Liposomal formulation for the oral hepatic delivery of drugs

Abstract

The present invention relates to liposomal compositions, comprising liposomes containing tetraether lipids (TELs), and further comprising the lipopeptide Myr-HBVpreS/2-48 (Myrcludex B) as part of said liposomes, as well as uses thereof for the prevention or treatment of hepatic disorders or diseases, and/or for the oral hepatic delivery of therapeutic and/or diagnostic agents.

Claims

1. A liposomal composition for oral administration comprising: (a) liposomes comprising glycerylcaldityltetraether (GCTE) in an amount of 4 to 6 mol-% based on the total lipid amount, egg phosphatidylcholine (E-PC; lecithin) in an amount of 80 to 90 mol-% based on the total lipid amount, and cholesterol in an amount of 5 to 15 mol-% based on the total lipid amount, wherein said liposomes exhibit a Z-Average measured by dynamic light scattering after dilution in aqueous medium of 100 to 250 nm and a polydispersity index (PDI) of at most 0.2, and (b) the lipopeptide Myr-HBVpreS/2-48 as part of said liposomes.

2. The liposomal composition for oral administration according to claim 1 for use in the prevention and/or treatment of a viral hepatitis.

Description

(1) The figures show:

(2) FIG. 1:

(3) Peptide drugs and other biologicals show poor oral availability with increasing size.

(4) FIG. 2:

(5) Degradation in the stomach and poor mucosa penetration are the main hurdles that prevent the oral availability of biologicals. Using TEL-liposomes, both hurdles can be overcome.

(6) FIG. 3:

(7) Comparison of standard lipids (lecithin; ester bonds) and TELs (ether bonds).

(8) FIG. 4:

(9) Comparison of the liposomal size (A) and the PDI (B) before/after lyophilisation using 100-500 mmol sucrose as lyoprotector. For this formulation the lowest possible concentration of sucrose is 300 mmol.

(10) FIG. 5:

(11) Determined blood levels of the control group (dashed line) and the TEL-liposomes group (full line).

(12) FIG. 6:

(13) Enrichment of different formulations of Myrcludex B in liver tissue [% ID/g] and [%/ID] after oral administration.

(14) FIG. 7:

(15) Distribution of Myrcludex B in Wistar rats after 5 h; injected dose: 500 l of the labeled free peptide.

(16) FIG. 8:

(17) Distribution of Myrcludex B in Wistar rats after 5 h; injected dose: 500 l of the standard liposomal formulation.

(18) FIG. 9:

(19) Distribution of Myrcludex B in Wistar rats after 5 h; injected dose: 500 l of the TEL-liposomal formulation.

(20) FIG. 10:

(21) (A) PET-images of .sup.68Ga labeled Myrcludex B (liposomal i.v.) 0-2 h post administration. (B) and (C) PET images of .sup.124I labeled Myrcludex B (liposomal oral) 0-24 h post administration.

(22) FIG. 11:

(23) Plot of liposomal size and PDI versus various ratios of cholesterol (meansSD; n3).

(24) FIG. 12:

(25) Plot of liposomal size and PDI versus various ratios of GCTE (meansSD; n3).

(26) FIG. 13:

(27) LC/MS-analysis of a) Myrcludex B and b) the tyrosine modified derivative.

(28) FIG. 14:

(29) Cryo-electron micrographs showing the lamellar structure of (a) Myrcludex B standard-liposomes and (b) Myrcludex B GCTE-liposomes. The micrographs show both mono-lamellar and oligo-lamellar liposomes. Comparing both formulations, no difference in liposomal lamellarity could be observed.

(30) FIG. 15:

(31) Comparison of liposomal size and PDI prior to/after freeze-drying using trehalose as lyoprotector at various molar ratios (meansSD; n3).

(32) FIG. 16:

(33) Comparison of liposomal size/PDI prior to/after freeze-drying using sucrose as lyoprotector for GCTE-liposomes at various ratios (meansSD; n5). Concentrations300 mmol provided promising results.

(34) FIG. 17:

(35) Uptake of Myrcludex B in liver tissue (meansSD; n=6) 3 h after oral administration. Both GCTE-liposomal formulations led to a significant increase in the uptake of Myrcludex B. In contrast, comparing the GCTE-liposomal groups, pretreatment with omeprazole (omep.) showed no significant difference in the uptake of Myrcludex B. Control (free peptide) and treatment groups were compared by the one-way ANOVA test and considered significant at *p<0.05, **p<0.01 and ***p<0.001.

(36) FIG. 18:

(37) Uptake of Myrcludex B in liver tissue (meansSD; n=3) 3 h after oral administration using various GCTE concentrations. In contrast to the 1 mol-% GCTE-liposomal formulation, the other two formulations (5 mol-% and 10 mol-%) showed a significant increase in the uptake of Myrcludex B compared with the standard liposomes. Control (standard liposomes) and treatment groups were compared by the one-way ANOVA test and considered significant at *p<0.05, **p<0.01 and ***p<0.001.

(38) FIG. 19:

(39) Comparison of the enrichment of Myrcludex B in the liver 3 h after oral administration of GCTE-liposomes and GCTE-liposomes containing additionally 1 mol-% and 10 mol-% of the bioenhancer cetylpyridinium chloride (meansSD; n=3).

(40) FIG. 20:

(41) Quantification of radiolabeled Myrcludex B in blood samples. Concentration of the free peptide and the peptide incorporated in standard- and GCTE-liposomes (meansSD; n=3) 0-6 h after oral administration.

(42) The present invention will be further illustrated by the following examples without being limited thereto.

EXAMPLES

(43) Material and Methods:

(44) Materials.

(45) Lecithin (EPC) was obtained from AppliChem GmbH (Darmstadt, Germany); tetraether lipids were isolated from S. acidocaldarius (DSM No. 639; ATCC No. 33909) as known in the art; 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); radioiodine was obtained from Perkin Elmer (Boston, USA), Triton X-100, cholesterol and all solvents were purchased from Sigma Aldrich (Taufkirchen, Germany).

(46) Isolation and Purification of GCTE and DGTE.

(47) Cell growth and lipid extraction were performed as known in the art. S. acidocaldarius was separated from the medium and lyophilized using a Delta 1-20 KD from Martin Christ (Osterode, Germany). Lipids were isolated by Soxhlet extraction with chloroform/methanol (2:1 v/v) as known in the art. The extracted solvent was removed by rotary evaporation. Afterwards, the lipid mixture was dissolved in a mixture of chloroform, methanol and hydrochloric acid (8:3:1 v/v). The mixture was heated for 3 days at 60 C. to cleave the lipid head groups.

(48) Finally, the lipids were extracted with chloroform/methanol (2:1) from the water phase. GCTE was separated by silica gel column chromatography with water/methanol (1:1 v/v) as first eluent (for prewashing the column), followed by water/methanol/chloroform (1:2.5:1 v/v) to remove unwanted lipids, and methanol/chloroform (1:1 v/v) to obtain the GCTE fraction.

(49) Peptide Synthesis and RadiolabelingSynthesis of Myrcludex B and Tyrosine-Modified Myrcludex B.

(50) Both peptides were manufactured by solid-phase synthesis using the fluorenylmethoxycarbonyl/tert-butyl (Fmoc/tBu) chemistry on an Applied Biosystems 433A peptide synthesizer as known in the art. The tyrosine analogue (the tyrosine is located at the C-terminus of the peptide) was produced for radiolabeling by iodination for animal trials as described below, while for all other trials, Myrcludex B was used.

(51) Peptide Synthesis and RadiolabelingRadiolabeling of Tyrosine-Modified Myrcludex B.

(52) For radiolabeling of the tyrosine-modified Myrcludex B, a 1 mM stock solution in water/dimethyl sulfoxide (DMSO) was prepared. The required amount of radioactive iodine-131 (.sup.131I) was added to a 1:1 mixture of 25 l of the stock solution and 25 l of a 0.25 M pH 7.5 phosphate buffer. Labeling was performed using the chloramine T method known in the art. The reaction mixture was purified by semi preparative HPLC as known in the art. Afterward, the purity of the radiolabeled compound was determined by radio-HPLC (Agilent 1100 series) using a Chromolith Performance RP-18e, 100-3 mm column applying a linear gradient of 0.1% TFA in water (eluent A) to 0.1% TFA in acetonitrile (eluent B) within 5 min; flow rate 2 ml/min; UV absorbance =214 nm; -detection.

(53) Lipid Analyses.

(54) A .sup.1H-NMR spectrum of TEL was acquired using an Avance II 400 system (Bruker BioSpin GmbH, Rheinstetten, Germany). An IR spectrum was acquired using a Nicolet Avatar 320 FT-IR spectrometer (Thermo Fisher Scientific GmbH, Dreieich, Germany). Mass spectrometry was performed with a TSQ 700 (Thermo Finnigan MAT, Bremen, Germany) system.

(55) LiposomesLipid Composition of Liposomes.

(56) For all experiments, two different liposomal formulations were examined. GCTE-containing liposomes (85 mol-% EPC, 10 mol-% cholesterol and 5 mol-% GCTE) were compared with standard liposomes (90 mol-% EPC, 10 mol-% cholesterol). The most promising GCTE-liposomal formulation with respect to size and PDI was determined by the preparation of liposomes using various amounts of EPC, cholesterol and GCTE (cf. Example 3, infra).

(57) Liposome Preparation.

(58) A lipid film composition containing 85 mol-% lecithin, 10 mol-% cholesterol and 5 mol-% GCTE was used. All lipids were dissolved in chloroform/methanol 9:1 (v/v). All liposomal formulations were prepared by the film method as known on the art using the DAC technology as known in the art, the latter using a SpeedMixer (DAC150FVZ Hauschild Engineering GmbH & Co. KG, Hamm, Germany). First of all, the lipids were dissolved in chloroform/methanol 9:1 (v/v) to obtain 100 mmol stock solutions while Myrcludex B was dissolved in chloroform/methanol 1:1 (v/v; 1 mmol stock). 25 l of the Myrcludex B stock solution was added to the lipid mixture; afterwards, the solution was dried by liquid nitrogen. The resulting lipid film was dried for 1 h in a vacuum chamber. Afterwards 20 mg of glass beads were added. The liposomes were prepared by speed mixing in 3 steps at 3540 rpm in a dual asymmetric centrifuge using a special vial holder as known in the art. Three runs were performed and different amounts of PBS were added (cf. Table 1, below).

(59) TABLE-US-00001 TABLE 1 Characteristic settings of the speed mixing process. Added Rotation in the volume speedmixer [min] Volume calculation of PBS [l] Run 1 30 min Overall lipid mass (mg) 1.5 28.4 Run 2 5 min Overall lipid mass (mg) 2.5 47.3 Run 3 1 min Total volume-1. volume-2. 127.0 volume
LiposomesEncapsulation Efficiency.

(60) The encapsulation efficiency of Myrcludex B was determined by reversed phase HPLC (Agilent 1100 Series) using a C18 column (Chromolith Performance RP-18e, 100-3 mm) applying a linear gradient of 0.1% TFA in water (eluent A) to 0.1% TFA in acetonitrile (eluent B) within 5 min (flow rate 2 ml/min; UV absorbance =214 nm). After the speed mixing process, the liposomes were divided into two parts with 100 l each. Part 1 was used to calculate the 100% value obtained by destroying the liposomes by the addition of 50 l 1% Triton X-100 and determining the area under the curve (AUC) of Myrcludex B by HPLC. Part 2 was purified by Sephadex G-25 gel filtration chromatography (NAP-5 columns) and quantified as part 1. In order to determine the potential loss of lipids on the NAP-5 columns during the purification of part 2, the concentration of cholesterol in the liposomal suspension was measured directly after the speedmixing process and after the purification using NAP-5 columns. For both measurements, the liposomes were dissolved 1:10 (v/v) in methanol. Cholesterol was quantified by HPLC applying an isocratic gradient of acetonitrile/methanol (80:20 v/v) within 15 min (flow rate 2 ml/min; UV absorbance =208 nm) on a RP-18 column. The concentration of cholesterol before and after the purification step was compared and the correction factor C was determined in order to include the loss of lipids on the NAP-5 columns into the calculation of the encapsulation efficiency. The encapsulation efficiency E (%) was calculated using the following equation:
E(%)=([AUC] Myrcludex B part 2/[AUC] Myrcludex B part 1)100%C
whereby [AUC] Myrcludex B part 2 is the concentration of Myrcludex B in the purified liposomal fraction and [AUC] Myrcludex B part 1 is the concentration of Myrcludex B in the liposomal suspension. C is the correction factor considering the loss of lipids on the NAP-5 columns.
Liposome AnalysesParticle Characterization; Particle Size, Polydispersity Index (PDI) and Zeta Potential.

(61) The particle size and PDI of the liposomes was determined using a Zetasizer Nano ZS from Malvern, while the encapsulation efficiency of Myrcludex B was determined by HPLC.

(62) More specifically, the particle size, PDI and zeta potential of all liposomal formulations were determined at room temperature using a Zetasizer Nano ZS from Malvern (Malvern Instruments Ltd., Worcestershire, United Kingdom). Size and PDI were measured after dilution to a lipid concentration of 0.076 mg/ml with a 10 mM phosphate buffer with a pH of 7.4 using the automatic mode. The zeta potential was determined after dilution to a lipid concentration of 0.95 mg/ml by a 50 mM phosphate buffer with a pH of 7.4. The default settings of the automatic mode of the Zetasizer Nano ZS from Malvern (Malvern Instruments Ltd., Worcestershire, United Kingdom) were the following: number of measurements=3; run duration=10 s; number of runs=10; equilibration time=60 s; refractive index solvent 1.330; refractive index polystyrene cuvette 1.590; viscosity=0.8872 mPa s; temperature=25 C.; dielectric constant=78.5 F/m; backscattering mode (173); automatic voltage selection; Smoluchowski equation.

(63) Liposome AnalysesParticle Characterization; Cryo-EM Micrographs.

(64) In order to determine the lamellar structure of the GCTE-liposomal formulation, samples were diluted to a concentration of 10 mg lipid per ml. Quantifoil grids (2/2) were glow discharged for 10 s in a H.sub.2 and O.sub.2 gas mixture. 3 l of samples was applied to the grid and blotted at 4 C. and 100% humidity for 8-10 s in a FEI Vitrobot. The grids were observed in a Krios microscope operated at 200 kV and liquid nitrogen temperature. The micrographs of the GCTE-liposomal sample were taken at 64,000 magnification as known in the art.

(65) Freeze Drying of LiposomesLong Term Storage Stability; Freeze-Drying Using Sucrose and Trehalose at Different Molar Ratios.

(66) All liposomal formulations were freeze dried in a Delta 1-20 KD from Christ. The main drying was carried out at 20 C. for two days with a following secondary drying at 0 C. for at least six hours. Best results were obtained by the use of 300-500 mM sucrose as lyoprotector (FIG. 4 A, B).

(67) More specifically, the main drying was carried out at 20 C. for 2 days followed by a secondary drying at 0 C. for at least 6 h. Sucrose or trehalose were used as lyoprotectors in a range of 100-500 mM as known in the art. Briefly, the liposomes were prepared as described above and the required amount of sucrose/trehalose was added. The liposomal suspension was partitioned into 50 l aliquots and freeze-dried. In order to assess the quality of the freeze-dried products, the liposomes were rehydrated with 50 l PBS and the size and PDI were determined.

(68) Freeze Drying of LiposomesLong Term Storage Stability; Recovery Rate of Myrcludex B after Freeze-Drying and Determination of Residual Moisture.

(69) To determine the recovery rate of intact Myrcludex B after the freeze-drying process, a sample of the rehydrated liposomes was purified by Sephadex G-25 column chromatography (NAP-5 columns) and 1:1 diluted with 1% Triton X-100. Myrcludex B was detected by HPLC using a C18 column (Chromolith Performance RP-18e, 100-3 mm) and compared with the unpurified rehydrated product (calculation analogous to LiposomesEncapsulation efficiency, supra). The residual moisture was determined by a moisture meter (Kern & Sohn GmbH, Balingen, Germany) using 100 mg of the freeze-dried liposomes by heating up to 120 C. in 90 s.

(70) Animal StudiesProof of Concept Study; Liver Accumulation of Myrcludex B Using Different Formulations.

(71) The animal study was performed according to local authorities using male Wistar rats with a body weight of about 250-270 g. In the proof of concept study, a tyrosine-modified analogue of the lipopeptide Myrcludex B was labeled with .sup.131I and incorporated into the liposomes. The organ distribution 3 h after oral administration was measured by direct counting of liver tissue. In the first part of the study, four groups (n=6) of Wistar rats were formed. While three groups of rats (free peptide, standard liposomes and GCTE-liposomes) were pretreated with suspended Antra MUPS (omeprazole) by gavage (10 mg per rat) the day before the experiment, one group received GCTE-liposomes without omeprazole pretreatment in order to examine whether the pretreatment method will increase the oral availability of Myrcludex B. In the second part of the study, three groups of Wistar rats (n=3) were formed and received three different liposomal GCTE concentrations (cf. Table 2, below) in order to evaluate the best GCTE-liposomal composition for the oral uptake of Myrcludex B.

(72) TABLE-US-00002 TABLE 2 Lipid composition of the three different GCTE-liposomal formulations EPC Cholesterol GCTE (mol-%) (mol-%) (mol-%) 1 mol-% GCTE-liposomes 89 10 1 5 mol-% GCTE-liposomes 85 10 5 10 mol-% GCTE-liposomes 80 10 10

(73) The rats were kept without food for 12 h before the experiment but with free access to water. Oral application took place by gavage. In the first part of the study, each rat of group 1 obtained a dose corresponding to 0.5 Mega Becquerel (MBq) of the labeled free peptide (negative control), while each rat of group 2 obtained a dose corresponding to 0.5 MBq of the standard liposomes and each rat of groups 3 and 4 obtained a dose corresponding to 0.5 MBq of the GCTE-liposomes. In the second part of the study, each rat of group 1 obtained a dose corresponding to 0.5 Mega Becquerel (MBq) of the 1 mol-% GCTE-liposomes while each rat of group 2 obtained a dose corresponding to 0.5 MBq of the 5 mol-% GCTE-liposomes and each rat of group 3 obtained a dose corresponding to 0.5 MBq of the 10 mol-% GCTE-liposomes. The rats were sacrificed after 3 h, the liver tissue was removed and weighed and the radioactivity was measured using a Berthold LB 951 G counter in comparison with standards. The liver-associated activity was related to the total injected dose (ID) and expressed as a percentage of the total injected dose per gram of tissue (% ID/g).

(74) Animal StudiesProof of Concept Study; Pharmakokinetic Study.

(75) For the pharmacokinetic study, 3 groups of Wistar rats (n=3) were treated as described above and blood samples were taken at 0.5, 1, 2, 3 and 6 h post administration. The amount of the radioactivity of the blood samples was measured using a Berthold LB 951 G counter.

(76) Animal StudiesProof of Concept Study; Statistical Analyses.

(77) Statistical data were processed using the Prism software (GraphPad Software, San Diego, Calif., USA) and presented as meanstandard deviation of the mean (S.D.). Control and treatment groups were compared by one-way ANOVA test using the Prism software and considered significant at *p<0.05, **p<0.01 and ***p<0.001.

Example 1

(78) Pharmacokinetic Study

(79) Myrcludex B has been labeled with .sup.125I and .sup.131I respectively in order to follow its organ distribution after oral administration (pharmacokinetic and biodistribution study) by direct counting of organ homogenates and blood samples.

(80) The size of these liposomes (determined by dynamic light scattering in a Malvern Zetasizer) was in the range of 150 to 170 nm. The use of more than 10 mol-% TEL lead to both, an increase in the particle size distribution and also to a decrease in the incorporation rate.

(81) In a first proof of concept study, the liposomes were administered to rats by gavage. Thereby, the tyrosin-modified lipopeptide Myrcludex B was labeled with .sup.131I in order to detect the peptide, and incorporated into the liposomes. The animals received either the free peptide or TEL-containing (5 mol-% TELs) liposomes. In comparison to the control group (free peptide), the blood levels of the liposomal group showed a threefold higher count rate indicating a much higher Myrcludex B uptake of the TEL-liposomal group (FIG. 5).

Example 2

(82) Labeled Myrcludex B and liposomes containing the same were prepared as in Example 1.

(83) For the biodistribution study, three groups of Wistar rats (three rats per group) were formed. The rats were kept without food for 12 h but with free access to water. One day before the experiment, the rats were pretreated with suspended Antra MUPS (omeprazole) by gavage. Oral application took place by gavage. Each rat of group 1 got 500 l of the labeled free peptide, while each rat of group 2 got 500 l of the standard liposomal preparation and each rat of group 3 got 500 l of the TEL-liposomal preparation.

(84) The control group in which each rat received 500 l of the labeled free peptide showed a liver uptake of Myrcludex B of only 0.33% initial dose (ID)/g compared with standards (FIG. 7). Group 2 (in which each rat received a conventional liposomal formulation consisting of 85% lecithin and 15% cholesterol) showed an increase in the liver uptake (0.56% ID/g) compared with standards (FIG. 8).

(85) In comparison to the standard liposomes group, the group which received TEL-liposomes showed by far the highest liver uptake of Myrcludex B (1.14% ID/g) (FIG. 9). Due to the fact that in general the weight of the liver of Wistar rats is about 6 to 8 g, in total a liver uptake of about 8 to 10% of the initial dose of Myrcludex B could be detected.

(86) FIG. 6 shows the comparison of the enrichment of Myrcludex B in liver tissue a) % ID/g and b) % ID 3 h after oral administration. The TEL-liposomal group showed the highest uptake of Myrcludex B (1.14% ID/g) compared 0.56% ID/g of the standard liposomal group and 0.33% ID/g of the group which got the free peptide.

(87) For a new approach of biodistribution studies, the peptide Myrcludex B was combined with the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). This combination allowed for an alternative labeling technique for Myrcludex B using .sup.68Ga as radionuclide. .sup.68Ga (half-live=68 min), as a positron-emitting agent, allowed the use of the modern molecular imaging method positron emission tomography (PET). Using PET, images of the biodistribution of the labeled peptide administered to rats, could be obtained (FIG. 10). In order to obtain PET images of orally administered liposomal Myrcludex B, the peptide was labeled by .sup.124I. PET images of i.v. administered liposomal Myrcludex B were obtained by chelation with DOTA/.sup.68Ga.

Example 3

(88) Determination of Particle Size and PDI of Different Liposomal Formulations

(89) In order to determine the best liposomal formulation with respect to size and PDI, liposomes were prepared using various amounts of the liposomal components EPC, cholesterol and GCTE. First, different amounts of cholesterol were mixed with the standard lipid EPC. The addition of more than 10 mol-% of cholesterol resulted in a moderate increase in liposomal size and PDI (FIG. 11).

(90) In the second step, the concentration dependency of GCTE (0-25 mol-%) was examined using x mol-% EPC and 10 mol-% cholesterol as standard lipid mixture for each GCTE concentration. The amount of EPC was calculated by the following equation:
Mol-% EPC=100 mol-%10 mol-% cholesterolx mol-% GCTE
with x mol-% GCTE=0-25

(91) The results showed nearly constant values of the liposomal size. In contrast, a significant increase in the PDI could be observed (FIG. 12).

(92) For fulfilling the requirement of small liposomal size accompanied by low PDI, a lipid mixture of 5 mol-% GCTE, 10 mol-% cholesterol and 85 mol-% EPC was considered to be the ideal liposomal formulation.

Example 4

(93) Isolation of Tetraether Lipids

(94) 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. 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 mass.

Example 5

(95) Peptide Synthesis and Radiolabeling

(96) Synthesis of Myrcludex B and the Tyrosine-Modified Myrcludex B Derivative.

(97) The lipopeptides could be obtained in high purity as determined by LC/MS on an Exactive mass spectrometer (Thermo Scientific).

(98) Myrcludex B and the tyrosine modified derivative were analyzed by mass spectrometry. The mass spectrum of Myrcludex B shows a main signal at

(99) $\frac{m}{z}=1800.64\left(z=3\right)$
which corresponds to the peak of Myrcludex B (molecular weight=5399 g/mol) while the mass spectrum of the derivative shows a main signal at

(100) $\frac{m}{z}=1854.87\left(z=3\right)$
which corresponds to its molecular weight of 5562 g/mol (FIG. 13).
Radiolabeling of the Tyrosine-Modified Myrcludex B.

(101) The .sup.131I-radiolabeling of the tyrosine modified Myrcludex B yielded the desired product in high purity (>95%) as determined by radio-HPLC. The labeling efficiency using the chloramine T method was 65% of the radioactivity applied.

Example 6

(102) Encapsulation Efficiency

(103) The recovery of lipids after purification by the NAP-5 columns was found to be 89.510.58%. This corresponds to a loss of lipids of 10.490.58%, resulting in a correction factor of lipid loss of C=1.12. The GCTE-liposomes containing Myrcludex B showed an encapsulation efficiency of 65.672.91% which is comparable with the determined value of the standard liposomes (63.102.02%). This emphasizes the high encapsulation efficiency of the DAC method compared with other common preparation methods, as previously shown for peptide drugs. An encapsulation efficiency of about 50% for 70 kDa FITC-Dextran using the speedmixing technology has been found, while, in contrast, other preparation methods for the incorporation of peptides into liposomes such as the film method followed by extrusion used for the encapsulation of octreotide only led to an encapsulation efficiency of 13.0% (m/m) [17].

Example 7

(104) Particle Characterization

(105) Particle Size, PDI and Zeta Potential.

(106) The DAC-method applied yielded Myrcludex B GCTE-liposomes with high homogeneity in size, PDI and encapsulation efficiency (cf. Table 3, below). Compared with the standard liposomes, size and PDI of the GCTE-liposomes showed a moderate increase while nearly no difference in the zeta potential could be detected (3.740.28 mV for standard liposomes and 4.200.48 mV for GCTE-liposomes). An increase in the amount of GCTE (up to 25 mol-%; for data see FIG. 12) led to an increase in the liposomal PDI, while, in contrast, the size remained nearly constant. The liposomes containing the bioenhancer CpCl (0-25 mol-%) showed constant values regarding the liposomal size (for data see Table 4, below) while the PDI increased when using 25 mol-% CpCl. This drastic increase might be traced back to differences in the phase transition temperature, as no agglomeration of liposomes could be observed.

(107) TABLE-US-00003 Size (nm) PDI Zeta potential (mV) GCTE-liposomes 140.7 4.3 0.156 0.010 4.20 0.48 Standard-liposomes 131.3 1.5 0.137 0.022 3.74 0.28

(108) TABLE-US-00004 TABLE 4 Particle characterization of 1-25 mol-% CpCl/GCTE-liposomes (means SD; n 5) Zeta potential Size (nm) PDI (mV) 1 mol-% CpCl/GCTE 127.72 2.13 0.164 0.016 7.29 0.61 10 mol-% CpCl/GCTE 129.89 5.34 0.204 0.035 17.13 0.74 25 mol-% CpCl/GCTE 144.99 10.06 0.494 0.050 29.50 1.47
Cryo-EM.

(109) The cryo-electron micrographs (FIG. 14) show the lamellar structure of a diluted sample of the Myrcludex B standard- and the Myrcludex B GCTE-liposomes. A mixture of mono- and oligolamellar liposomes could be detected, while previously mostly multi-lamellar structures for GCTE-liposomes were found by using higher amounts of GCTE. Therefore, the lamellarity of GCTE-liposomes seems to be dependent on both the amount of GCTE and also the liposomal preparation technique.

Example 8

(110) Long Term Storage Stability

(111) Freeze-Drying Using Sucrose and Trehalose as Lyoprotectors at Different Molar Ratios.

(112) The freeze-drying of liposomes containing sucrose and trehalose in different molar ratios as lyoprotectors resulted in a comparable size and PDI for certain molar ratios of sucrose when compared to the data measured prior to the freeze-drying process (FIG. 16). Regarding this liposomal formulation, the minimal concentration of sucrose should be at least 300 mM. These results are in accordance with previous findings that the best protecting effect for a liposomal formulation consisting of EPC and cholesterol uses at least 0.4 M sucrose. An increase in the concentration of the lyoprotector (shown for 500 mM sucrose) does not provide better results regarding size and PDI of the liposomes. Regarding both lyoprotectors, sucrose provided better results compared with trehalose (data see FIG. 15). For this reason, 300 mM sucrose was used for the following determination of the recovery rate of intact Myrcludex B and also for the measurement of the residual moisture after the lyophilization process.

(113) Recovery Rate of Intact Myrcludex B after Freeze-Drying and Residual Moisture.

(114) After the freeze-drying process the recovery rate of intact Myrcludex B incorporated in the GCTE-liposomes was 83.31.3%. The remaining Myrcludex B (14.81.6%) could be detected as the intact peptide. It was removed using a NAP-5 column.

(115) In order to ensure the long term stability of the lyophilized liposomes, low residual moisture has to be achieved. The residual moisture of the Myrcludex B liposomal formulation using sucrose (300 mM) as lyoprotector was 4.21.5%. These findings are in accordance with previous findings determining a residual moisture of 2-4% for liposomes consisting of EPC and cholesterol using sucrose and trehalose as lyoprotectors. In order to determinate the long term stability of the lyophilized product, samples were stored at 20 C. for 3 months. After resuspension, the size, PDI and recovery rate were measured. All values were comparable with the values determined directly after the freeze-drying process (Table 5).

(116) TABLE-US-00005 TABLE 5 Comparison of the rehydrated GCTE-liposomes directly and 3 months after freezedrying (means SD; n P .3). Recovery of Size (nm) PDI Myrcludex B (%) After freeze-drying 145.9 4.0 0.156 0.017 83.3 1.3 After 3 months 146.1 3.7 0.163 0.014 82.7 1.6

Example 9

(117) Proof of Concept Study: Animal Trials

(118) Liver Accumulation of Myrcludex B Using Different Formulations.

(119) The first part of the animal studies showed a significant increase in the enrichment of Myrcludex B in liver tissue (FIG. 17) using GCTE-liposomes (1.14% ID/g) when compared with standard liposomes (0.56% ID/g) and the labeled free peptide (0.33% ID/g). Considering an average liver weight of approximately 6-8 g in a 250 g Wistar rat, the results show that at least 7% of the initial dose of Myrcludex B had been absorbed. This highlights the strong increase in the oral availability of Myrcludex B by the use of GCTE-liposomes. Further, it was examined if pretreatment with omeprazole for the 5 mol-% GCTE-liposomal formulation would lead to an increase in the oral availability of Myrcludex B. However, there was no significant difference apparent between the pretreated and the not pretreated group (FIG. 17).

(120) In the second part of the animal studies three different GCTE concentrations were compared. In contrast to the 5 mol-% and 10 mol-% GCTE-liposomal formulations, for the 1 mol-% GCTE-liposomal formulation no significant increase in the oral availability of Myrcludex B in comparison with the standard liposomes could be observed (FIG. 18). Furthermore, there was no significant difference between the 5 mol-% and the 10 mol-% GCTE-liposomal formulations. For this reason, 5 mol-% of GCTE seems to be sufficient for the stabilizing effect of the tetraether lipids.

(121) The addition of the bioenhancer cetylpyridinium chloride (CpCl; 1-10 mol-%) to the GCTE-liposomal formulation didn't show a significant increase in the oral availability of Myrcludex B (FIG. 19).

(122) In particular, it was tested if the addition of bioenhancers would lead to an increase in the oral availability of Myrcludex B. For this purpose, 1-25 mol-% of the bioenhancer cetylpyridinium chloride (CpCl) were added to the GCTE lipid mixture and the size, PDI and the zeta potential of the liposomes were determined. While the 1 mol-% and 10 mol-% CpCl/GCTE-liposomes showed comparable values regarding size and PDI, a high increase in the PDI of the 25 mol-% CpCl/GCTE-liposomes (cf. Table 4, supra) could be observed.

(123) With respect to the high PDI of the 25 mol-% CpCl/GCTE liposomes, only the liver uptake of the 1 mol-% and 10 mol-% CpCl/GCTE-liposomes in male Wistar rats was determined and compared with the GCTE formulation. Regarding both formulations containing the bioenhancer CpCl, no significant increase in the oral availability of Myrcludex B could be observed (FIG. 19).

(124) Pharmakokinetic Study.

(125) The blood samples of the GCTE-liposomes group (AUC=3550) showed a significant increase in the uptake 0-6 h after oral administration compared to the standard liposomes (AUC=2175) and the labeled free peptide group (AUC=1705; FIG. 20).

(126) The animal trials highlight the potential of the GCTE-formulation for the oral application of Myrcludex B. In particular, a 3.5-fold increase in the oral availability of Myrcludex B could be shown. Furthermore, using sucrose as lyoprotector, it could be shown that long term storage of the GCTE-liposomes by freeze-drying and rehydration can be enabled without destroying the incorporated peptide drug.

(127) Besides the use of GCTE-liposomes, there exists a plentitude of other attempts to enhance the oral availability of macromolecular drugs. When compared with liposomes bearing surface modificationsrecently the most common strategy for oral peptide deliverye.g. coating of liposomes with thiolated chitosan or chitosan-aprotinin, the GCTE formulation shows the big advantage that no coupling step is required. This enables a faster and more reliable liposomal production.

CONCLUSION

(128) In the present invention, 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 drug. Taken together, this study shows that the encapsulation of Myrcludex B into GCTE-liposomes led to a significant improvement in the oral uptake independent of pretreatment with omeprazole.