Research paper
A liposomal formulation for the oral application of the investigational hepatitis B drug Myrcludex B

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Abstract

The aim of this study was the development of a liposomal formulation containing specific tetraether lipids for the oral administration of the investigational hepatitis B peptide drug Myrcludex B. For this purpose, tetraether lipids were extracted from the extremophilic archaeon Sulfolobus acidocaldarius and purified in order to obtain the desired glycerylcaldityltetraether lipids (GCTE). Myrcludex B was synthesized by solid-phase synthesis and incorporated into liposomes containing 5 mol% of GCTE. These liposomes showed a size, polydispersity index and zeta potential comparable to the standard liposomes. Cryo-EM micrographs of both liposomal formulations displayed low lamellarity, the prerequisite for high drug loading capacity. Long term storage of the GCTE-liposomes was achieved by freeze-drying using 100–500 mM sucrose or trehalose as lyoprotectors. The lyophilized product showed high stability with a recovery rate of 82.7 ± 1.6% of intact Myrcludex B observed after storage for 3 months at −20 °C as compared to a recovery rate of 83.3 ± 1.3% directly after the freeze-drying process. In vivo, the GCTE-liposomal formulation led to substantial enhancement of the liver uptake of iodine-131-labeled Myrcludex B in Wistar rats. 3 h after oral application, approximately 7% of the initial dose (corresponding to a 3.5-fold increase compared to the free peptide) could be detected in the liver. In summary, the GCTE-liposomes enabled efficient oral administration of Myrcludex B and provided long term storage by freeze-drying.

Introduction

Oral drug delivery is the most advantageous way of drug application [1], in particular for the treatment of chronic diseases such as hepatitis B, which demands long-term drug administration [2]. Despite the achievements of modern medicine, annually up to one million people die due to hepatitis infections and their detrimental consequences [3], [4]. Until now, targeted therapy of chronic HBV infections is limited to viral polymerase inhibitors [5]. This illustrates the importance for adequate hepatitis B therapeutics. Previous findings showed that Myrcludex B, a virus entry inhibitor which specifically accumulates in the liver, can block HBV entry in vitro and in vivo [6], [7]. Myrcludex B is a linear myristoylated peptide consisting of 47 amino acids corresponding to the amino acids 2–48 of the hepatitis B virus (HBV) preS surface protein [8]. Although already proceeded into phase II clinical trials, the application of Myrcludex B is hampered due to its limitation to subcutaneous application, causing increased medical costs and inconvenience for the patients. This highlights the necessity to establish an oral formulation of Myrcludex B. However, many drugs, particularly peptides (including Myrcludex B) and other macromolecular drugs such as proteins and antibodies, show both, an extremely poor stability under the acidic conditions in the stomach after oral administration and insufficient absorption across the gastrointestinal barrier [9]. To overcome this problem, different approaches to improve the oral availability of macromolecular drugs have been proposed in the past years [10] including solid lipid nanoparticles [11], nano- or micro-emulsions [12], polymeric micelles [13], or surface-modified liposomes [14], [15], [16]. Despite these major efforts, all formulations available so far have not been convincing due to their instability in the gastrointestinal tract or low mucosa penetration. Nevertheless, a significant increase in the oral availability using liposomal formulations can be achieved by the combination of conventional phospholipids (e.g. EPC), cholesterol and the so-called tetraether lipids, specific lipids derived from archaea, e.g. the extremophilic archaeon Sulfolobus acidocaldarius. Several studies showed that these tetraether lipids can improve the liposomal stability in the gastrointestinal tract and also facilitate mucosal penetration [17] (Fig. 1). Stabilization of the liposomal membrane by the addition of tetraether lipids in order to provide a controlled release system was also tested by Mahmoud et al. [18] who examined chlorin e6 based liposomes for photodynamic therapy. Two further studies found a significant increase in the oral availability of macromolecular drugs (octreotide; human growth hormone) using tetraether lipid liposomes [17], [19]. Recently, Jensen et al. [20] could show that liposomes containing lipids from Sulfolobus islandicus withstand intestinal bile salts.

To overcome the obstacles described, a liposomal formulation containing particular tetraether lipids, the so-called glycerylcaldityltetraether lipids, for improved oral administration of the HBV inhibitor Myrcludex B was established. Furthermore, conventional liposomal formulations show the disadvantage, that long-term stability is not fulfilled [21]. This can be achieved by freeze-drying as shown by Miyajima and van Winden [22], [23]. Based on this work, the Myrcludex B GCTE-liposomes were freeze-dried in the presence of sucrose and trehalose as lyoprotectors at various molar ratios. The integrity of GCTE-liposomes was assessed. Their performance with regard to oral availability of Myrcludex B using various GCTE-concentrations was evaluated in vivo in an animal study with Wistar rats. Furthermore, it was studied whether omeprazole pretreatment, in order to raise the pH in the stomach, would increase the oral availability of Myrcludex B. In addition, the bioenhancer cetylpyridinium chloride (CpCl) was incorporated into the GCTE-liposomes in order to examine whether the oral uptake of Myrcludex B can be increased according to Parmentier et al. [19].

Section snippets

Materials

Lecithin (EPC) was obtained from AppliChem GmbH (Darmstadt, Germany); tetraether lipids were isolated from S. acidocaldarius (DSM No. 639; ATCC No. 33909) as described in 2.2; glass beads (0.75–1.0 mm) were purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany); NAP™-5 columns were obtained from GE Healthcare (Buckinghamshire, UK); Antra MUPS® (omeprazole) was purchased from Astra Zeneca GmbH (Wedel, Germany); silica gel 60 (0.063–0.200 mm) was obtained from Merck (Darmstadt, Germany);

Isolation of tetraether lipids

The isolation procedure yielded the purified tetraether lipids with only slight variations in the number of pentyl rings (3–5) in the lipophilic chains as determined by mass spectrometry and thin-layer chromatography in comparison with standards (data not shown). These findings are comparable with the results shown by [17]. The number of pentyl rings is influenced by the temperature during cultivation of the archaea. Approximately 1 g of tetraether lipids could be obtained per 400 g of wet cell

Conclusion

In this project an oral delivery system for the investigational hepatitis B drug Myrcludex B could be established by the use of GCTE-liposomes. The film method with subsequent dual asymmetric centrifugation enabled the fast and reproducible liposomal preparation. The GCTE-liposomes showed high homogeneity in size, PDI and encapsulation efficiency. The long term storage of the liposomes could be achieved by freeze-drying using sucrose as lyoprotector without destroying the incorporated peptide

Acknowledgment

We thank the DFG (Deutsche Forschungsgemeinschaft; Grant No. MI 684/2-1) for financial support; Sonja V. Albers (Albert-Ludwigs-Universität Freiburg) for the kind provision of the S. acidocaldarius cell extract and Roger Sandhoff (German Cancer Research Center) for his expert support in the MS-analysis of the tetraether lipids.

References (33)

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