Pharmaceutical Nanotechnology
In vivo evaluation of an oral self-microemulsifying drug delivery system (SMEDDS) for leuprorelin

https://doi.org/10.1016/j.ijpharm.2014.05.047Get rights and content

Abstract

The objective of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) for the model peptide drug leuprorelin to prove a protective effect against luminal enzymatic metabolism. In order to incorporate leuprorelin into microemulsion droplets (o/w), the commercially available hydrophilic leuprolide acetate was modified by hydrophobic ion paring with sodium oleate. The obtained hydrophobic leuprolide oleate was dissolved in the SMEDDS formulation (30% (m/m) Cremophor EL, 30% (m/m) Capmul MCM, 10% (m/m) propylene glycol and 30% (m/m) Captex 355) in a concentration of 4 mg/g showing a mean droplet size of 50.1 nm when dispersed in a concentration of 1% (m/v) in phosphate buffer pH 6.8. The microemulsion was able to shield leuprolide oleate from enzymatic degradation by trypsin and α-chymotrypsin, so that after 120 min 52.9% and 58.4%, respectively, of leuprolide oleate were still intact. Leuprolide acetate dissolved in an aqueous control solution was completely metabolized by trypsin within 60 min and by α-chymotrypsin within 5 min. Moreover, an in vivo study in rats showed a 17.2-fold improved oral bioavailability of leuprolide oleate SMEDDS compared to a leuprolide acetate control solution. This is the first time, to our knowledge, that hydrophobic ion pairing is utilized in order to incorporate a peptide drug in SMEDDS and evidence of a protective effect of oil-in-water (o/w) microemulsion droplets against enzymatic degradation of a peptide drug was provided. According to these results, the system could be likely a novel platform technology to improve the oral bioavailability of peptide drugs.

Introduction

The oral administration of peptide and protein drugs remains a significant challenge for pharmaceutical researcher due to several physiological barriers limiting gastrointestinal absorption. The rapid degradation by luminal enzymes is one of the main problems that needs to be overcome to enhance systemic uptake (Guo et al., 2004). An increasingly popular approach to improve the bioavailability of peptide drugs via the oral route are self-microemulsifying drug delivery systems (SMEDDS).

SMEDDS are isotropic mixtures of oil(s), one or more surfactants and a co-surfactant (or co-solubilizer) (Gursoy and Benita, 2004). Dispersion of these mixtures in an aqueous environment leads to transparent or slightly bluish, thermodynamically stable oil-in-water (o/w) microemulsions with a droplet size range from 10 to 300 nm (Sarciaux et al., 1995, Gursoy and Benita, 2004, Anton and Vandamme, 2011). As just a gentle agitation is required to emulsify these formulations, the digestive motility of stomach and intestine is sufficient after oral application (Charman et al., 1992, Shah et al., 1994, Constantinides, 1995). Among many factors contributing to the improved oral bioavailability are the large surface area, permeation enhancement and protection against luminal enzymatic degradation. Although the latter point is often mentioned in the literature, to our knowledge, it is not yet demonstrated. In addition, it was rarely successful to incorporate peptides into o/w microemulsion droplets due to their generally hydrophilic nature.

Leuprorelin (leuprolide acetate) is a synthetic gonadotropin-releasing hormone (GnRH) analogue used in the treatment of sex hormone-related disorders such as advanced prostate cancer, endometriosis and precocious puberty (Plosker and Brogden, 1994, Kutscher et al., 1997). The highly water-soluble nonapeptide has two ionizable basic side chains, imidazole group of histidine (pKa  6.0) and guanidine group of arginine (pKa  13.0) (Choi and Park, 2000). As most peptide drugs, its bioavailability is low, and it is usually given intramuscularly as depot injection (e.g., Lupron®). The Transport studies already showed that inhibition of proteolytic enzymes could improve the intestinal absorption of leuprorelin (Guo et al., 2004).

Therefore, it was the aim of the study to prove a protective effect of a SMEDDS for the model peptide drug leuprorelin against metabolism by intestinal enzymes. In order to incorporate leuprorelin into the lipophilic core of the SMEDDS droplets, the commercially available hydrophilic leuprolide acetate was modified by hydrophobic ion paring with sodium oleate to obtain the hydrophobic leuprolide oleate. As the peptide drugs in general show poor permeability across intestinal membranes, a formulation with permeation enhancing properties seems appropriate for the preparation of leuprorelin SMEDDS. Therefore, leuprolide oleate SMEDDS were prepared employing a formulation with a permeation enhancing effect. Increased permeation for the hydrophilic macromolecular compound fluorescein isothiocyanate–dextran 4 (FD4) via tight junction opening was shown in a previous study (Hintzen et al., 2013); as leuprolide acetate is also mainly absorbed by the paracellular route (Guo et al., 2004) this formulation seems suitable.

Section snippets

Materials

Capmul MCM (mono/diglycerides of caprylic acid, HLB = 5–6) and Captex 355 (caprylic/capric triglyceride) was supplied by Abitec Corporation, USA. Cremophor EL (non-ionic emulsifier obtained by causing ethylene oxide to react with castor oil in a molar ratio of 35 to 1, HLB = 12–14) was purchased from BASF, Germany. Propylene glycol was obtained from Gatt-Koller, Absam, Austria. Acetonitrile and water for HPLC analysis were purchased from Avantor Performance Materials, Netherlands. Trifluoroacetic

Hydrophobic ion pairing of leuprorelin

The hydrophobic ion pair exchange of leuprolide acetate with sodium oleate led to precipitation in aqueous media. In the following, this precipitate could be separated from the water soluble fraction by centrifugation. The extent of ion pairing was determined by measuring residual leuprolide in the water phase. As shown in Fig. 1, the leuprolide water solubility decreased up to a molar ratio of 3:1 and increased with further increasing the concentration of oleate. Although leuprolide has two

Conclusion

This is the first time, to our knowledge, that hydrophobic ion pairing of a peptide drug was used in order to embed a peptide drug successful into microemulsion droplets. Moreover, the self-microemulsifying drug delivery system for leuprorelin proved that SMEDDS can shield peptides from degradation by intestinal proteases. Furthermore, a sustained release of leuprorelin could be demonstrated that avoids an initial rapid degradation of the drug. These considerations could be confirmed by an in

Acknowledgement

The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007–2013) under grant agreement n° 280761.

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