Pharmaceutical Nanotechnology
Improvement of the intestinal membrane permeability of low molecular weight heparin by complexation with stem bromelain

https://doi.org/10.1016/j.ijpharm.2006.06.042Get rights and content

Abstract

The aim of this study was to investigate the influence of the proteolytic enzyme bromelain on the permeation of heparin across the gastrointestinal epithelial barrier. Stability of the complex and effect of heparin on the enzymatic activity of bromelain was analysed photometrically by measuring bromelain enzymatic activity in complex with the heparin. In vitro permeation studies were performed with Caco-2 cell monolayer and rat small intestinal mucosa in Ussing-type chambers, respectively. Results revealed that enzymatic activity of bromelain remained uninfluenced by the immobilization of heparin on it. Transport studies across Caco-2 cell monolayer and rat small intestine showed that the permeation of heparin could be significantly increased in presence of bromelain. In the study with Caco-2 cells, the most effective molar ratio of bromelain to heparin was 2:1, leading to 6.7-fold improvement in uptake, whereas the molar ratio 1:1 showed the highest permeation enhancing effect in the study on intestinal mucosa. This study provides evidence that heparin and bromelain form stable complexes leading to a significantly improved uptake of heparin.

Introduction

Heparin is a water soluble, negatively charged polysaccharide which has been successfully used in the therapy of myocardial infarction, pulmonary embolism and deep vein thrombosis. The major disadvantage of heparin is its poor bioavailability when administered orally due to its inability to pass the gastrointestinal epithelial barrier. The epithelial cells lining of the gastrointestinal tract represent major barrier to absorption of many orally administered drugs (Jackson, 2005). For that reason heparin has been regularly dosed parenterally which is less convenient way of dosing for patients. For many years, researches have been attempting to develop an effective heparin formulation capable of passing the gastrointestinal barrier. Gastrointestinal barrier is, among others, represented by mucous gel layer covering epithelial cells and extracellular matrix. On the one hand, mucous gel layer comprises three-dimensional network of glycoproteins. On the other hand extracellular matrix comprises tight junctions, whose function is regulated by different transmembrane and intracellular proteins (Ho et al., 2000, Ho et al., 2004).

Recently, different compounds have been reported to enhance the intestinal permeation of heparin, including organic bases (Caramazza et al., 1991, DalPozzo et al., 1989), spermine and lysine salts (Morton et al., 1981) or [N-8(-2-hydroxy-benzoyl)amino]caprylate (SNAC) which led in combination with heparin to a significant increase of the intestinal absorption of heparin in rats (Leone-Bay et al., 1998). All these approaches are based on the improvement of the lipophilic properties of the heparin delivery system diminishing at the same time its hydrophilic properties and favoring transport through the lipid bilayer. Proteolytic enzymes are, in contrary, capable of degrading the extracellular matrix components (Boudjennah and Pagano, 1998) such as proteins of tight junctions. They also exhibit a strong mucolytic activity by cleaving within the amino acid sequence of mucus glycoproteins (Bernkop-Schnürch et al., 2004). In previous study by our research group bromelain could be identified as permeation enhancer for low molecular size compounds like sodium-fluoresce and fluoroisothiocyanate-dextran across rat small intestine (Guggi and Bernkop-Schnürch, 2005). Bromelain is a general term for enzymes derived from fruit, stem and leaves of Ananas comosus. Its main components are cysteine proteases and smaller amounts of other enzymes such as amylase, acid phosphatase, peroxidase, and cellulase (Rowan, 1990). Within this study stem protease has been chosen, representing a basic glycoprotein with a molecular weight of 28 kDa.

The aim of the present study was to improve the membrane permeability properties of negatively charged heparin by complexing it with the positively charged proteolytic enzyme bromelain, as illustrated in Fig. 1. Studies with heparin–bromelain complexes were performed via spectrophotometry. The effect of bromelain on the permeation of heparin was investigated with human intestinal epithelium in cell culture and with freshly excised rat small intestine.

Section snippets

Materials

Low molecular weight heparin (LMWH, 105 IU/mg, average MW 4.125 kDa) was purchased from ICN Biomedicals; bromelain (EC 3.4.22.32; 3.14 units/mg protein), heparin-acrylic beads saline suspension, Nα-CBZ-l-lysine p-nitrophenyl ester and HEPES [N-(2-hydroxyethyl)piperizine-N-(2-ethanesulfonic acid)] were purchased from Sigma, Vienna, Austria.

Photometric analysis

Bromelain–heparin binding studies are based on the decrease in bromelain concentration in solution due to the addition of heparin being immobilized to acrylic

Photometric analysis

After being incubated with bromelain solution for 1 h, heparin acrylic beads were removed from the solution by centrifugation. The amount of bromelain bound to 940 mg of heparin-acrylic beads in sodium acetate buffer at pH 5.5 was plotted against the equilibrium concentration (Fig. 2). When bromelain concentration was below the binding capacity of the heparin acrylic beads a linear relationship was found. The average percentage of bromelain bound to heparin acrylic beads amounted 97.7 ± 1.2%.

Influence of heparin on bromelain activity at different pHs

An

Discussion

The results of this study provide the proof of concept that permeation of heparin through the intestinal membrane can be enhanced by its complexation with proteolytic enzyme bromelain. According to Capila and Linhardt (2002) interactions of heparin with proteins are mainly based on ionic interactions. Clusters of positively charged basic amino acids on proteins form ion pairs with spatially defined negatively charged sulfo- and carboxyl groups on the heparin chain. Further on, hydrogen bonds

Conclusion

Within this study, it was shown that heparin complexates with bromelain without influencing its enzymatic activity of bromelain. The stability of the complex at different pH levels was confirmed. Further on, it was demonstrated that bromelain increases the paracellular transport of heparin across both Caco-2 monolayer and rat small intestinal mucosa. Since bromelain exerts a mild and reversible effect on tight junctions, using bromelain as permeation enhancer could represent a promising novel

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