RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Improved Oral Bioavailability of Human Growth Hormone by a Combination of Liposomes Containing Bio-Enhancers and Tetraether Lipids and Omeprazole 2 ¨ ¨ JOHANNES PARMENTIER,1 GOTZ HOFHAUS,2 SILKE THOMAS,3 LAURA CLUSA CUESTA,1 FELIX GROPP,4 RASMUS SCHRODER, 3 1 KLAUS HARTMANN, GERT FRICKER 1

Institute of Pharmacy and Molecular Biotechnology, Department of Pharmaceutical Technology and Biopharmacy, University of Heidelberg, Heidelberg, Germany 2 Cellnetworks, University of Heidelberg, Heidelberg, Germany 3 ¨ p¨adiatrische Endokrinologie and Diabetologie, Frankfurt am Main, Germany Praxis fur 4 Bernina Plus GmbH, Planegg, Germany Received 14 May 2014; revised 23 September 2014; accepted 23 September 2014 Published online 20 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24215

ABSTRACT: Liposomes for the oral delivery of human growth hormone (hGH) containing bio-enhancers and tetraether lipids were prepared by dual asymmetric centrifugation. Cetylpyridinium chloride (CpCl), D-␣-tocopheryl polyethylene glycol 400 succinate, phenylpiperazine, sodium caprate or octadecanethiol were used as permeation enhancers. In vitro data showed that oligolamellar vesicles with average size in the range of 200–250 nm were formed. Performance of the formulations was investigated both ex vivo by confocal microscopy scans of sections of rat small intestine and in vivo by comparing the area under the plasma curve of hGH after oral or subcutaneous (s.c.) application. The microscopic data reveal an interaction between the liposomal formulation and the intestinal mucus layer. Particularly one formulation, which was designed to be mucus penetrative by addition of a high quantity of TPGS 400 and a ␨-potential close to 0 mV, showed a very strong mucus association in the duodenum and jejunum. Vesicles with CpCl 33% (mol/mol) led to a relative hGH bioavailability of 3.4% C 2014 Wiley Periodicals, Inc. compared with s.c. control, whereas free hGH administered orally showed a bioavailability of only 0.01%.  and the American Pharmacists Association J Pharm Sci 103:3985–3993, 2014 Keywords: dual asymmetric centrifugation; lipogelosomes; archae; imaging methods; oral absorption; proteins; liposomes; permeation enhancers

INTRODUCTION Oral peptide and protein delivery remains one of the major tasks in drug delivery, despite the considerable amount of attention paid to this field over the last years.1,2 On the contrary, because of recent advances in biotechnology, the number of available protein drugs is constantly increasing and therapeutic use becomes more cost-effective.3 For long and repeated use, oral application is by far the most convenient route for drug delivery. However, proteins, when applied orally, are easily degraded because of low-stomach pH and digestive enzymes, and their size and hydrophilicity lead to a poor intestinal absorption.4,5 Absorption enhancers, such as small molecule carriers, surfactants and enzyme inhibitors, but also particulate systems, for instance, nanoparticles and liposomes, were used to stabilise proteins in the gastrointestinal tract (GIT) and to improve permeation through the intestinal epithelium.4,6–11 Liposomes represent a highly versatile delivery system in terms of composition, size and production method with a good biocompatibility and were already investigated for the oral delivery of peptide drugs.12–14 Nevertheless, they have some principal drawbacks as oral delivery system for macromolecules such as proteins. Liposomes exhibit a limited stability in the

Correspondence to: Gert Fricker (Telephone: +49-6221-548330; Fax: +496221-545971; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 103, 3985–3993 (2014)

 C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

GIT and have to be stabilised, for example by the use of phospholipids with high phase transition,15 membrane-spanning lipids16 or polymer coating.17 Moreover, conventionally prepared liposomes usually show rather low encapsulation efficiencies for hydrophilic drugs.12,18 This can be overcome by the use of vesicular phospholipid gels (VPGs). These are semi-solid, aqueous phospholipid dispersions that exhibit high drug loading for hydrophilic molecules independent of their charge or size.19,20 These VPGs can be either prepared by high-pressure homogenisation or by dual asymmetric centrifugation (DAC), a technique recently introduced for VPG preparation by Massing et al.21,22 Recently, we showed that liposomes containing bioenhancers can improve the permeation of a macromolecule in vitro 23 and that the tetraether lipid glycerylcaldityl tetraether (GCTE) can stabilise liposomes containing bio-enhancers in simulated gastrointestinal fluids.24 One goal of the present study was to test the suitability of the DAC technology for the preparation of liposomes containing GCTE and different bio-enhancers and to characterise their physical properties. In addition, the fate of fluorescently labelled liposomes containing bio-enhancers and GCTE in the rat intestine was investigated ex vivo by confocal microscopy to gain a better understanding of the correlation between bioavailability improvement and liposome interaction with the mucus barrier and mucosa. The suitability of this type of liposomes for oral protein delivery was examined in rats with human growth hormone (hGH), a 191 amino acids protein with a molecular weight of

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approximately 22 kDa, as model drug. In paediatric use, hGH has to be administered daily over a long time period, which makes an oral delivery form desirable, particularly regarding an application to younger children.25 Although we could show in a previous study that liposomes stabilised with GCTE do not exhibit leakage of macromolecules at pH 2, experimental results suggest that small, charged molecules such as protons can permeate into the aqueous core of the liposomes,24 which could lead to the denaturation of encapsulated hGH. Thus, to overcome this stability issue, animals were pre-treated before the administration of liposomal encapsulated hGH either with the proton-pump-inhibitor omeprazole to raise stomach pH or liposomes were further stabilized by incorporating 15% gelatine in the vesicle core or the VPGs were freeze-dried and encapsulated in enteric-coated capsules.

MATERIAL AND METHODS Materials EPC was gifted from Lipoid (Ludwigshafen, Germany). GCTE was provided from Bernina Plus (Planegg, Germany). D-"-Tocopheryl polyethylene glycol 400 succinate (TPGS 400) was supplied by Eastman (Kingsport, Tennessee). Cetylpyridinium chloride (CpCl) was purchased from Roth (Karlsruhe, Germany). Cholesterol (Chol), sodium caprate (Capr), FITC-dextran (70 kDa), octadecanethiol (OT) and 1phenylpiperazine (PP) were purchased from Sigma–Aldrich (Taufkirchen, Germany). hGH (Genotropin ) was obtained from Pfizer Pharma (Berlin, Germany) and omeprazole from 1A Pharma (Oberhaching, Germany). Rhodamine–dipalmitoyl phosphatidylethanolamine (Rh-DPPE) was purchased from Otto Nordwald (Hamburg, Germany). LB gelatine was provided by Gelita AG (Eberbach, Germany). All other chemicals were obtained in the highest purity from the usual commercial sources. R

Liposome Preparation For both the ex vivo and the in vivo study, liposomes were prepared in a similar way by DAC according to a method previously described.21 The lipid composition of all formulations for the bioavailability study is listed in Table 1. In case of the microscopic study, the same compositions were used with only a small amount (0.25%) of the fluorescent lipid Rh-DPPE added to the formulation without changing the ratio of the other lipids. Lipid mixtures were prepared beforehand in excess. Therefore, a total amount of 1 g of the different lipids and enhancers were weighed in the exact ratio needed for the different formulations into a round-bottom flask. Approximately 20 mL of a chloroform–methanol (9:1) mixture was added to dissolve all substances. Subsequently, the solvent was removed in a ro¨ tary evaporator (Rotavapor-R; Buchi Labortechnik AG, Flawil, Table 1. Ratio (mol/mol) of the Different Lipids and Bio-Enhancers in the Tested Liposomal Formulations EPC GCTE Chol TPGS CpCl 1-PP Caprate OT CpCl-pos CpCl-neutr PP/OT PP/Capr

4 4 4 4

1 2 1 1

2.4 4 3.6 2

4 1.2 4

3.6 3

3 5 5

1.2 5

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Switzerland) and the films were kept for an additional 12 h under high vacuum to remove all solvent traces. For confocal microscopy, 25 mg of lipids mixed in the desired molar ratio with 0.25% (m/m) Rh-DPPE were weighed into a 2-mL cup together with approximately 80 mg of glass beads (1 mm diameter). 37.5 :L of phosphate-buffered saline (PBS) pH 7.4 (NaCl 137 mM, KCl 2.7 mM, KH2 PO4 1.5 mM, Na2 HPO4 8.1 mM) were added, and the cups were mixed for 30 min at 3540 rpm in a dual asymmetric centrifuge (DAC 150 FVZ; Hauschild, Hamm, Germany) using a special vial holder as described by Hirsch et al.26 Finally, the VPGs were further diluted with 0.5 mL of PBS in a 30 s mixing step. As for the in vivo experiments, larger batches were prepared, and the mixing protocol was slightly modified. hGH was reconstituted with distilled water to a concentration of 80 mg/mL. One-hundred milligram of lipids was weighed into a 2-mL cup; 250 mg of glass beads (1.5 mm diameter) and 150 :L of hGH 80 mg/mL were added. If mentioned, 15% of gelatine (m/V) was added to the hGH solution prior to liposome formation. Subsequently, the cups were mixed as described above, whereas this time the mixing process was stopped every 10 min and the mixer was allowed to cool down for 20 min to avoid overheating of the lipid protein mixture. VPGs were further diluted with PBS to achieve a protein concentration of 16 mg/mL. In one case, the formulation was freeze-dried after mixing. To remove the glass beads, the VPG was centrifuged through an 80-:m polyamide monofil filter (NeoLab Migge Laborbedarf-Vertriebs GmbH, Heidelberg, Germany) fixed on the top of a 2-mL cup at 16.1 rcf for 1 min. Subsequently, the formulation was dried in a Delta 1–20 KD freeze-drier (Christ, Osterode am Harz, Germany) under following conditions: −40◦ C for 6 h (freezing), −30◦ C for 40 h (primary drying) and 15◦ C for 8 h (secondary drying). The dried gel was encapsulated into PCcaps size 9 capsules (Capsugel, Cambridge, UK) and subsequently capsules were enteric coated by dipping in a Eudragit L 100 (Evonik, Darmstadt, Germany) solution in acetone/isopropanol/water followed by a short heating period with a common hairdryer to allow film formation. R

R

Size and ␨-Potential Determination Liposomes were diluted with PBS to an appropriate concentration and Z-average and polydispersity index (PDI) was determined using a Zetasizer 3000 HS (Malvern, Works, UK) in the automatic mode. Values for each formulation were calculated from three main runs consisting of 10 sub-runs. In terms of .-potential, particles were diluted in PBS and values were calculated from 10 measurements. R

Determination of hGH Encapsulation Two-hundred micro-litre of liposome dispersion was given on a Sepharose CL-4B column to separate non-encapsulated hGH. Liposomes were further diluted 1:10 with Triton-X 1% in PBS, as control-uncolumned vesicles were diluted 1:100 with Triton-X 1% in PBS. hGH concentration was determined by HPLC with a Dionex UltiMate 3000 system (Dionex, Idstein, Germany) using an Acclaim 120 C18 5-:m column (4.6 × 250 mm2 ) at 50◦ C and a UV PDA detector. Flow was kept constant during the run at 1 mL/min with 20% water plus 0.05% trifluoroacetic acid (TFA) and 80% acetonitrile plus 0.05% TFA as mobile phase. hGH concentration was determined at 218 nm against a calibration curve. R

R

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Table 2.

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Size, PDI, .-Potential and hGH Encapsulation Efficiency of the Tested Liposomal Formulations After Preparation by DAC Z-Average (nm, Mean ± SEM) PDI (Mean ± SEM) .-Potential (mV, Mean ± SEM) Encapsulation Efficiency (%, Mean ± SEM)

CpCl-pos CpCl–neutr PP/OT PP/Capr

229.7 202.6 240.7 244.2

± ± ± ±

12.8 18.9 61.9 4.3

0.29 0.08 0.11 0.17

± ± ± ±

0.03 0.02 0.05 0.037

Encapsulation (E%) of hGH in the different liposomal formulation has been calculated as follows: E% =

Ccol 100% Cuncol

where Ccol and Cuncol are the concentration of the drug in the liposomes after and before separation, respectively. Cryo-Transmission Electron Microscopy The concentrated liposome suspension was applied to a glow discharged Quantifoil specimen support grid, blotted from one side in a humidified atmosphere for 4 s using a Vitrobot (FEI, Hillsboro, Oregon), and plunged into liquid ethane. Grids were mounted under liquid nitrogen on a Gatan 3500 cold stage ¨ (Gatan, Munchen, Germany). The stage was transferred on a Zeiss 923 (Sesam; Carl Zeiss SMT, Oberkochen, Germany) electron microscope equipped with a field emission gun operated at 200 kV and an in column corrected Omega filter with a slit width of 50 eV. Zero loss images were recorded with a 4k × 4k Tietz camera (Gauting, Germany) at about 10–20 m under-focus at a magnification of 50,000×. Confocal Microscopy Animal studies were performed according to the guidelines of the local authorities using male Wistar rats (body weight 250– 280 g). Animals were maintained under standard diurnal conditions with free access to food and water. Prior to 16 h of the experiment, they were kept without food, but with free access to water and glucose 5% (w/v) solution. Liposomes corresponding to 60 mg lipid per kilogram body weight fluorescently labelled with Rh-DPPE were administered orally by gavage. After 45 min, a solution of FITC-dextran 70 kDa 4% (w/v) in saline solution was given in the rat tail vein, corresponding to 80 mg of FITC-dextran per kilogram body weight, to visualize blood vessels under the microscope. Animals were sacrificed after a further 15 min by cervical dislocation under CO2 narcosis. The small intestine was exercised and rinsed amply with saline solution; subsequently, the intestine was filled with a small amount of sucrose 66% (w/v) in water and the tissue was frozen in isopentane on dry ice. Twenty-five microgram slices were cut in a cryostat at −28◦ C (CM 3050 S; Leica, Bensheim, Germany), and then mounted onto surface-treated glass slides (Superfrost plus; Fisher, Pittsburgh, Pennsylvania) and finally embedded in Aquatex (Merck, Darmstadt, Germany). Confocal laser scanning microscopy was performed by a Leica TCS SP 5 system (Leica Microsystems, Mannheim, Germany) using the corresponding Leica software. To allow a sufficient separation of the two fluorescent dyes, sequential scans were carried out, the first one with 476 nm excitation and 510–545 nm emission wavelength, the second with 561 nm excitation and 650–700 nm emission wavelength. R

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41.0 5.1 − 13.7 − 6.1

± ± ± ±

1.2 0.5 0.42 0.5

31.2 24.0 18.1 13.8

± ± ± ±

0.5 2.1 0.6 1.4

hGH Pharmacokinetic Study Male Wistar rats (body weight 250–280 g) were treated prior to the experiments as described above. Either 0.5 mL of hGH in liposomal dispersion or in solution both corresponding to 8 mg hGH or two capsules containing together 7 mg of hGH were applied to each rat in groups of six. In terms of the dispersed formulations, rats were pre-treated 14 and 2 h before application with 10 mg of omeprazole per rat dispersed in 0.5 mL PBS (pH 9.0) to raise the stomach pH. Before the application of enteric-coated capsules containing the freeze-dried formulation the rats were not pre-treated with omeprazole. As control, 0.3 mg of hGH (16 mg/mL) prepared according to the instruction leaflet provided with the commercial drug were injected in the loose skin of the neck area of six rats. Blood samples were collected after 0.5, 1, 2, 4 and 6 h from the tail vein ¨ into lithium/heparin-coated Microvettes (Sarstedt, Numbrecht, Germany). Plasma was separated by centrifugation at 1000g for 10 min in an Eppendorf centrifuge 5415 C (Eppendorf, Hamburg, Germany) and stored at −80◦ C until further use. Prior to quantification, plasma samples were diluted with Multi Diluent 2 (Siemens Diagnostics, Erlangen, Germany) to achieve the minimal volume needed for testing. hGH amount was determined by a Immulite 2000 device (Siemens Diagnostics) using the corresponding kit from Siemens for hGH quantification. Statistical Analysis The area under the plasma curve (AUC) was estimated using the trapezoidal rule. Bioavailability was determined by the ratio of AUC values after peroral (p.o.) and subcutaneous (s.c.) administration, respectively, following the equation: AUCp.o. doses.c. 100 = %bioavailability. AUCs.c. dosep.o. All values are given as means ± SEM. Treatment and control group were compared by one-way Student’s t-test. Differences were considered significant at *p < 0.05, **p < 0.01 and ***p < 0.001. Plots and statistical analysis were made using the software Prism (GraphPad Software, San Diego, California). R

RESULTS Liposome Properties Composition and properties of the liposomal formulations can be seen in Tables 1 and 2. All vesicles had a Z-average between 200 and 250 nm and a PDI below 0.3. The electron micrographs for CpCl-pos, PP/OT and PP/Capr exhibited a mixture of uni/oligo-lamellar and multi-lamellar vesicles (see Fig. 1). CpClneutr liposomes exhibited a smaller Z-average and seemed to contain a higher amount of uni-lamellar vesicles compared with the other formulations. The vesicles containing only CpCl as Parmentier et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3985–3993, 2014

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Figure 1. Cryo-transmission electron microscopy pictures of (a) CpCl-pos, (b) CpCl-neutr, (c) PP/OT, and (d) PP/Capr liposomal formulations at a magnification of 50,000×.

enhancer had the highest [31.2% (±0.5%)] encapsulation efficiency for hGH. The PP/Capr formulation could only encapsulate around 13.8% (±1.4%) of hGH added during liposome formation. .-Potentials for both CpCl containing formulations were positive, whereas the charge could be reduced in terms of the CpClneutr particles to approximately 5 mV. The other two formulations exhibited a slightly negative .-potential, which was more pronounced in terms of the PP/OT liposomes. Confocal Microscopy To investigate the uptake of liposomes and their interaction with the mucus barrier and mucosa, the phospholipid membrane was labelled with rhodamine and blood vessels were coloured with FITC-dextran 70 kDa to allow a better orientation in the cross-section of the small intestine. Representative overlay pictures of bright field, red and green channel for each formulation and a control can be seen in Figure 2. Blood vessels in the villi and sub-mucosa can be identified in all sections by their green fluorescence. All formulations showed either in duodenum or jejunum an interaction with the mucus layer visible in a red fluorescence. The strongest signal could be seen for the CpCl-neutr formulation, followed by the two other formulations containing TPGS 400 in the lipid bilayer. Pharmacokinetic Study

show that the bioavailability of liposomal encapsulated hGH was comparable to free hGH after administration to untreated rats (see Fig. 5). The freeze-dried liposomal gel encapsulated into enteric-coated hard gelatine capsules led to hGH plasma levels below the detection limit.

DISCUSSION Protein drugs have unfavourable physicochemical properties for oral delivery, for example large hydrodynamic diameter, susceptibility to enzyme degradation and the tendency to undergo denaturation caused by low pH or high ion strength.27 Larger peptides (more than 12 amino acids) are degraded in the stomach within minutes and also exhibit only a very short half-life in intestinal fluids and faecal slurry.28–30 According to Lipinski’s rule of five, low permeation across the intestinal wall can be expected for most proteins, caused by their size and hydrophilicity.31 To overcome these obstacles, proteins can either be modified chemically or can be delivered in a suitable vehicle or both.32 An oral delivery system for proteins should cover following aspects: 1. Protect the protein from denaturation and enzymatic degradation in the stomach and intestine. 2. Improve the permeability of the protein across the intestinal barrier.

Human growth hormone plasma levels were determined after oral application of liposomes and compared with plasma levels after s.c. injection of hGH and oral application of free hGH (see Fig. 3). Independently from the way of application, plasma concentration reached its peak at the first time point of 0.5 h or in terms of the CpCl-pos formulation at 1 h. Plasma levels of the protein administered orally in CpCl-neutr and PP/Capr liposomes and in solution, respectively, were close to the lower detection limit. AUC were calculated after normalisation of the plasma curves to body weight and encapsulation efficiency of the vesicular formulations and AUC obtained after s.c. application of hGH was set as 100% bioavailability (see Fig. 4). CpClpos liposomes displayed with 3.37% the highest and the hGH solution with 0.01% the lowest relative bioavailability after oral application. However, it should be mentioned that rats were only pre-treated with omeprazole in the case of the vesicular hGH and not in case of the hGH solution. The encapsulation in PP/Capr and CpCl-neutr liposomes, respectively, had nearly no effect on the bioavailability. To gain a better understanding of the influence of the stomach passage on the bioavailability of hGH, the most promising formulation (CpCl-pos) was administered either modified or unmodified to rats not pre-treated with omeprazole. The AUC data

Previous studies demonstrated that liposomes can increase in principal the bioavailability of proteins,33,34 but these approaches were never transferred into clinical studies. Further improvement of liposomal formulations to increase their stability and uptake seems desirable. We could recently show that liposomes containing tetraether lipids can improve the oral bioavailability of the peptide drug octreotide.35 Tetraether lipids are known to stabilise phospholipid vesicles against bile salts and pancreatin and are used in liposomes for oral delivery of proteins, but also for vaccine delivery.36–38 Even when liposomes are stable in size and distribution under acidic conditions, they are still susceptible to leakage of smaller molecules.24 Protons and small ions can pass the lipid bilayer through transient pores without disrupting the entire integrity of the liposomes.39 The release of a macromolecule in the stomach from liposomes might be negligible, but diffusion of protons into vesicles can cause denaturation of encapsulated proteins. To overcome this problem, animals were pre-treated with omeprazole to raise the stomach pH. Indeed, hGH orally administered in the same liposomal formulation (CpCl-pos) either with or without omeprazole treatment resulted in a 100-fold different bioavailability. Moreover, the CpCl-pos formulation

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Figure 2. Confocal microscopy scans of duodenum, jejunum and ileum 1 h after application of PBS (control) or different liposomal formulations containing 0.25% (m/m) Rh-DPPE. Blood vessels were coloured with FITC-dextran 70 kDa and appear in green.

did exhibit a relative bioavailability similar to hGH in solution (0.03% vs. 0.01%), when both were administered without omeprazole. This shows that it is not sufficient to only increase the uptake of hGH by the use of bio-enhancers or other means, but that the protein has to be protected against denaturation during the stomach passage. Commonly, an acid-labile drug is easily protected by enteric coating of the final drug form. Liposomes can be transferred by freeze-drying into a powder, but the need for stabilisers and matrix formers leads to a high bulkiness of the final product.40 Unfortunately, because of the small size of capsules applicable to rats, the encapsulation of a sufficient amount of hGH in a conventional freeze-dried li-

posomal dispersion was not possible and only one freeze-fried VPG formulation was tested in the present study that did not show any efficiency in delivering hGH. Size measurements of the dried VPGs after reconstitution in buffer suggest an aggregation and destabilisation of the liposomes because of the freeze-drying procedure (data not shown). In addition, freezedried VPGs have a high density and are not easily reconstituted in the intestinal fluid. Another approach to improve the stability of liposomes in general is the processing of gelatine 15% (m/V), together with the lipids during vesicle preparation. Liposomes filled with a gelled substance in the aqueous compartment were first

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Figure 3. Plasma levels of hGH 0.5, 1, 2, 4 and 6 h after oral application of liposomal encapsulated hGH. Animals administered 0.3 mg/rat of hGH s.c. or 8 mg/rat of hGH orally were used as control groups. (+Omep) or (−Omep) indicates whether animals were pre-treated with omeprazole or not. Values represent means ± SEM with n = 6.

˘ described by Hauton et al.41 Degim et al.42 could improve the glucose-lowering effect of liposomal encapsulated orally administered insulin around double by adding methyl cellulose to the formulation and increasing the viscosity of the inner lumen of the vesicles. The formulation tested in the present study contained an equal amount of CpCl to the CpCl-pos formulation but no GCTE and was given to rats without omeprazole treatment. This formulation exhibited an AUC around double of the AUC of CpCl-pos liposomes, which were administered to rats not treated with omeprazole. Although a certain permeation-enhancing effect of liposomes as such can be expected because of their particulate nature,43 a further improvement of their uptake or of the encapsulated drug seems to be desirable. Many types of bio-enhancers are described in the literature44,45 and several, mostly bile salts, were already employed in liposomes.46–48 TPGS 400, CpCl, and OT were tested in liposomal formulations by our group in previous studies,23,24 1-PP and caprate were described by other groups as effective bio-enhancers.49–51 The modes of action of absorption enhancers are manifold and one substance might act over different ways, for example increasing permeability of cell membranes, opening of tight junctions, increasing mucus and cell adhesion or shielding the surface charge of the drug molecule.44 In addition to the use of absorption enhancers, it is possible to design particles in a way that they can penetrate more easily through the mucus and therefore increase the residence time of the drug and also its bioavailability.52–54 The key parameter for this mucus-penetrative ability seems to be the .-potential, which should be close to zero. Furthermore, short PEG chains help to shield the particle surface and allow the particles to slip more easily through the mucus. Indeed, in the present study, the CpCl-neutr particles that were designed to be mucus penetrative by the addition of TPGS 400 and .potential close to 0 mV showed the strongest mucus interaction, but this did not result in an increased bioavailability of encap-

sulated hGH. Although it was shown that mucus-penetrating nanoparticles have a positive effect on the activity of acyclovir after vaginal delivery,55 there are to our knowledge no studies proving the efficacy of this type of particles for oral delivery.

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Figure 4. Area under the plasma curve over 6 h of liposomal encapsulated hGH after oral application (8 mg/rat). Animals administered 0.3 mg/rat of hGH s.c. or 8 mg/rat of hGH orally were used as control groups. (+Omep) or (−Omep) indicates whether animals were pre-treated with omeprazole or not. Values on top of the bars indicate the relative bioavailability compared with s.c. control. Values represent means ± SEM with n = 6. Groups were compared with free oral hGH as control group by one-way Student’s t-test with *p < 0.05, **p < 0.01, ***p < 0.001.

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In this case, liposomes would protect the protein during the GI passage and deliver the bio-enhancers with the therapeutic protein together close to the site of absorption. Further studies will be necessary to gain a better understanding of the underlying absorption mechanisms of liposomal encapsulated protein drugs.

CONCLUSION

Figure 5. Area under the plasma curve over 6 h of liposomal encapsulated or free hGH (8 mg/rat) or freeze-dried liposomes in a capsule (7 mg/rat) after oral application. All tested liposomes contained the same amount of CpCl corresponding to the CpCl-pos formulation. The “Gel–CpCl-pos” was prepared with 15% gelatine in the aqueous medium and GCTE was replaced with EPC. (+Omep) or (−Omep) indicates whether animals were pre-treated with omeprazole or not. The values on top of the bars indicate the relative bioavailability compared to s.c. control. Values represent means ± SEM with n = 6. n.d., not detectable.

In the present study, we tested liposomes containing bioenhancers and tetraether lipids prepared by DAC for the oral delivery of hGH. This technique is a fast an easy method to prepare VPGs with high encapsulation efficiency for hGH. In further studies, this method should be optimised to minimise degradation of protein during the mixing procedure. The most effective formulation concerning the oral delivery of hGH contained CpCl as bio-enhancer and led to a bioavailability of around 3.4%. Liposomes with .-potential close to 0 mV and a high quantity of TPGS 400 in the lipid bilayer showed in the microscopic pictures the strongest interaction with mucus, but could enhance the bioavailability of hGH only slightly. One major challenge for the oral delivery of proteins by liposomes seems to be the stomach passage. To overcome the instability under acidic conditions, the development of an enteric-coated drug form for liposomes is desirable, for example a freeze-dried liposome formulation in a hard gelatine capsule.

ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the Phospholipid Research Center (Heidelberg, Germany). We would also like to thank Mr. Andrew Fussel for the critical review of the paper.

Nevertheless, in several studies mucus-adhesive particle were more efficient in oral drug delivery than particles with no particular mucus interaction.9,56,57 This could be seen as contradictory to our findings. As in the present study, other factors apart from the mucus penetration influenced the oral bioavailability of hGH, for example liposome stability, further studies are necessary to investigate the benefits of mucus-penetrative liposomes for oral protein delivery. Several publications suggest that liposomes can increase the oral bioavailability of proteins by permeating the intestinal barrier as intact vesicles with the protein still encapsulated.34,58–60 However, these findings might not apply to all liposomal formulations and types of protein drugs and the uptake of intact particles might only be one mode of action among others. Indeed, previous findings showed that if a particle is taken up across the intestinal tract or not, depends on its properties, such as size, charge, surface structure or composition.43,61 In the present study, a co-localisation of green and red fluorescence indicating a possible uptake of liposomes into the blood system was not very pronounced. This is in good agreement with a previous study, where radio-labelled tetraether lipids incorporated into liposomes were hardly taken up into the blood system after oral administration.35 Considering the fact that distinct plasma levels could be reached for oral hGH in CpClpos liposomes, it could be hypothesised that the uptake of intact vesicles with encapsulated hGH is not the only way of absorption. It was discussed in a previous publication that encapsulated proteins could be released from liposomes stuck in the mucus layer and cross the intestinal barrier as free drug.62

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