Polyethylene glycol-coated liposomes for oral delivery of recombinant human epidermal growth factor

Abstract

The present study was to investigate the feasibility of oral delivery of recombinant human epidermal growth factor (rhEGF). Polyethylene glycol (PEG)-coated liposomes containing rhEGF was prepared and evaluated for their stability and permeability in Caco-2 cells. In the animal study, we also determined plasma concentration and gastric ulcer healing effect after oral administration of rhEGF liposomes or the solution. Encapsulation of rhEGF into liposomes, suppressed the degradation in Caco-2 cell homogenate compared with the solution. The flux of rhEGF from dipalmitoylphosphatidylcholine (DPPC) liposome across Caco-2 cell monolayer from the apical to basolateral side was three times greater than that from phosphatidylcholine (PC) liposome or the solution. After oral administration of rhEGF liposomes or the solution in rats, the area under the concentration–time curve (AUC) of rhEGF increased 1.7- and 2.5-fold for PC and DPPC liposomes, respectively. The gastric ulcer healing effect was significantly increased in DPPC liposome compared with PC liposome and the solution. The enhanced curative ratio of rhEGF encapsulated into DPPC liposome may be due to the resistance to enzyme degradation, higher permeability and increased plasma AUC. Therefore, PEG-coated liposomes containing rhEGF could be used as an oral delivery formulation with enhanced encapsulation efficiency.

Introduction

Human epidermal growth factor (rhEGF) is a single-chain polypeptide containing 53 amino acid residues (MW=6045) and three disulfide bridges (Senderoff et al., 1994). rhEGF stimulates the proliferation and differentiation of epithelial tissues, such as the intestinal mucosa, corneal epithelial tissue, lung and trachea epithelial (Carpenter and Cohen, 1979). Moreover, rhEGF also inhibits gastric acid secretion (Bower et al., 1975, Elder et al., 1975, Gregory, 1975, Konturek et al., 1981b) and protect gastroduodenal mucosa against tissue injury induced by ulcergenic agents (Konturek et al., 1981a, Kirkegaard et al., 1983). We have reported that oral bioadhesive gels containing rhEGF was effective against induced acute and chronic gastric ulcers in rat, but the healing efficacy was not complete (Han et al., 1998b). When rhEGF was orally administered, the bioavailability is very low (1–5%) because of its liability to enzyme and poor gastrointestinal membrane transport ability (Han et al., 1998a). Therefore, most of peptides and proteins including rhEGF, can be mostly administered by the injection formulation. In order to enhance their oral bioavailability, many strategies have been tested, such as a co-administration of absorption enhancers which increase the permeability of peptides through the intestinal membrane (Morishita et al., 1993, Hovgaard et al., 1995), a synthesis of their stable derivatives (Bruce and Susanna, 1995) and the development of special dosage forms by utilizing the micro- or nano-particles as a drug carriers (Desai et al., 1996, Jenkins et al., 1994, Takeuchi et al., 2001). However, absorption enhancers showed several safety problems in their clinical use. The bioactivity of peptides might be reduced due to the derivatization process. On the contrary, a drug carrier system has many advantages. First of all, it is possible to control the release rate of peptides from particles after oral administration. Liposomes are one of the most potent candidates for such carrier systems. However, the efficacy of liposomes as an oral delivery system for peptide drugs has not yet been established. Many researchers have investigated the stability of liposomes in the GI tract (Iwanaga et al., 1997, Iwanaga et al., 1999, Moribe et al., 1999). Liposomes coated with polyethylene glycol 2000 or the sugar chain of mucin found to be resistant to their digestion by bile salts and were useful for an oral delivery system for peptide drugs (Iwanaga et al., 1997, Iwanaga et al., 1999). Furthermore, liposomes with polyethylene glycol (PEG)-derivatized lipid have been reported to increase encapsulation efficiency of nystatin (Moribe et al., 1999).

In this study, we prepared PEG-coated liposomes containing rhEGF and evaluated for their stability and the permeation of rhEGF across Caco-2 cells. Furthermore, we also investigated the in vivo absorption and gastric ulcer healing effect after oral administration of rhEGF liposomes to examine the feasibility of PEG-coated liposomes as an oral delivery system of peptides.