jiaotong University, No. 76 Yanta West Road, Xi'an, Shaanxi 710061, China. Tel.: +86 29 82655139; fax: +86 29 82655139. E-mail addresses: [email protected], [email protected] (Y. Gao). http://dx.doi.org/10.1016/j.ijpharm.2014.03.010 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
extent of oral absorption of LMWH was sometimes not enough for its therapeutic effect owing to the characteristics of LMWH such as hydrophilicity, anionic surface charge and the large molecular weight, which led to the poor membrane permeability (Jaques, 1980; Norris et al., 1998; Ross and Toth, 2005). Therefore, various strategies had been investigated to improve the oral absorption of LMWH including preparing of microparticle (Lanke et al., 2009; Meissner et al., 2007; Paliwal et al., 2011a), nanoparticle (Hoffart et al., 2006; Leung, 2012) and complexation (Lee et al., 2001; Yang et al., 2006; Grabovac and Bernkop-Schnürch, 2006), delivering by carrier system (Kast et al., 2003; Ito et al., 2006; Paliwal et al., 2011a; Schmitz et al., 2005) and using of absorption enhancers such as glycyrrhetinic acid (Motlekar et al., 2006), fatty acids and their salts (Mori et al., 2004), sodium caprate (Motlekar et al., 2005) and chitosan and its derivatives (Thanou et al., 2001a,b; Thanou et al., 2007). Unfortunately, the oral formulation of LMWH is still not available commercially up to now. Chitosan oligomers are derivatives of chitosan and can be prepared by complete deacetylation from chitosan. Fig. 1 shows the chemical structure and physicochemical properties of chitosan oligomers. As shown in Fig. 1, different types of chitosan oligomers
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(0.1%, 0.5%, 1.0%, w/v) of each type of chitosan oligomers as absorption enhancers were added to LMWH solutions. In certain experiment, chitosan oligomers including dimer, tetramer and hexamer with the concentration of 0.1%, 0.5%, 1.0% (w/v), were prepared respectively to evaluate the intestinal membrane toxicity of chitosan oligomers and Triton X-100 (3.0%, v/v) was used as the positive control. 2.3. Intestinal absorption studies in rats
Fig. 1. Chemical structure and physicochemical properties of various chitosan oligomers.
including chitosan dimer, tetramer and hexamer are named according to the number of sugar rings in their chemical structures. These chitosan oligomers are of remarkable water-soluble characteristics and relative low molecular weight (Fig. 1), compared with conventional chitosan and its derivatives, which generally have poor solubility in water at physiological pH and consequently are limited in the clinic development. Previous studies reported that chitosan oligomers, as a novel type of absorption enhancer, were capable of increasing the intestinal absorption of insulin and FD4 (Gao et al., 2008) and also improving the pulmonary absorption of interferon-a in rats (Yamada et al., 2005). In the present study, further investigations were continuously performed to examine whether these chitosan oligomers could improve the oral bioavailability of anticoagulant LMWH based on the beneficial prevention and treatment of venous thrombo-embolism in clinic. In this regard, chitosan oligomers including chitosan dimer, tetramer and hexamer were used to investigate their absorption enhancing effects on the intestinal absorption of LMWH by an in situ closed loop method in rats. In addition, we also examined the intestinal membrane toxicity of chitosan oligomers by the morphological observation and determining biological markers such as LDH and protein released from the intestine epithelium. Finally, the mechanistic hypothesis that chitosan oligomers exert their absorption enhancing effects possibly through the electrostatic interactions between positively charged chitosan oligomers and negatively charged LMWH were tested by measuring zeta potential of each dosing solution of LMWH with or without chitosan oligomers and quantitative analysis of interactions between LMWH and chitosan oligomers using azure A assay method. 2. Materials and methods 2.1. Materials Low molecular weight heparin (LMWH, Anti-factor Xa 109 U/mL, mean M.W. 5266 Da) was provided from Nanda Pharm. Co. (Nanjing, China). Chitosan oligomers were supplied by Huicheng Bio. Co. (Shanghai, China). Chromogenix Coamatic Heparin Kit was obtained from Instrumentation Lab. Co. (Bedford, MA, USA). LDH assay Kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and protein assay Kit (BCA method) was obtained from Applygen Technologies Inc. (Beijing, China), using bovine serum albumin (BSA) as a standard. Azure A and Triton X-100 were purchased from Aladdin Industrial Inc. (Shanghai, China) and Amresco (Solon, OH, USA), respectively. All other reagents were of analytical grade. 2.2. Preparation of drug solution Dosing solutions of LMWH were prepared in PBS (pH 7.4) to yield a final concentration of 1000 IU/mL and varying concentrations
Studies on the intestinal absorption of LMWH were performed by an in-situ closed loop method in rats, as reported previously (Asada et al., 1995; Fetih et al., 2005; Lin et al., 2011). The experiments were carried out in accordance with the guidelines of the Animal Ethics Committee at Xi'an Jiaotong University. Male SD rats (8–10 weeks, 250–280 g) were fasted overnight but water was freely available. Prior to the experiment, the animals were anesthetized with sodium pentobarbital (40 mg/kg body weight, i.p.) and the anesthesia was maintained with additional doses of anesthetic solution as needed throughout the experiment. The rats were placed under a heating lamp to maintain the body temperature around 37 C. The intestine was exposed through the midline abdominal incision. After ligating the bile duct, the intestine (small intestine or large intestine) was cannulated with polyethylene tubing and then flushed by PBS (pH 7.4). The remaining buffer solution was expelled with air and the distal part of the intestine was closed by clipping a forceps. Each dosing solution of LMWH with or without chitosan oligomers, kept at 37 C, was introduced into the lumen of the intestinal loop through an opening in the proximal cannulation of the intestine, which was then closed by clipping with another forceps. The jugular vein was exposed and blood samples were collected into heparined syringe at predetermined time intervals up to 240 min. Samples were immediately centrifuged at 12,000 rpm for 5 min to obtain the plasma fraction, which was then kept in ice until determination. 2.4. Assessment of intestinal membrane toxicity and morphological examination PBS (pH 7.4), chitosan oligomers (0.1%, 0.5%, 1.0%, w/v) and Triton X-100 (3.0%, v/v) were injected into the intestinal loop respectively with a similar method used in the intestinal absorption experiment. And then 4 h after administration, the perfusate in the intestine was withdrawn for the evaluation of membrane toxicity by determining the release amount of protein and the activity of LDH. Finally, the intestines exposed of PBS (pH 7.4) and various chitosan oligomers were excised, fixed with 4% buffered paraformaldehyde and embedded in paraffin blocks. The sections of intestines cut from the paraffin blocks with 5 mm thick were stained with hematoxylin and eosin (H and E) to evaluate intestinal damage by light microscopy (Nikon Eclipse 80i, Japan). 2.5. Analytical methods Absorption of LMWH was measured by determining the antifactor Xa activity present in rat plasma with a colorimetric assay according to the protocol of Chromogenix Coamatic Heparin Kit. Samples and controls were evaluated based on the standard curve of LMWH prepared by diluting the standard LMWH with normal plasma, obtained from untreated rats and used as a negative control to account for the impact of endogenous ant-factor Xa that otherwise led to false positive results. The area under the plasma anti-factor Xa activity versus time curve (AUC0–240 min) was calculated by the linear trapezoidal rule and the absorption enhancing ratio of LMWH by chitosan
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oligomers was calculated as follows: Absorption enhancement ratio ¼
AUCwith enchancer AUCcontrol ðLMWH aloneÞ
The concentration of protein was assayed with BCATM Protein Assay Kit using bovine serum albumin as the standard (Applygen Tech. Inc., China) and the activity of LDH was assayed with LDH test kit (Nanjing Jiancheng Co., China). Absorption enhancers used in this experiment did not interfere with the assay of protein and LDH. 2.6. Measurements of zeta potential Zeta potentials of dosing solutions of LMWH with or without various chitosan oligomers were measured by electrophoretic laser doppler anemometry at 25 C with a Malvern Zetasizer – nano zs90 (Malvern Instruments, UK). The viscosity and dielectric constant of deionized water were used as calculation parameters. 2.7. Quantitative analysis of interactions between chitosan oligomers and LMWH Further studies were performed to quantify the electrostatic interactions between chitosan oligomers and LMWH by a colorimetric assay with azure A blue dye, as described previously (Cadene et al., 1995; Rawat et al., 2007). Firstly, a series of test samples were prepared: (a) LMWH solutions with the increasing concentrations of 0.5, 1.0, 2.5, 5.0, 10, 20 mM; (b) 0.1%, 0.5%, 1.0% (w/ v) of chitosan oligomers; (c) various LMWH solutions containing different types and varying concentrations of chitosan oligomers. And then in each test, 150 mL of azure A solution (150 mM) was mixed with 50 mL of the test sample in a 96-well microplate and the resulting mixture was incubated for 30 min at room temperature. The final samples were then measured by microplate reader (Bio-tek, synergyHT, USA) at 595 nm. 2.8. Statistical analysis Results were depicted as the mean S.E. and statistical significance was evaluated with analysis of variance (ANOVA) for multiple comparisons with the minimum level of significance with P < 0.05. 3. Results and discussion 3.1. Effects of chitosan oligomers on the intestinal absorption of LMWH in different intestinal regions of rats Effects of different type and varying concentration of chitosan oligomers on the absorption of LMWH from small intestine and large intestine were examined by an in situ closed loop method in
rats. Fig. 2 shows the plasma anti-factor Xa activity-time profiles of LMWH with or without chitosan oligomers after intestinal administration to rats. As shown in Fig. 2, the absorption of LMWH from small intestine was greatly increased in the presence of chitosan hexamer and tetramer, and the absorption enhancing effect of chitosan hexamer was a little larger than that of tetramer. However, chitosan dimer did not show obvious enhancing ability in the absorption of LMWH after small intestinal administration to rats (Fig. 2A). In contrast, in the large intestine, the absorption of LMWH was significantly increased by chitosan dimer, tetramer and hexamer (Fig. 2B). After an evaluation on the efficiency of various chitosan oligomers, chitosan hexamer with the most enhancing ability was selected as an optimal absorption enhancer and varying concentrations (0.1%, 0.5%, 1.0%, w/v) of chitosan hexamer were used to examine the influence of concentration on the absorption enhancing ability of chitosan oligomer, and the data were plotted in Fig. 3. It was found that plasma anti-factor Xa activities were greatly influenced by the concentration of chitosan hexamer and the largest absorption enhancing effect was observed in the presence of 0.5% (w/v) chitosan hexamer after co-administration of LMWH to small intestine (Fig. 3A) and large intestine (Fig. 3B). Overall, all above data demonstrated that chitosan hexamer at the concentration of 0.5% (w/v) showed the greatest absorption enhancing effect among different types of chitosan oligomers and varying concentrations of chitosan hexamer. These findings are well correlated with previous studies that chitosan hexamer showed the greatest absorption enhancing effects for improving the intestinal absorptions of FD4 and insulin (Gao et al., 2008) and also enhancing the pulmonary absorption of interferon-a in rats (Yamada et al., 2005). In addition, these enhancing effects of chitosan hexamer were also influenced by its concentration and the maximal enhancing effects were observed at the concentration of 0.5% (w/v). Based on these findings, it was concluded that there exists an optimal concentration for chitosan oligomers to show the greatest absorption enhancing ability for improving the intestinal or pulmonary absorptions of poorly absorbable drugs in rats. The area under anti-factor Xa in rat plasma versus time curve from 0 to 4 h with or without various chitosan oligomers (AUC0–240 min) and relative absorption enhancement ratio were summarized in the Table 1. We found that the absorption of LMWH alone was limited both in the small intestine and large intestine. However, after co-administration of LMWH with different chitosan oligomers to rats, the AUC values of LMWH were greatly increased in different degree, and the largest AUC value was obtained in the presence of 0.5% (w/v) chitosan hexamer with relative absorption enhancing ratios of 9.2 and 13.2 in the small intestine and large intestine, respectively, more effective comparing to many conventional enhancers studied previously. The general rank order of absorption-enhancing ability of these chitosan oligomers was 0.5%
Fig. 2. Plasma anti-factor Xa activity-time profiles of LMWH in the presence or absence of different type of chitosan oligomers after administration to rat small intestine (A) and large intestine (B). Results are expressed as the mean S.E. of at least three experiments.
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Fig. 3. Plasma anti-factor Xa activity-time profiles of LMWH in the presence or absence of varying concentration of chitosan hexamer after administration to rat small intestine (A) and large intestine (B). Results are expressed as the mean S.E. of at least three experiments. Table 1 Pharmacokinetic parameters of LMWH after intestinal administration with or without chitosan oligomers. Small intestine
LMWH Dimer (0.5%) Tetramer (0.5%) Hexamer (0.1%) Hexamer (0.5%) Hexamer (1.0%)
Large intestine
AUC0–240 min (Anti-factor Xa, IUmin/mL)
Enhancement ratio
AUC0–240 min(Anti-factor Xa, IUmin/mL)
Enhancement ratio
10.97 2.29 17.36 3.86 64.35 9.62 29.60 6.31 100.54 15.47 52.73 6.47
– 1.6 5.9** 2.7* 9.2** 4.8**
5.87 1.25 25.32 2.41 60.32 8.27 21.93 4.92 77.17 9.35 30.47 4.86
4.3** 10.3** 3.7* 13.2** 5.2**
Results are expressed as the mean S.E. of at least three experiments. ** P < 0.01; *P < 0.05, compared with the LMWH.
chitosan hexamer >0.5% chitosan tetramer >1.0% chitosan hexamer >0.5% chitosan dimer 30.1% chitosan hexamer, but the absorptionenhancing abilities of these chitosan oligomers in the large intestine were generally a little larger than those in the small intestine. This result was a little different with previous studies that absorption enhancing effects of chitosan oligomers for improving the intestinal absorption of FD4 in the jejunum was a little larger than that in the colon (Gao et al., 2008), which may be due to the difference in the physicochemical characteristics of model drugs used in these studies, although further studies are necessary to elucidate this discrepancy in regional difference for the absorption enhancing effects of chitosan oligomers. It was reported that activity of anti-factor Xa in plasma required for reaching 50% of anti-thrombotic effect was 0.12 IU/mL and only 0.2 IU/mL or higher levels of plasma anti-factor Xa could induce anti-thrombotic effects in male Sprague Dawley rats (Bianchini et al., 1995). In the present study, the plasma anti-factor Xa levels could not be detected when delivering of chitosan oligomers alone to rat intestine (data not shown) and the intestinal administration of LMWH alone (4000 IU/kg) only produced lower levels of plasma anti-factor Xa and failed to attain therapeutic levels in rats. However, after intestinal administration of LMWH with chitosan oligomers to rats, a significant increase in the plasma anti-factor Xa activity was obtained compared with control group (LMWH alone). Furthermore, increasing levels of plasma anti-factor Xa above 0.2 IU/mL were observed 1 h after intestinal delivering of LMWH with chitosan hexamer and tetramer (Figs. 2 and 3). From this standpoint, it is suggested that chitosan oligomers are promising to become the ideal absorption enhancer for improving the oral absorption of LMWH formulations applied in clinic.
potency and toxicity (Whitehead et al., 2008). Therefore, evaluation of membrane damage induced by absorption enhancer becomes an important issue before its application in the clinic. Recently, some biological markers including protein and LDH released from intestinal epithelium cells are usually used to evaluate the membrane toxicity of these enhancers (Gao et al., 2008; Hamid et al., 2009; Lin et al., 2011). It is well known that protein is one of the components of biological membranes and LDH is the cytosolic enzyme, and consequently the presences of protein and/or LDH in the perfusion of intestine are generally regarded as evidence of cell membrane toxicity. Therefore, in the present study, biological markers including protein and LDH released from intestinal epithelial cells were measured to confirm the safety of chitosan oligomers after their intestinal administrations to rats. As is evident in Fig. 4, 4 h after intestinal administration, the amount of protein and activity of LDH for each chitosan oligomer group were similar with that for the control (PBS, pH 7.4) and meanwhile a significant difference in the levels of protein and LDH was obtained when comparing with Triton X-100 (3.0%, v/v), used as a positive control. On the other hand, morphological observation of the intestinal mucosa 4 h after the exposure of various chitosan oligomers to rat intestine was also performed by light microscopy and the pictures were shown in Fig. 5. We found that no inflammatory cells and necrotic or damaged cells were observed in stained intestinal mucosa receiving different type and varying concentration of chitosan oligomers (Fig. 5). Taken together, these results demonstrated that chitosan oligomers including chitosan dimer, tetramer and hexamer did not induce remarkable membrane damage to the intestine over the concentration range of 0.1–1.0% (w/v) and could be used safely as an absorption enhancer.
3.2. Evaluation of intestinal membrane toxicity
3.3. Measurements of zeta potential
Great attentions have recently been paid to the potential membrane toxicity caused by absorption enhancer because of its inherent toxicity (Maher et al., 2009) and the direct link between
Fig. 6 showed the zeta potentials of LMWH in the presence or absence of different type of chitosan oligomers with varying concentration. It was found that zeta potential of LMWH alone was
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Fig. 4. The amount of protein and LDH activity released from intestinal epithelium cells at 4 h after intestinal administration of chitosan oligomers to rats. Each data represents the mean S.E. of 3–5 samples. ** P < 0.01, compared with control group.
negative with a value of 31.7. However, the magnitude of negative charge of LMWH gradually declined when various chitosan oligomers were added to LMWH solution, indicating the charge neutralization of negative LMWH by chitosan oligomers with
positive charge. This observation well agrees with the fact that the negative surface charge of the drug can be neutralized by the addition of a positively charged polymer, and the net charge will gradually decreased with the increasing amount of the oppositely charge polymer. Furthermore, it was also reported that surface charge of polyelectrolyte complexes in a sense depends on the drug-polymer ratios (Buchhammer et al., 2003; Dash et al., 1999; Paliwal et al., 2011a). In addition, it was interestingly noticed from Fig. 6 that zeta potential of LMWH solution in the presence of 0.5% (w/v) chitosan hexamer was less negative than that in the presence of 1.0% (w/v) chitosan hexamer. Similar result was also found by Yang et al. that zeta potential of PEI-1000 KDa at the concentration of 0.25% was almost equal to that of 0.5% PEI-1000 KDa and the selfassociate/aggregation of PEI polymers was attributed to this phenomenon (Yang et al., 2006). Similarly, chitosan hexamer, used in the present study, might self-associate at higher concentration, consequently resulting in an overall charge neutralization. These data substantiated our hypothesis of a possible electrostatic interaction between negatively charged LMWH and positively charged chitosan oligomers. 3.4. Colormetric assay for interactions between LMWH and chitosan oligomers
Fig. 5. Morphological observation of small intestine (A) and large intestine (B) at 4 h after intestinal administration of PBS (pH 7.4), various chitosan oligomers (0.1%, 0.5%, 1.0%, w/v) and Triton X-100 (3.0%, w/v) to rats, respectively.
An azure A colormetric assay was performed to measure the extent of electrostatic interactions between LMWH and chitosan oligomers. This method had previously been used to determine peracetylated sulfoglycolipids (Tadano-Aritomi and Ishizuka, 1983) and heparin concentration in biological samples (Gundry et al.,1984)
Fig. 6. Zeta potential of LMWH with or without various chitosan oligomers. Data represents the mean S.E., n = 12. * P < 0.05, ** P < 0.01, compared with control group.
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Fig. 7. Absorbance of azure A in the presence of various chitosan oligomers at 595 nm (A) and percentage of free LMWH with various chitosan oligomers (B). Data represents the mean S.E., n 3.
and to study the interactions between neutrophil elastase and heparin (Cadene et al., 1995). Azure A, a positively charged phenothiazine dye, gives a strong absorbance at 595 nm, but its absorbance will decrease upon binding with negatively charged sulfate groups of the heparin due to the formation of purple colored complex, which produces a negative shift in lmax. Therefore, the azure A assay can be used to quantitatively measure the heparin or LMWH on the base of the linear relationship of the absorption of azure A at 595 nm and the concentration of LMWH. As is evident in Fig. 7A that there was no significant change in the absorbance of azure A at 595 nm when the different type and varying concentration of chitosan oligomers were added to the azure A solutions. This result can be interpreted that chitosan oligomers do not possess sulfate group binding with the azure A, therefore no obvious difference in the absorbance of azure A was observed. The amount of free LMWH in each LMWH solution with or without chitosan oligomers was determined with the same method and the data are plotted in Fig. 7B. We found that amount of free LMWH in LMWH solution was in a sense dependent on the type of chitosan oligomers and the percentage of free LMWH in the LMWH solution containing chitosan hexamer was a little higher than that containing chitosan tetramer and dimer, indicating that the electrostatic interaction between chitosan hexamer and LMWH was larger than that between chitosan tetramer or dimer and LMWH. This tendency was well consistent with the result of absorption enhancing studies on chitosan oligomers, which demonstrated clearly that absorption enhancing potential of chitosan hexamer for improving intestinal absorption of LMWH was greater than that of chitosan tetramer and dimer (Fig. 2). The reason for the superior performance of chitosan hexamer in comparison to chitosan tetramer and dimer was not fully understood. However, it may be plausible that amino group contents of chitosan oligomers with different types perhaps influence drug absorption from intestinal epithelium. It is well known that the surface of mucus membrane is generally negative due to the existence of sialic acid and consequently the coulombic repulsion effect will be generated between the negatively charged cell membranes and negative LMWH, which probably contributes in part to the poor oral bioavailability of LMWH. Therefore, combination of negative LMWH with positive charged chitosan oligomers, especially chitosan hexamer which has relative more cationic charges (Fig. 6) and stronger electrostatic interaction with LMWH (Fig. 7B) due to the larger numbers of amino groups in its structure compared to chitosan tetramer and dimer (Fig. 1), may attenuate to a larger extent above coulombic repulsion effect, thereby triggering the more efficient absorption enhancing effect on the intestinal absorption of LMWH. Similar results were also observed in the case of PAMAM dendrimers as absorption enhancers on pulmonary absorption, which reported the good liner correlation between the zeta potential caused by amino group of PAMAM dendrimers and their pulmonary absorption enhancing
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effects (Dong et al., 2010). Therefore, the content of amino group in the molecules of chitosan oligomers might be one important factor for the absorption enhancing effects of chitosan oligomers although further studies are required to elucidate it in the future. In addition, reduction degree in the amount of free LMWH varied with the concentration of chitosan oligomers and the largest reducing amount of free LMWH was obtained at the concentration of 0.5% (w/v) chitosan hexamer (Fig. 7B). Similar results were also reported that the level of free LMWH (enoxaparin) linearly decreased upon addition of positively charged PEI (Yang et al., 2006), PAMAM dendrimers (Bai et al., 2006) or poly-L-argine (Rawat et al., 2007) to LMWH solution. Therefore, these data are consistent with the data on zeta potential and further verified the existence of electrostatic interactions proposed between the positively charged chitosan oligomers and the negatively charged LMWH. 4. Conclusion In conclusion, chitosan oligomers, especially chitosan hexamer, could significantly improve the intestinal absorption of negatively charged LMWH most likely via electrostatic interactions and meanwhile did not induce obvious damage to the intestinal membranes, suggesting that chitosan oligomers would be a promising absorption enhancer with high efficiency and low toxicity, therefore could be applied hopefully to increase the oral bioavailability of various LMWH formulations. Acknowledgments The work was supported by grants from the National Natural Science Foundation of China (81102382) and the Fundamental Research Funds for the Central Universities (to Yang Gao and Hailong Zhang). References Asada, H., Douen, T., Waki, M., Adachi, S., T, Muranishi, S., 1995. Absorption characteristics of chemically modified-insulin derivatives with various fatty acids in the small and large intestine. Journal of Pharmaceutical Sciences 84, 682–687. Bai, S., Thomas, C., Ahsan, F., 2006. Dendrimers as a carrier for pulmonary delivery of enoxaparin, a low-molecular weight heparin. Journal of Pharmaceutical Sciences 96, 2090–2106. Bianchini, P., Bergonzini, G.L., Parma, B., Osima, B., 1995. Relationship between plasma antifactor Xa activity and the antithrombotic activity of heparins of different molecular mass. Haemostasis 25, 288–298. Buchhammer, H.-M., Mende, M., Oelmann, M., 2003. Formation of mono-sized polyelectrolyte complex dispersions: effects of polymer structure, concentration and mixing conditions. Colloids and Surfaces A: Physicochemical and Engeering Aspects 218, 151–159. Cadene, M., Boudier, C., de Marcillac, G.D., Bieth, J.G., 1995. Influence of low molecular mass heparin on the kinetics of neutrophil elastase inhibition by mucus proteinase inhibitor. Journal of Biological Chemistry 270, 13204–13209. Dash, P.R., Read, M.L., Barrett, L.B., Wolfert, M.A., Seymour, L.W., 1999. Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery. Gene Therapy 6, 643–650. Dong, Z.Q., Hamid, K.A., Gao, Y., Lin, Y.L., Katsumi, H., Sakane, T., Yamamoto, A., 2010. Polyamidoamine dendrimers can improve the pulmonary absorption of insulin and calcitonin in rats. International Journal of Pharmaceutics 100, 1866–1878. Fetih, G., Habib, F., Okada, N., Fujita, T., Attia, M., Yamamoto, A., 2005. Nitric oxide donors can enhance the intestinal transport and absorption of insulin and [Asu1,7]-eel calcitonin in rats. Journal of Controlled Release 106, 287–297. Gao, Y., He, L., Katsumi, H., Sakane, T., Fujita, T., Yamamoto, A., 2008. Improvement of intestinal absorption of insulin and water-soluble macromolecular compounds by chitosan oligomers in rats. International Journal of Pharmaceutics 358, 70–78. Grabovac, V., Bernkop-Schnürch, A., 2006. Improvement of the intestinal membrane permeability of low molecular weight heparin by complexation with stem bromelain. International Journal of Pharmaceutics 326, 153–159. Gundry, S.R., Klein, M.D., Drongowski, R.A., Kirsh, M.M., 1984. Clinical evaluation of a new rapid heparin assay using the dye azure A. American Journal of Surgery 148, 191–194.
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Motlekar, N.A., Srivenugopal, K.S., Wachtel, M.S., Youan, B.B.C., 2006. Evaluation of the oral bioavailability of low molecular weight heparin formulated with glycyrrhetinic acid as permeation enhancer. Drug Development Research 67, 166–174. Norris, D.A., Puri, N., Sinko, P.J., 1998. The effect of physical barriers and properties on the oral absorption of particulates. Advanced Drug Delivery Reviews 34, 135–154. Paliwal, R., Paliwal, S.R., Agrawal, G.P., Vyas, S.P., 2011a. Biomimetic solid lipid nanoparticles for oral bioavailability enhancement of low molecular weight heparin and its lipid conjugates: in vitro and in vivo evaluation. Molecular Pharmacology 8, 1314–1321. Rawat, A., Yang, T., Hussain, A., Ahsan, F., 2007. Complexation of a poly-L-arginine with low molecular weight heparin enhances pulmonary absorption of the drug. Pharmaceutical Research 25, 936–948. Ross, B.P., Toth, I., 2005. Gastrointestinal absorption of heparin by lipidization or coadministration with penetration enhancers. Current Drug Delivery 2, 277–287. Schmitz, T., Leitner, V.M., Bernkop-Schnurch, A., 2005. Oral heparin delivery: design and in vivo evaluation of a stomach-targeted mucoadhesive delivery system. Journal of Pharmaceutical Sciences 94, 966–973. Tadano-Aritomi, K., Ishizuka, I., 1983. Determination of peracetylated sulfoglycolipids using the azure A method. Journal of Lipid Research 24, 1368–1375. Thanou, M., Nihot, M.T., Jansen, M., Verhoef, J.C., Junginger, H.E., 2001a. Mono-Ncarboxymethyl chitosan (MCC), a polyampholytic chitosan derivative, enhances the intestinal absorption of low molecular weight heparin across intestinal epithelia in vitro and in vivo. Journal of Pharmaceutical Sciences 90, 38–46. Thanou, M., Verhoef, J.C., Junginger, H.E., 2001b. Enhancement by chitosan and its derivatives. Advanced Drug Delivery Reviews 52, 117–126. Thanou, M., Henderson, S., Kydonieus, A., Elson, C., 2007. N-Sulfonato-N,Ocarboxymethylchitosan: a novel polymeric absorption enhancer for the oral delivery of macromolecules. Journal of Controlled Release 117, 171–178. Weltermann, A., Kyrle, P.A., Eichinger, S., 2003. Novel anticoagulants for the prevention and treatment of venous thromboembolish. Wiener Medizinische Wochenschrift 153, 426–433. Whitehead, K., Karr, N., Mitragotri, S., 2008. Safe and effective permeation enhancers for oral drug delivery. Pharmaceutical Research 25, 1782–1788. Yamada, K., Okada, M.O.N., Fujita, T., Yamamoto, A., 2005. Chitosan oligomers as potential and safe absorption enhancers for improving the pulmonary absorption of interferon-a in rats. Journal of Pharmaceutical Sciences 94, 2432–2440. Yang, T.Z., Hussain, A., Bai, S.H., Khalil, I.A., Harashima, H., Ahsan, F., 2006. Positively charged polyethylenimines enhance nasal absorption of the negatively charge drug, low molecular weight heparin. Journal of Controlled Release 115, 289–297.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Absorption enhancing effects of chitosan oligomers on the intestinal absorption of low molecular weight heparin in rats Hailong Zhang a , Jie Mi a , Yayu Huo a , Xiaoyan Huang a , Jianfeng Xing a , Akira Yamamoto b , Yang Gao a, * a b
School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi'an 710061, China Department of Pharmaceutics, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 December 2013 Received in revised form 13 February 2014 Accepted 2 March 2014 Available online 11 March 2014
Absorption enhancing effects of chitosan oligomers with different type and varying concentration on the intestinal absorption of low molecular weight heparin (LMWH) were examined by an in situ closed loop method in different intestinal sections of rats. Chitosan hexamer with the optimal concentration of 0.5% (w/v) showed the highest absorption enhancing ability both in the small intestine and large intestine. The membrane toxicities of chitosan oligomers were evaluated by morphological observation and determining the biological markers including amount of protein and activity of lactate dehydrogenase (LDH) released from intestinal epithelium cells. There was no obvious change both in levels of protein and LDH and morphology in the intestinal membrane between control and various chitosan oligomers groups, suggesting that chitosan oligomers did not induce any significant membrane damage to the intestinal epithelium. In addition, zeta potentials became less negative and amount of free LMWH gradually decreased when various chitosan oligomers were added to LMWH solution, revealing that electrostatic interaction between positively charged chitosan oligomers and negative LMWH was included in the absorption enhancing mechanism of chitosan oligomers. In conclusion, chitosan oligomers, especially chitosan hexamer, are safe and efficient absorption enhancers and can be used promisingly to improve oral absorption of LMWH. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Absorption enhancer Intestinal absorption Chitosan oligomers Low molecular weight heparin (LMWH) Membrane toxicity Electrostatic interaction
1. Introduction Low molecular weight heparin (LMWH), an anionic polysaccharide, is preferred clinically as an improved anticoagulant compared to un-fractionated heparin for the prevention and treatment of deep vein thrombosis (DVT) and pulmonary embolism through catalyzing the inactivation of factors Xa and IIa by binding to anti-thrombin, which ultimately leads to the inhibition of the clotting cascade (Hirsh et al., 1998; Paliwal et al., 2011a; Weltermann et al., 2003). However, it has been recognized that the clinic application of LMWH is limited because of the poor oral bioavailability and consequently the requirement for daily subcutaneous injection although it provides a predictable anticoagulant response. Drug delivery via oral route remains the most preferred administration mode due to several advantages including convenient application, patient compliance and low cost. However, the
* Corresponding author at: Xi'an Jiaotong University, No. 76 Yanta West Road, Xi'an, Shaanxi 710061, China. Tel.: +86 29 82655139; fax: +86 29 82655139. E-mail addresses: [email protected], [email protected] (Y. Gao). http://dx.doi.org/10.1016/j.ijpharm.2014.03.010 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
extent of oral absorption of LMWH was sometimes not enough for its therapeutic effect owing to the characteristics of LMWH such as hydrophilicity, anionic surface charge and the large molecular weight, which led to the poor membrane permeability (Jaques, 1980; Norris et al., 1998; Ross and Toth, 2005). Therefore, various strategies had been investigated to improve the oral absorption of LMWH including preparing of microparticle (Lanke et al., 2009; Meissner et al., 2007; Paliwal et al., 2011a), nanoparticle (Hoffart et al., 2006; Leung, 2012) and complexation (Lee et al., 2001; Yang et al., 2006; Grabovac and Bernkop-Schnürch, 2006), delivering by carrier system (Kast et al., 2003; Ito et al., 2006; Paliwal et al., 2011a; Schmitz et al., 2005) and using of absorption enhancers such as glycyrrhetinic acid (Motlekar et al., 2006), fatty acids and their salts (Mori et al., 2004), sodium caprate (Motlekar et al., 2005) and chitosan and its derivatives (Thanou et al., 2001a,b; Thanou et al., 2007). Unfortunately, the oral formulation of LMWH is still not available commercially up to now. Chitosan oligomers are derivatives of chitosan and can be prepared by complete deacetylation from chitosan. Fig. 1 shows the chemical structure and physicochemical properties of chitosan oligomers. As shown in Fig. 1, different types of chitosan oligomers
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(0.1%, 0.5%, 1.0%, w/v) of each type of chitosan oligomers as absorption enhancers were added to LMWH solutions. In certain experiment, chitosan oligomers including dimer, tetramer and hexamer with the concentration of 0.1%, 0.5%, 1.0% (w/v), were prepared respectively to evaluate the intestinal membrane toxicity of chitosan oligomers and Triton X-100 (3.0%, v/v) was used as the positive control. 2.3. Intestinal absorption studies in rats
Fig. 1. Chemical structure and physicochemical properties of various chitosan oligomers.
including chitosan dimer, tetramer and hexamer are named according to the number of sugar rings in their chemical structures. These chitosan oligomers are of remarkable water-soluble characteristics and relative low molecular weight (Fig. 1), compared with conventional chitosan and its derivatives, which generally have poor solubility in water at physiological pH and consequently are limited in the clinic development. Previous studies reported that chitosan oligomers, as a novel type of absorption enhancer, were capable of increasing the intestinal absorption of insulin and FD4 (Gao et al., 2008) and also improving the pulmonary absorption of interferon-a in rats (Yamada et al., 2005). In the present study, further investigations were continuously performed to examine whether these chitosan oligomers could improve the oral bioavailability of anticoagulant LMWH based on the beneficial prevention and treatment of venous thrombo-embolism in clinic. In this regard, chitosan oligomers including chitosan dimer, tetramer and hexamer were used to investigate their absorption enhancing effects on the intestinal absorption of LMWH by an in situ closed loop method in rats. In addition, we also examined the intestinal membrane toxicity of chitosan oligomers by the morphological observation and determining biological markers such as LDH and protein released from the intestine epithelium. Finally, the mechanistic hypothesis that chitosan oligomers exert their absorption enhancing effects possibly through the electrostatic interactions between positively charged chitosan oligomers and negatively charged LMWH were tested by measuring zeta potential of each dosing solution of LMWH with or without chitosan oligomers and quantitative analysis of interactions between LMWH and chitosan oligomers using azure A assay method. 2. Materials and methods 2.1. Materials Low molecular weight heparin (LMWH, Anti-factor Xa 109 U/mL, mean M.W. 5266 Da) was provided from Nanda Pharm. Co. (Nanjing, China). Chitosan oligomers were supplied by Huicheng Bio. Co. (Shanghai, China). Chromogenix Coamatic Heparin Kit was obtained from Instrumentation Lab. Co. (Bedford, MA, USA). LDH assay Kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and protein assay Kit (BCA method) was obtained from Applygen Technologies Inc. (Beijing, China), using bovine serum albumin (BSA) as a standard. Azure A and Triton X-100 were purchased from Aladdin Industrial Inc. (Shanghai, China) and Amresco (Solon, OH, USA), respectively. All other reagents were of analytical grade. 2.2. Preparation of drug solution Dosing solutions of LMWH were prepared in PBS (pH 7.4) to yield a final concentration of 1000 IU/mL and varying concentrations
Studies on the intestinal absorption of LMWH were performed by an in-situ closed loop method in rats, as reported previously (Asada et al., 1995; Fetih et al., 2005; Lin et al., 2011). The experiments were carried out in accordance with the guidelines of the Animal Ethics Committee at Xi'an Jiaotong University. Male SD rats (8–10 weeks, 250–280 g) were fasted overnight but water was freely available. Prior to the experiment, the animals were anesthetized with sodium pentobarbital (40 mg/kg body weight, i.p.) and the anesthesia was maintained with additional doses of anesthetic solution as needed throughout the experiment. The rats were placed under a heating lamp to maintain the body temperature around 37 C. The intestine was exposed through the midline abdominal incision. After ligating the bile duct, the intestine (small intestine or large intestine) was cannulated with polyethylene tubing and then flushed by PBS (pH 7.4). The remaining buffer solution was expelled with air and the distal part of the intestine was closed by clipping a forceps. Each dosing solution of LMWH with or without chitosan oligomers, kept at 37 C, was introduced into the lumen of the intestinal loop through an opening in the proximal cannulation of the intestine, which was then closed by clipping with another forceps. The jugular vein was exposed and blood samples were collected into heparined syringe at predetermined time intervals up to 240 min. Samples were immediately centrifuged at 12,000 rpm for 5 min to obtain the plasma fraction, which was then kept in ice until determination. 2.4. Assessment of intestinal membrane toxicity and morphological examination PBS (pH 7.4), chitosan oligomers (0.1%, 0.5%, 1.0%, w/v) and Triton X-100 (3.0%, v/v) were injected into the intestinal loop respectively with a similar method used in the intestinal absorption experiment. And then 4 h after administration, the perfusate in the intestine was withdrawn for the evaluation of membrane toxicity by determining the release amount of protein and the activity of LDH. Finally, the intestines exposed of PBS (pH 7.4) and various chitosan oligomers were excised, fixed with 4% buffered paraformaldehyde and embedded in paraffin blocks. The sections of intestines cut from the paraffin blocks with 5 mm thick were stained with hematoxylin and eosin (H and E) to evaluate intestinal damage by light microscopy (Nikon Eclipse 80i, Japan). 2.5. Analytical methods Absorption of LMWH was measured by determining the antifactor Xa activity present in rat plasma with a colorimetric assay according to the protocol of Chromogenix Coamatic Heparin Kit. Samples and controls were evaluated based on the standard curve of LMWH prepared by diluting the standard LMWH with normal plasma, obtained from untreated rats and used as a negative control to account for the impact of endogenous ant-factor Xa that otherwise led to false positive results. The area under the plasma anti-factor Xa activity versus time curve (AUC0–240 min) was calculated by the linear trapezoidal rule and the absorption enhancing ratio of LMWH by chitosan
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oligomers was calculated as follows: Absorption enhancement ratio ¼
AUCwith enchancer AUCcontrol ðLMWH aloneÞ
The concentration of protein was assayed with BCATM Protein Assay Kit using bovine serum albumin as the standard (Applygen Tech. Inc., China) and the activity of LDH was assayed with LDH test kit (Nanjing Jiancheng Co., China). Absorption enhancers used in this experiment did not interfere with the assay of protein and LDH. 2.6. Measurements of zeta potential Zeta potentials of dosing solutions of LMWH with or without various chitosan oligomers were measured by electrophoretic laser doppler anemometry at 25 C with a Malvern Zetasizer – nano zs90 (Malvern Instruments, UK). The viscosity and dielectric constant of deionized water were used as calculation parameters. 2.7. Quantitative analysis of interactions between chitosan oligomers and LMWH Further studies were performed to quantify the electrostatic interactions between chitosan oligomers and LMWH by a colorimetric assay with azure A blue dye, as described previously (Cadene et al., 1995; Rawat et al., 2007). Firstly, a series of test samples were prepared: (a) LMWH solutions with the increasing concentrations of 0.5, 1.0, 2.5, 5.0, 10, 20 mM; (b) 0.1%, 0.5%, 1.0% (w/ v) of chitosan oligomers; (c) various LMWH solutions containing different types and varying concentrations of chitosan oligomers. And then in each test, 150 mL of azure A solution (150 mM) was mixed with 50 mL of the test sample in a 96-well microplate and the resulting mixture was incubated for 30 min at room temperature. The final samples were then measured by microplate reader (Bio-tek, synergyHT, USA) at 595 nm. 2.8. Statistical analysis Results were depicted as the mean S.E. and statistical significance was evaluated with analysis of variance (ANOVA) for multiple comparisons with the minimum level of significance with P < 0.05. 3. Results and discussion 3.1. Effects of chitosan oligomers on the intestinal absorption of LMWH in different intestinal regions of rats Effects of different type and varying concentration of chitosan oligomers on the absorption of LMWH from small intestine and large intestine were examined by an in situ closed loop method in
rats. Fig. 2 shows the plasma anti-factor Xa activity-time profiles of LMWH with or without chitosan oligomers after intestinal administration to rats. As shown in Fig. 2, the absorption of LMWH from small intestine was greatly increased in the presence of chitosan hexamer and tetramer, and the absorption enhancing effect of chitosan hexamer was a little larger than that of tetramer. However, chitosan dimer did not show obvious enhancing ability in the absorption of LMWH after small intestinal administration to rats (Fig. 2A). In contrast, in the large intestine, the absorption of LMWH was significantly increased by chitosan dimer, tetramer and hexamer (Fig. 2B). After an evaluation on the efficiency of various chitosan oligomers, chitosan hexamer with the most enhancing ability was selected as an optimal absorption enhancer and varying concentrations (0.1%, 0.5%, 1.0%, w/v) of chitosan hexamer were used to examine the influence of concentration on the absorption enhancing ability of chitosan oligomer, and the data were plotted in Fig. 3. It was found that plasma anti-factor Xa activities were greatly influenced by the concentration of chitosan hexamer and the largest absorption enhancing effect was observed in the presence of 0.5% (w/v) chitosan hexamer after co-administration of LMWH to small intestine (Fig. 3A) and large intestine (Fig. 3B). Overall, all above data demonstrated that chitosan hexamer at the concentration of 0.5% (w/v) showed the greatest absorption enhancing effect among different types of chitosan oligomers and varying concentrations of chitosan hexamer. These findings are well correlated with previous studies that chitosan hexamer showed the greatest absorption enhancing effects for improving the intestinal absorptions of FD4 and insulin (Gao et al., 2008) and also enhancing the pulmonary absorption of interferon-a in rats (Yamada et al., 2005). In addition, these enhancing effects of chitosan hexamer were also influenced by its concentration and the maximal enhancing effects were observed at the concentration of 0.5% (w/v). Based on these findings, it was concluded that there exists an optimal concentration for chitosan oligomers to show the greatest absorption enhancing ability for improving the intestinal or pulmonary absorptions of poorly absorbable drugs in rats. The area under anti-factor Xa in rat plasma versus time curve from 0 to 4 h with or without various chitosan oligomers (AUC0–240 min) and relative absorption enhancement ratio were summarized in the Table 1. We found that the absorption of LMWH alone was limited both in the small intestine and large intestine. However, after co-administration of LMWH with different chitosan oligomers to rats, the AUC values of LMWH were greatly increased in different degree, and the largest AUC value was obtained in the presence of 0.5% (w/v) chitosan hexamer with relative absorption enhancing ratios of 9.2 and 13.2 in the small intestine and large intestine, respectively, more effective comparing to many conventional enhancers studied previously. The general rank order of absorption-enhancing ability of these chitosan oligomers was 0.5%
Fig. 2. Plasma anti-factor Xa activity-time profiles of LMWH in the presence or absence of different type of chitosan oligomers after administration to rat small intestine (A) and large intestine (B). Results are expressed as the mean S.E. of at least three experiments.
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Fig. 3. Plasma anti-factor Xa activity-time profiles of LMWH in the presence or absence of varying concentration of chitosan hexamer after administration to rat small intestine (A) and large intestine (B). Results are expressed as the mean S.E. of at least three experiments. Table 1 Pharmacokinetic parameters of LMWH after intestinal administration with or without chitosan oligomers. Small intestine
LMWH Dimer (0.5%) Tetramer (0.5%) Hexamer (0.1%) Hexamer (0.5%) Hexamer (1.0%)
Large intestine
AUC0–240 min (Anti-factor Xa, IUmin/mL)
Enhancement ratio
AUC0–240 min(Anti-factor Xa, IUmin/mL)
Enhancement ratio
10.97 2.29 17.36 3.86 64.35 9.62 29.60 6.31 100.54 15.47 52.73 6.47
– 1.6 5.9** 2.7* 9.2** 4.8**
5.87 1.25 25.32 2.41 60.32 8.27 21.93 4.92 77.17 9.35 30.47 4.86
4.3** 10.3** 3.7* 13.2** 5.2**
Results are expressed as the mean S.E. of at least three experiments. ** P < 0.01; *P < 0.05, compared with the LMWH.
chitosan hexamer >0.5% chitosan tetramer >1.0% chitosan hexamer >0.5% chitosan dimer 30.1% chitosan hexamer, but the absorptionenhancing abilities of these chitosan oligomers in the large intestine were generally a little larger than those in the small intestine. This result was a little different with previous studies that absorption enhancing effects of chitosan oligomers for improving the intestinal absorption of FD4 in the jejunum was a little larger than that in the colon (Gao et al., 2008), which may be due to the difference in the physicochemical characteristics of model drugs used in these studies, although further studies are necessary to elucidate this discrepancy in regional difference for the absorption enhancing effects of chitosan oligomers. It was reported that activity of anti-factor Xa in plasma required for reaching 50% of anti-thrombotic effect was 0.12 IU/mL and only 0.2 IU/mL or higher levels of plasma anti-factor Xa could induce anti-thrombotic effects in male Sprague Dawley rats (Bianchini et al., 1995). In the present study, the plasma anti-factor Xa levels could not be detected when delivering of chitosan oligomers alone to rat intestine (data not shown) and the intestinal administration of LMWH alone (4000 IU/kg) only produced lower levels of plasma anti-factor Xa and failed to attain therapeutic levels in rats. However, after intestinal administration of LMWH with chitosan oligomers to rats, a significant increase in the plasma anti-factor Xa activity was obtained compared with control group (LMWH alone). Furthermore, increasing levels of plasma anti-factor Xa above 0.2 IU/mL were observed 1 h after intestinal delivering of LMWH with chitosan hexamer and tetramer (Figs. 2 and 3). From this standpoint, it is suggested that chitosan oligomers are promising to become the ideal absorption enhancer for improving the oral absorption of LMWH formulations applied in clinic.
potency and toxicity (Whitehead et al., 2008). Therefore, evaluation of membrane damage induced by absorption enhancer becomes an important issue before its application in the clinic. Recently, some biological markers including protein and LDH released from intestinal epithelium cells are usually used to evaluate the membrane toxicity of these enhancers (Gao et al., 2008; Hamid et al., 2009; Lin et al., 2011). It is well known that protein is one of the components of biological membranes and LDH is the cytosolic enzyme, and consequently the presences of protein and/or LDH in the perfusion of intestine are generally regarded as evidence of cell membrane toxicity. Therefore, in the present study, biological markers including protein and LDH released from intestinal epithelial cells were measured to confirm the safety of chitosan oligomers after their intestinal administrations to rats. As is evident in Fig. 4, 4 h after intestinal administration, the amount of protein and activity of LDH for each chitosan oligomer group were similar with that for the control (PBS, pH 7.4) and meanwhile a significant difference in the levels of protein and LDH was obtained when comparing with Triton X-100 (3.0%, v/v), used as a positive control. On the other hand, morphological observation of the intestinal mucosa 4 h after the exposure of various chitosan oligomers to rat intestine was also performed by light microscopy and the pictures were shown in Fig. 5. We found that no inflammatory cells and necrotic or damaged cells were observed in stained intestinal mucosa receiving different type and varying concentration of chitosan oligomers (Fig. 5). Taken together, these results demonstrated that chitosan oligomers including chitosan dimer, tetramer and hexamer did not induce remarkable membrane damage to the intestine over the concentration range of 0.1–1.0% (w/v) and could be used safely as an absorption enhancer.
3.2. Evaluation of intestinal membrane toxicity
3.3. Measurements of zeta potential
Great attentions have recently been paid to the potential membrane toxicity caused by absorption enhancer because of its inherent toxicity (Maher et al., 2009) and the direct link between
Fig. 6 showed the zeta potentials of LMWH in the presence or absence of different type of chitosan oligomers with varying concentration. It was found that zeta potential of LMWH alone was
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Fig. 4. The amount of protein and LDH activity released from intestinal epithelium cells at 4 h after intestinal administration of chitosan oligomers to rats. Each data represents the mean S.E. of 3–5 samples. ** P < 0.01, compared with control group.
negative with a value of 31.7. However, the magnitude of negative charge of LMWH gradually declined when various chitosan oligomers were added to LMWH solution, indicating the charge neutralization of negative LMWH by chitosan oligomers with
positive charge. This observation well agrees with the fact that the negative surface charge of the drug can be neutralized by the addition of a positively charged polymer, and the net charge will gradually decreased with the increasing amount of the oppositely charge polymer. Furthermore, it was also reported that surface charge of polyelectrolyte complexes in a sense depends on the drug-polymer ratios (Buchhammer et al., 2003; Dash et al., 1999; Paliwal et al., 2011a). In addition, it was interestingly noticed from Fig. 6 that zeta potential of LMWH solution in the presence of 0.5% (w/v) chitosan hexamer was less negative than that in the presence of 1.0% (w/v) chitosan hexamer. Similar result was also found by Yang et al. that zeta potential of PEI-1000 KDa at the concentration of 0.25% was almost equal to that of 0.5% PEI-1000 KDa and the selfassociate/aggregation of PEI polymers was attributed to this phenomenon (Yang et al., 2006). Similarly, chitosan hexamer, used in the present study, might self-associate at higher concentration, consequently resulting in an overall charge neutralization. These data substantiated our hypothesis of a possible electrostatic interaction between negatively charged LMWH and positively charged chitosan oligomers. 3.4. Colormetric assay for interactions between LMWH and chitosan oligomers
Fig. 5. Morphological observation of small intestine (A) and large intestine (B) at 4 h after intestinal administration of PBS (pH 7.4), various chitosan oligomers (0.1%, 0.5%, 1.0%, w/v) and Triton X-100 (3.0%, w/v) to rats, respectively.
An azure A colormetric assay was performed to measure the extent of electrostatic interactions between LMWH and chitosan oligomers. This method had previously been used to determine peracetylated sulfoglycolipids (Tadano-Aritomi and Ishizuka, 1983) and heparin concentration in biological samples (Gundry et al.,1984)
Fig. 6. Zeta potential of LMWH with or without various chitosan oligomers. Data represents the mean S.E., n = 12. * P < 0.05, ** P < 0.01, compared with control group.
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Fig. 7. Absorbance of azure A in the presence of various chitosan oligomers at 595 nm (A) and percentage of free LMWH with various chitosan oligomers (B). Data represents the mean S.E., n 3.
and to study the interactions between neutrophil elastase and heparin (Cadene et al., 1995). Azure A, a positively charged phenothiazine dye, gives a strong absorbance at 595 nm, but its absorbance will decrease upon binding with negatively charged sulfate groups of the heparin due to the formation of purple colored complex, which produces a negative shift in lmax. Therefore, the azure A assay can be used to quantitatively measure the heparin or LMWH on the base of the linear relationship of the absorption of azure A at 595 nm and the concentration of LMWH. As is evident in Fig. 7A that there was no significant change in the absorbance of azure A at 595 nm when the different type and varying concentration of chitosan oligomers were added to the azure A solutions. This result can be interpreted that chitosan oligomers do not possess sulfate group binding with the azure A, therefore no obvious difference in the absorbance of azure A was observed. The amount of free LMWH in each LMWH solution with or without chitosan oligomers was determined with the same method and the data are plotted in Fig. 7B. We found that amount of free LMWH in LMWH solution was in a sense dependent on the type of chitosan oligomers and the percentage of free LMWH in the LMWH solution containing chitosan hexamer was a little higher than that containing chitosan tetramer and dimer, indicating that the electrostatic interaction between chitosan hexamer and LMWH was larger than that between chitosan tetramer or dimer and LMWH. This tendency was well consistent with the result of absorption enhancing studies on chitosan oligomers, which demonstrated clearly that absorption enhancing potential of chitosan hexamer for improving intestinal absorption of LMWH was greater than that of chitosan tetramer and dimer (Fig. 2). The reason for the superior performance of chitosan hexamer in comparison to chitosan tetramer and dimer was not fully understood. However, it may be plausible that amino group contents of chitosan oligomers with different types perhaps influence drug absorption from intestinal epithelium. It is well known that the surface of mucus membrane is generally negative due to the existence of sialic acid and consequently the coulombic repulsion effect will be generated between the negatively charged cell membranes and negative LMWH, which probably contributes in part to the poor oral bioavailability of LMWH. Therefore, combination of negative LMWH with positive charged chitosan oligomers, especially chitosan hexamer which has relative more cationic charges (Fig. 6) and stronger electrostatic interaction with LMWH (Fig. 7B) due to the larger numbers of amino groups in its structure compared to chitosan tetramer and dimer (Fig. 1), may attenuate to a larger extent above coulombic repulsion effect, thereby triggering the more efficient absorption enhancing effect on the intestinal absorption of LMWH. Similar results were also observed in the case of PAMAM dendrimers as absorption enhancers on pulmonary absorption, which reported the good liner correlation between the zeta potential caused by amino group of PAMAM dendrimers and their pulmonary absorption enhancing
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effects (Dong et al., 2010). Therefore, the content of amino group in the molecules of chitosan oligomers might be one important factor for the absorption enhancing effects of chitosan oligomers although further studies are required to elucidate it in the future. In addition, reduction degree in the amount of free LMWH varied with the concentration of chitosan oligomers and the largest reducing amount of free LMWH was obtained at the concentration of 0.5% (w/v) chitosan hexamer (Fig. 7B). Similar results were also reported that the level of free LMWH (enoxaparin) linearly decreased upon addition of positively charged PEI (Yang et al., 2006), PAMAM dendrimers (Bai et al., 2006) or poly-L-argine (Rawat et al., 2007) to LMWH solution. Therefore, these data are consistent with the data on zeta potential and further verified the existence of electrostatic interactions proposed between the positively charged chitosan oligomers and the negatively charged LMWH. 4. Conclusion In conclusion, chitosan oligomers, especially chitosan hexamer, could significantly improve the intestinal absorption of negatively charged LMWH most likely via electrostatic interactions and meanwhile did not induce obvious damage to the intestinal membranes, suggesting that chitosan oligomers would be a promising absorption enhancer with high efficiency and low toxicity, therefore could be applied hopefully to increase the oral bioavailability of various LMWH formulations. Acknowledgments The work was supported by grants from the National Natural Science Foundation of China (81102382) and the Fundamental Research Funds for the Central Universities (to Yang Gao and Hailong Zhang). References Asada, H., Douen, T., Waki, M., Adachi, S., T, Muranishi, S., 1995. Absorption characteristics of chemically modified-insulin derivatives with various fatty acids in the small and large intestine. Journal of Pharmaceutical Sciences 84, 682–687. Bai, S., Thomas, C., Ahsan, F., 2006. Dendrimers as a carrier for pulmonary delivery of enoxaparin, a low-molecular weight heparin. Journal of Pharmaceutical Sciences 96, 2090–2106. Bianchini, P., Bergonzini, G.L., Parma, B., Osima, B., 1995. Relationship between plasma antifactor Xa activity and the antithrombotic activity of heparins of different molecular mass. Haemostasis 25, 288–298. Buchhammer, H.-M., Mende, M., Oelmann, M., 2003. Formation of mono-sized polyelectrolyte complex dispersions: effects of polymer structure, concentration and mixing conditions. Colloids and Surfaces A: Physicochemical and Engeering Aspects 218, 151–159. Cadene, M., Boudier, C., de Marcillac, G.D., Bieth, J.G., 1995. Influence of low molecular mass heparin on the kinetics of neutrophil elastase inhibition by mucus proteinase inhibitor. Journal of Biological Chemistry 270, 13204–13209. Dash, P.R., Read, M.L., Barrett, L.B., Wolfert, M.A., Seymour, L.W., 1999. Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery. Gene Therapy 6, 643–650. Dong, Z.Q., Hamid, K.A., Gao, Y., Lin, Y.L., Katsumi, H., Sakane, T., Yamamoto, A., 2010. Polyamidoamine dendrimers can improve the pulmonary absorption of insulin and calcitonin in rats. International Journal of Pharmaceutics 100, 1866–1878. Fetih, G., Habib, F., Okada, N., Fujita, T., Attia, M., Yamamoto, A., 2005. Nitric oxide donors can enhance the intestinal transport and absorption of insulin and [Asu1,7]-eel calcitonin in rats. Journal of Controlled Release 106, 287–297. Gao, Y., He, L., Katsumi, H., Sakane, T., Fujita, T., Yamamoto, A., 2008. Improvement of intestinal absorption of insulin and water-soluble macromolecular compounds by chitosan oligomers in rats. International Journal of Pharmaceutics 358, 70–78. Grabovac, V., Bernkop-Schnürch, A., 2006. Improvement of the intestinal membrane permeability of low molecular weight heparin by complexation with stem bromelain. International Journal of Pharmaceutics 326, 153–159. Gundry, S.R., Klein, M.D., Drongowski, R.A., Kirsh, M.M., 1984. Clinical evaluation of a new rapid heparin assay using the dye azure A. American Journal of Surgery 148, 191–194.
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