Eudragit RS 100 microparticles containing 2-hydroxypropyl-β-cyclodextrin and glutathione: Physicochemical characterization, drug release and transport studies

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Abstract

The aim of this study was to encapsulate glutathione (GSH) alone or in combination with hydroxypropyl-β-cyclodextrin (HP-β-CD) in Eudragit RS 100 microparticles (MPs), and to evaluate these novel delivery systems for oral administration of the considered tripeptide. The MPs were prepared by an O/O emulsion–solvent evaporation method according to a multilevel experimental design involving the volume of liquid paraffin, the HP-β-CD amount, and the drug/polymer ratio as independent variables. The effects of these parameters on particle size, entrapment efficiency, and drug release were investigated.

Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) analysis and differential scanning calorimetry (DSC) studies were performed to evaluate possible interactions between GSH and Eudragit RS 100 polymer and to characterize the physical state of drug within the MPs. The release profiles of GSH from MPs were examined in vitro at pH 1.2, 6.8. and 7.4 using the USP III (BioDis) dissolution apparatus. In general, a slow and zero-order release of GSH from MPs at pH 1.2 occurred, while at higher pH values considerable amounts of glutathione disulfide (i.e., GSSG) were observed. The enzymatic stability and the intestinal permeability of some GSH-containing MPs were assessed by using pepsin, α-chymotrypsin, γ-glutamyl-transpeptidase and everted frog intestinal sac methodology, respectively. The results suggest that GSH-loaded Eudragit RS 100 MPs containing HP-β-CD represent a new sustained GSH delivery system useful for the oral administration of the examined tripeptide.

Introduction

Glutathione (γ-glutamylcysteinylglycine, GSH) is an ubiquitous nucleophilic tripeptide involved in several biological functions such as metabolism, catalysis, and transport. It also has a role in the protection of cells against reactive oxygen species and xenobiotics as a scavenger of free radicals (Meister, 1991). Consistent with this last function, GSH concentrations are high in organs frequently exposed to xenobiotics such as the kidney, liver, lungs and intestine, but are low in plasma and urine (Deleve and Kaplowitz, 1991). GSH also plays an important role in the activation of T lymphocytes. In HIV-positive patients, systemic GSH and cysteine deficiency has been linked to an increase in virus replication (Mihm et al., 1991). GSH depletion has also been observed both in lung and neurological disorders such as acute respiratory and Parkinson's diseases (Rahman and Mc Knee, 2000, Jenner, 1996). It has been suggested that GSH may also play a role in cancer prevention (Flagg et al., 1994). As is the case for the majority of approved peptide and protein therapeutics, the clinical use of GSH is limited only to parenteral administration for approved indications such as the treatment of alcohol and drug poisoning and protection against toxicity induced by cytotoxic chemotherapy, radiation trauma, or AIDS-associated cachexia (Deleve and Kaplowitz, 1991, Shigesawa et al., 1992, Townsend et al., 2003). Rapid enzymatic degradation by γ-glutamyl-transpeptidases and γ-glutamyl-cyclotransferases, however, brings the drug plasma concentration to negligible levels within a few minutes of intravenous administration of high doses of GSH. Therefore, the development of dosage forms enabling GSH administration by alternative routes (such as oral or pulmonary) may provide increased clinical value to GSH in terms of bioavailability, patient compliance and duration of therapeutic effect. In order to be administered by the oral route, peptides (and proteins) need to be protected. As with other peptides, GSH can suffer both chemical and enzymatic hydrolysis by exopeptidases during gastrointestinal transit following oral administration, leading to the corresponding free aminoacids. The thiol group of GSH can be oxidized both enzymatically and non-enzymatically at pH values greater than 7 (Camera and Picardo, 2002) giving rise to the corresponding glutathione disulfide (GSSG), which is devoid of radical scavenging activity. Microencapsulation of a small peptide such as GSH in a polymeric system able to protect against chemical and/or enzymatic degradation, however, may enable the retention of therapeutic properties following oral administration.

The aim of this study was to encapsulate GSH alone or together with hydroxypropyl-β-cyclodextrin (HP-β-CD) in Eudragit RS 100 microparticles (MPs), and to evaluate these novel GSH delivery systems for oral administration. In this regard, it should be noted that (to the best of our knowledge) only one report on GSH encapsulation in poly(isobutylcyanoacrilate) nanoparticles has been published by Couvreur and coworkers (Gate et al., 2001). Eudragit RS 100 is a poly(ethyl acrylate, methyl-methacrylate, and chlorotrimethyl-ammoniumethylmethacrilate) co-polymer. It is insoluble at physiological pH but undergoes swelling in water (Pignatello et al., 2002). Eudragit RS 100 is commonly used for the enteric coating of tablets and the preparation of controlled-release drug forms, and represents a good material for the dispersion of drugs. Cyclodextrins (CDs) have been widely employed in pharmaceutical field to increase the solubility of poorly water-soluble drugs and to improve their dissolution rate. Due to their cone-shaped structure, these oligosaccharides can form inclusion complexes entrapping (either partially or entirely) a variety of drugs. CD complexation of some drugs (including peptides) increases their water solubility, chemical stability and absorption. Indeed, recent results from the literature highlight the enhancement of drug oral bioavailability by hydrophilic CDs through the inhibition of P-glycoprotein activity (Haeberlin et al., 1996, Irie and Uekama, 1999, Arima et al., 2004). In this context, it is interesting to note that GSH is able to act itself as a permeation enhancer, very probably by inhibition of protein tyrosine phosphatase which results in the opening of the tight junctions (Kafedjiiski et al., 2005). As for the ability of CDs to complex with biological molecules, it is well recognized that most peptide or protein compounds are hydrophilic, and only those with hydrophobic side chains can interact with CD molecules. In the case of GSH, there is a thiol group that might be incorporated in the CD cavity, as observed for the –SH group of the angiotensin-converting enzyme inhibitor captopril with α-CD (Ikeda et al., 2002). If this is the case, the oxidative degradation of GSH leading to the corresponding disulfide GSSG would be markedly suppressed. Recently, CDs have been incorporated into drug delivery systems such as microspheres, nanospheres and polymeric films, modifying both drug release and drug loading. It is now clear that incorporation of drug/CD mixtures or complexes into polymeric matrices (including swellable ones, Quaglia et al., 2001) can improve hydration of the matrix and modify drug solubility and diffusivity, leading to an enhanced or retarded drug release from the polymeric system.

In this study, to avoid an unacceptably high HP-β-CD dose, MPs were prepared by using GSH and oligosaccharide in different weight ratio (1/0–1/3), and their physicochemical properties (such as particle size, drug entrapment efficiency, and drug release kinetics) were investigated. Furthermore, we attempted to identify which factors influence these properties. For this purpose, a screening experimental design was used to plan and perform the experiments. Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) analysis, and differential scanning calorimetry (DSC) studies were also performed to characterize the physical state of drug within the MPs. Finally, the physical and enzymatic stability, as well as the intestinal permeability, of some GSH-containing MPs was assessed.

Section snippets

Materials

Reduced GSH, glutathione disulfide (GSSG), Span 80, perchloric acid (70%), Pepsin A (from porcine stomach mucosa), α-Chymotrypsin (TLCK treated from bovine pancreas Type VII), and γ-glutamyl-transpeptidase (from equine kidney Type VI, 11 U/mg) were purchased from Sigma–Aldrich (Milan, Italy). Eudragit RS 100 (Röhm GmbH & Co.) was kindly donated from Rofarma (Gaggiano, Milan, Italy). Liquid paraffin (density measured at 20 °C 0.840 kg/dm3; viscosity measured at 20 °C 31.31 cPs) and Magnesium Stearate

Results and discussion

The development of oral GSH sustained delivery systems may provide an increased clinical value over the currently used injectable dosage forms. The main obstacles to oral administration are the very efficient barriers of the gastrointestinal tract and the risk of oxidation of GSH to GSSG. In this work, we decided to encapsulate GSH alone or dispersed with HP-β-CD in MPs based on Eudragit RS 100. This insoluble (but swellable in water) polymer may protect the GSH from proteolytic degradation and

Conclusions

In this work, GSH was successfully encapsulated in Eudragit RS-100-based MPs containing HP-β-CD by an O/O emulsification solvent evaporation method. Formulations were planned and performed according to an experimental design and were characterized by encapsulation efficiency, size, morphology and in vitro release profiles. It was found that the optimal parameters for GSH delivery from MPs are both a high CD level and a low drug/polymer ratio. A combination of these parameters gives rise to

Acknowledgements

Thanks are due to Roquette Italia (Cassano Spinola, Italy) for the kind gifts of HP-β-CD. This work was supported by a grant from Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR).

References (22)

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