Abstract
Liposomes which comprise sphingomyelin in the lipid bilayer. The liposomes are configured to cross the blood-brain barrier for the treatment of neuro-degenerative diseases and spinal cord injuries. The liposomes are essentially free of ganglioside. A method of producing liposomes is also disclosed along with use of liposome as a medicament.
Claims
1. Liposomes comprising: a lipid bilayer comprising sphingomyelin, and an inner aqueous compartment; wherein the lipid bilayer is essentially free of ganglioside, wherein the liposomes are completely free of surface modifications, wherein the liposomes have a mean diameter between 10 and 50 nm, and wherein the liposomes are capable of crossing the blood brain barrier.
2. The liposomes according to claim 1, wherein the liposomes additionally comprise cholesterol.
3. The liposomes according to claim 1, further comprising at least one active component which is optionally encapsulated in the inner aqueous compartment of the liposomes.
4. The liposomes according to claim 3, wherein the active component is selected from the group consisting of: cholinesterase-inhibitors; dopamine agonist; resveratrol; and nicotinic derivatives.
5. The liposomes according to claim 3, wherein at least one active component comprises gangliosides, and wherein the gangliosides are encapsulated in the inner aqueous compartment of the liposome.
6. The liposomes according to claim 1, contained in a liposomal formulation, wherein the liposomal formulation has a polydispersity index <0.15.
7. The liposomes according to claim 1, contained in a liposomal formulation, wherein the liposomes of the liposomal formulation have a mean relative circularity of 0.98 to 1.
8. The liposomes according to claim 1, wherein the liposomes are contained in a liposomal formulation, wherein at least 90% of the liposomes of the liposomal formulation are unilamellar.
9. The liposomes according to claim 3, wherein the liposomes are utilized as a medicament.
10. The liposomes according to claim 3, for the treatment of amyotrophic lateral sclerosis, wherein the treatment comprises the oral or intravenous administration of the liposomes.
11. The liposomes according to claim 1, wherein the liposomes are obtained by: a) providing lipids and cholesterol in an organic solvent, b) adding an aqueous liquid, and c) sonicating to enable liposome formation.
12. A method for producing the liposomes of claim 2, comprising: a) providing lipids and cholesterol in an organic solvent wherein the lipids include sphingomyelin, b) adding an aqueous liquid, and c) sonicating to enable liposome formation.
13. The method according to claim 12, wherein the organic solvent used in a) is selected from the group consisting of: ethanol, methanol, chloroform, and mixtures thereof.
14. The method according to claim 12, wherein the aqueous liquid used in b) is selected from the group consisting of: water, aqueous buffer solution, and aqueous glycine-solution.
15. The method according to claim 12, wherein at least one of the organic solvent and the aqueous liquid comprises an active component.
16. The method according to claim 15, wherein the method is for producing liposomes for the treatment of neurodegenerative diseases and spinal cord injuries.
Description
(1) The invention will be further outlined in the following:
(2) FIG. 1: In vivo biodistribution of liposomes comprising sphingomyelin labelled with ICG according to the invention.
(3) FIG. 2: In vivo biodistribution of liposomes comprising sphingomyelin labelled with DiR according to the invention.
(4) FIG. 3: In vivo biodistribution of liposomes comprising sphingomyelin and GM1 labelled with DiR as comparative example to FIG. 2.
(5) FIG. 4: Graphic representation of the biodistribution analysis in the brain, spinal cord, liver and spleen from the in vivo biodistribution images of FIGS. 2 and 3.
(6) FIG. 5: Characterization of the liposomes without surface modification by cryoTEM: (A) Visualization low magnification, (B) Visualization high magnification, (C) Qualitative assessment, (D) Quantitative diameter distribution.
(7) FIG. 6: Quantitative characterisation of the liposomes without surface modification by cryoTEM. (A) circularity distribution, (B) lamellarity diagram.
(8) FIG. 7: Characterization of size stability of the liposomes over time, measured by dynamic light scattering DSC.
(9) FIG. 8: Characterization of polydispersity stability of the liposomes over time, measured by dynamic light scattering DSC.
(10) FIGS. 9/10: In-vivo fluorescence of different liposomal formulations in spinal cord and brain.
(11) FIG. 11: In-vivo fluorescence of liposomal formulations with different lipid compositions in the brain
(12) FIG. 1 shows the in vivo biodistribution of sphingomyelin liposomes labelled with Indocyaninegreen (ICG). Mice were treated intravenously with liposomes carrying near-infrared dye and biodistribution was analysed 24 hours post-injection. Analysis was performed with a GE HealthCare eXplore Optix. Signals of the ICG were found in brain (A) and the spinal cord (B). Total liposome lipid injection was 45 mg/kg carrying 1:200 weight-to-weight ICG. Further signals could be found in the clearance organs liver (C) and spleen (D), indicating that after treatment the liposomes can be removed from the body.
(13) FIG. 2 shows the in vivo biodistribution of sphingomyelin liposomes labelled with DiR. Different mice A, B, C were treated intravenously with liposomes carrying near-infrared dye and biodistribution was analysed 24 and 48 hours post-injection in a ventral view and a dorsal view. Analysis was performed with an optical imaging system, IVIS Spectrum of Perkin Elmer. Signals of the DiR were found in the brain (circle) and spinal cord (rectangle). Total liposome lipid injection was 15 mg/kg carrying 50 μg/ml DiR. Further signals could be found in the clearance organs liver (plain arrow) and spleen (doted arrow), indicating that after treatment the liposomes can be removed from the body. The fluorescence scale is termed in the following unit: total Radiant efficiency [p/s]/[μW/cm.sup.2].
(14) FIG. 3 shows a comparative example of the in vivo biodistribution of a liposome with sphingomyelin and GM1, labelled with DiR. Mice were treated intravenously with liposomes carrying near-infrared dye and biodistribution was analysed 24 and 48 hours post-injection. Analysis was performed with an optical imaging system, IVIS Spectrum of Perkin Elmer. Signals of the DiR were found in brain (circle) and the spinal cord (rectangle). Total liposome lipid injection was 25 mg/kg carrying 50 μg/ml DiR. Further signals could be found in the clearance organs liver (plain arrow) and spleen (doted arrow), indicating that after treatment the liposomes can be removed from the body. The fluorescence scale is termed in the following unit: total Radiant efficiency [p/s]/[μW/cm.sup.2]. Even though the liposomes are found in the same organs as the liposomes presented in FIG. 2, the biodistribution is less distinct compared to the essentially GM1-free liposomes in FIG. 2.
(15) FIG. 4 shows a graphic representation of the biodistribution analysis in the brain, spinal cord, liver and spleen from the in vivo biodistribution images of FIG. 2 and FIG. 3. FIG. 4A shows the normalised fluorescence of the biodistribution of the liposome without ganglioside in four different tissues: brain, spinal cord, liver and spleen. The biodistribution is displayed for two different time points: 24 and 48 hours. The bars represent the standard deviation to the mean. FIG. 4B shows the normalised fluorescence of the biodistribution of the liposome with ganglioside.
(16) It was surprisingly found, that the in vivo biodistribution of the liposome essentially lacking ganglioside (FIG. 4A) is higher than the in vivo biodistribution of the liposome comprising ganglioside (FIG. 4B).
(17) FIG. 5 shows the characterization of the liposomes without surface modification. liposomes were visualized using Cryo Transmission Electron Microscope JEOL JEM-2100F and a TVIPS Tem-Cam camera (JEOL Ltd., Japan). FIG. 5A shows an image of the liposomes at low magnification (20000×). FIG. 5B shows the liposomes at high magnification (80000×). FIG. 5C shows a qualitative assessment done by ocular/visual observation of the liposomal distribution. FIG. 5D shows the size distribution of the liposomes of this invention. In order to quantify the mean diameter of the liposomes N(liposomes)=5128 were analysed (Vironova Analyzer Software, Vironova, Sweden). The mean diameter of the liposomes is 30.46 nm with a standard deviation of 10.10 nm.
(18) FIG. 6 comprises two further tests for a quantitative characterisation of the liposomes without surface modification by cryoTEM (Vironova Analyzer Software, Vironova, Sweden). FIG. 6A shows the circularity distribution of 5128 liposomes. FIG. 6B shows the lamellarity grade of the liposomal distribution. 98% of 5128 liposomes have been characterised as unilamellar.
(19) FIGS. 7 and 8 show the size and polydispersity stability of liposomes according to the invention over time, measured by dynamic light scattering. The liposomal formulations were obtained according to the method described above, by using sphingomyelin and cholesterol in a 1:1 molar ratio. The liposomes were completely free of gangliosides, surface modifications, and did not comprise or encapsulate an active component. The liposomal formulations were stored in PBS at a pH-value of 6.8 and a temperature of 4° C. Size and polydispersity were determined by DLS standard methods. It shall be noted that the values measured by dynamic light scattering are slightly higher than the values obtainable by cryoTEM due to the impact of the hydrodynamic radius of liposomes on DLS measurements. A diameter of 60 nm as indicated in FIG. 7 corresponds to a mean diameter in the range of 10 and 50 nm when measured by Cryo Transmission Electron Microscopy.
(20) The dotted curve shows the results of a small scale production batch of liposomal formulation as described above, while the dashed curve shows the results of an upscale production, i.e. a batch size of 2 litres. As can be seen from FIGS. 7 and 8, both the size and polydispersity of the liposomal formulations from Q3 2017 to Q3 2018, i.e. during storage time of one year, remained essentially unchanged.
(21) FIGS. 9 and 10 show relative in-vivo fluorescence of different liposomal formulations in the spinal cord and brain of mice. In both charts, the liposomes of groups 1 to 4 were obtained according to the method described above, by using only sphingomyelin and cholesterol in a 1:1 molar ratio. Gr. 5 is a control group of free DiR in PBS. In Gr. 1, synthetic sphingomyelin was used and GM1 was comprised in the liposomes. In Gr. 2, synthetic sphingomyelin was used and the liposome was completely free from surface modifications, in particular free from GM1. In Gr. 3, sphingomyelin of animal origin was used and GM1 was comprised in the liposomes. In Gr. 4 sphingomyelin of animal origin was used and the liposome was completely free from surface modifications, in particular free from GM1. For all four test groups, DiR was added as a labelling agent. The measurements were performed by NIR imaging technique.
(22) FIGS. 9 and 10 show the accumulation of the four different kinds of liposomes in the spinal cord and brain respectively in 0.1 h, 4 h, 24 h and 48 h post-injection. It can be seen that the presence of GM1 does not significantly affect the ability of the liposomes to target the central nervous system. The same holds true for the use of synthetic sphingomyelin compared to the use of sphingomyelin of animal origin.
(23) FIG. 11 shows the relative in-vivo fluorescence of liposomal formulations with different lipid compositions in the brain of mice. The liposomes of groups 1 to 3 were obtained according to the method described above by using lipids and cholesterol in a 1:1 molar ratio. In group 1, phosphatidylcholine and sphingomyelin in combination were used as lipids. In group 2, phosphatidylcholine alone was used as a lipid. In group 3, sphingomyelin alone was used as a lipid. In all three test groups, DiR was added to the formulations as a labelling agent. The measurements were performed by NIR imaging technique.
(24) FIG. 11 shows the accumulation of the three different kinds of liposomes in the brain of mice after 24 h and 48 h post-injection. It can be seen that the composition consisting of sphingomyelin and cholesterol alone results in superior longevity of circulation and CNS bioavailability of the liposome compared to the other variants (Grps 1 and 2).
