LIPOSOMES AND USES THEREOF
20220313607 · 2022-10-06
Assignee
Inventors
- Francesca RE (Milano (MI), IT)
- Massimo Ernesto MASSERINI (Milano (MI), IT)
- Marco Antonio Ercole SARDINA (Milano (MI), IT)
Cpc classification
A61P43/00
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61K9/1272
HUMAN NECESSITIES
A61K47/28
HUMAN NECESSITIES
A61K47/24
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
International classification
A61K9/127
HUMAN NECESSITIES
A61K47/24
HUMAN NECESSITIES
A61K47/28
HUMAN NECESSITIES
Abstract
The present invention relates to a liposome containing a lipid or lipid mixture, phosphatidic acid and/or cardiolipin and apolipoprotein E for use in the treatment and/or prevention of amyloidosis, wherein the amyloidosis is not Alzheimer's disease and pharmaceutical compositions containing the same.
Claims
1. A method for the treatment and/or prevention of amyloidosis, wherein said amyloidosis is not Alzheimer' s disease and is selected from the group consisting of: senile systemic amyloidosis, familial amyloid polyneuropathy, dialysis-related amyloidosis, reactive amyloidosis, cerebral amyloid angiopathy, prion diseases, Finnish type amyloidosis, leptomeningeal amyloidosis, Familial visceral amyloidosis, Primary cutaneous amyloidosis, Prolactinoma, Familial corneal amyloidosis, Senile amyloid of atria of heart, Medullary carcinoma of the thyroid LECT2 amyloidosis, type 2 diabetes mellitus, end stage renal failure in patients with type 1 and 2 diabetes mellitus and diabetic kidney disease, comprising administering an effective amount of a liposome comprising: a lipid or lipid mixture; phosphatidic acid and/or cardiolipin and apolipoprotein E or a fragment thereof to a patient in need thereof and wherein said lipid or lipid mixture is selected from the group consisting of: sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and cholesterol.
2. The method according to claim 1 wherein the lipid is a mixture of sphingomyelin and cholesterol.
3. The method according to claim 1, wherein the apolipoprotein E is any of the two isoforms E2, E3 of ApolipoproteinE or a fragment thereof.
4. The method according to claim 1, wherein said apolipoprotein E includes, at its C-terminal, a cystein-ending tripeptide.
5. The method according to claim 1, wherein the liposome further comprises at least one PEG (polyethyleneglycol) molecule, PEO (poly-ethylene-oxide) molecule, POE (poly-oxy-ethylene) molecule, PDO (Polydioxanone) molecule or a mixture thereof, the average molecular mass of the PEG molecule optionally being above 1 kDa but less than 1 lkDa.
6. The method according to claim 5 wherein the PEG molecule is selected from the group consisting of : methylpolyethyleneglycol-1,2-distearoyl-phosphatidyl ethanolamine conjugate (MPEG-2000-DSPE); monomethoxypolyethylene glycol (MPEG-OH), monomethoxypolyethylene glycol-succinate (MPEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MPEG-S -NHS), monomethoxypolyethylene glycol-amine (MPEG-NH2), monomethoxypolyethylene glycol-tresylate (MPEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MPEG-IM); or mixtures thereof.
7. The method according to claim 1, wherein the phosphatidic acid is present in 1-20% molar percentage.
8. The method according to claim 1, wherein the apolipoprotein E is present in 1-5% molar percentage.
9. The method according to claim 1, wherein said liposome consists of: 46.25 mol % cholesterol 46.25 mol % sphingomyelin 1.25-1.5% mol mal-PEG-PE linked to mApoE 1.25-1.0% mol mal-PEG-PE free; and 5 mol% phosphatidic acid wherein the sum of the % of mal-PEG-PE free and % mal-PEG-PE linked to mAPOE is 2.5% wherein mal-PEG-PE is 1,2 stearoyl-sn-glycero-3- phosphoethanolamine-N- [maleimide(poly(ethylene glycol)-2000)] and mAPOE is SEQ ID. No. 1.
10. The method according to claim 1, wherein the liposome has an average size <200 nm.
11. The method according to claim 1, wherein the liposome has a PDI <0.2.
12. The method according to claim 1, wherein the liposome decreases amyloid protein aggregation and/or increases amyloid protein disaggregation.
13. The method according to claim 12 wherein the amyloid protein is selected from the group consisting of: Transthyretin , β.sub.2microglobulin, amylin, amyloid light chain, Serum amyloid A protein, Gelsolin, Cystatin C, ApoA1, Fibrinogen alfa chain, LYZ (Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase), OSMR (Oncostatin-M specific receptor subunit beta also known as the Oncostatin M receptor), Integral membrane protein 2B (ITM2B or BRI2), prolactin, LECT2 protein, keratoepithelin (Transforming growth factor, beta-induced, 68kDa, also known as TGFBI (initially called BIGH3, BIG-H3), calcitonin, atrial natriuretic factor and prion protein.
14. A method for the treatment and/or prevention of amyloidosis, wherein said amyloidosis is not Alzheimer's disease and is selected from the group consisting of: senile systemic amyloidosis, familial amyloid polyneuropathy, dialysis-related amyloidosis, reactive amyloidosis, cerebral amyloid angiopathy, prion diseases such as Creutzfeldt-Jakob disease (humans), BSE or “mad cow disease” (cattle), and scrapie, Finnish type amyloidosis, leptomeningeal amyloidosis, Familial visceral amyloidosis, Primary cutaneous amyloidosis, Prolactinoma, Familial corneal amyloidosis, Senile amyloid of atria of heart, Medullary carcinoma of the thyroid LECT2 amyloidosis, type 2 diabetes mellitus, end stage renal failure in patients with type 1 and 2 diabetes mellitus and diabetic kidney disease, comprising administering a pharmaceutical composition comprising a liposome comprising a lipid or lipid mixture; phosphatidic acid and/or cardiolipin and apolipoprotein E or a fragment thereof, together with one or more pharmaceutically acceptable excipients and, optionally, further active agents to a patient in need thereof.
15. The method of claim 1, wherein the reactive amyloidosis is accompanied by rheumatoid arthritis and/or atherosclerosis.
16. The method of claim 3, wherein the fragment is one of: a) the amino acid sequence 100-200 of ApoE; b) within the amino acid sequence 120-170 of ApoE; or c) the sequence 141-150 of ApoE or a dimer thereof.
17. The method of claim 4, wherein the cysteine-ending tripeptide is CWG.
18. The method of claim 4, wherein the apolipoprotein E has the sequence CWGLRKLRKRLLR or is a dimer thereof.
19. The method of claim 7, wherein molar percentage for the phosphatidic acid is 1-10%.
20. The method of claim 8, wherein the molar percentage for the apolipoprotein is 1-3%.
Description
[0056] The present invention will be illustrated by means of non-limiting examples in reference to the following figures.
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DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods
[0067] The following reagents were purchased from Sigma- Aldrich: THIOFLAVIN T used as stain for amyloid (ThT , code T3516-25G), cholesterol Sigma grade (Chol, code C8667-5G), brain Sphingomyelin (SM, Code 860062P), Dimyristoylphosphatidic acid (PA, Code 830845P), Distearoyl-phospatidylethanolam ine-Polyetyleneglycl-maleim ide (DSPE-PEG-MAL, Code 880126P), 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, Code 105228), Phenylmethylsulfonyl fluoride (PMSF, Code 10837091001). All other common reagents, resins for columns, solvent, reagents for electrophoresis and syntheses were also purchased from Sigma-Aldrich.
[0068] The Peptide CWGLRKLRKRLLR-NH2 (mApoE, Code 822594, SEQ ID No. 1) was purchased by KareBay Biochem — NJ USA. Trypsin Gold-Mass Spec Grade was purchased by Promega (code V5280).
Proteins
[0069]
TABLE-US-00002 Human Beta-Amyloid (1-40): (Aβ-40, Code AS-24236, SEQ ID No. 2) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV was purchased from AnaSpect Inc. Recombinant TTR variant TTR_S52P: (SEQ ID No. 3) MKHHHHH HPMSDYDIPT TENLYFE GAM GPTGTGESKC PLMVKVLDAV RGSPAINVAV HVFRKAADDT WEPFASGKTS EPGELHGLTT EEEFVEGIYK VEIDTKSYWK ALGISPFHEH AEVVFTANDS GPRRYTIAAL LSPYSYSTTA VVTNPKE was expressed and purified as described previously [Verona G. et al. 2017 Sci. Rep. 7, 182-187]. Recombinant β2m variants: β2m_D76N (SEQ ID No. 4) MIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKV EHSDLSFSKDWSFYLLYYTEFTPTEKNEYACRVNHVTLSQPKIVKWDRDM and β2m_ΔN6 (SEQ ID No. 5) MIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLS FSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM were expressed and purified as described previously [Valleix S. et al. Engl. J. Med., 2012, 366, 2276-2283; Verdone G. et al, Protein Sci. 2002, 11, 487-499; Esposito G. et al. Protein Sci. 2000, 9:831-45]. The truncated amyloidogenic form of Serum Amyloid A, SAA(1-76): (SEQ ID No. 6) RSFFSFLGEA FDGARDMWRA YSDMREANYI GSDKYFHARG NYDAAKRGPG GAWAAEAISDARENIQRFFG HGAEDS was obtained by synthesis, as follows.
Briefly, three peptides:
TABLE-US-00003 Peptide 1 (SEQ ID No. 7) H-RSFFSFLGEAFDG-NHNH.sub.2 (segment 1-13) Peptide 2 (SEQ ID No. 8) H-CRDMWRAYSDMREANYIGSDKYFHARGNYDA-NHNH.sub.2 (segment 14-14) Peptide 3 (SEQ ID No. 9) H-CKRGPG GAWAAEAISDARENIQRFFGHGAEDS-NH.sub.2 (segment 45-76)
were synthesized through standard Fmoc-SPPS; each peptide was purified and subsequently the three fragments were assembled in sequence via hydrazide-based native chemical ligation method of peptide to obtain the complete protein SAA(1-76) [Zheng, J.S et al. Nature Protocol 2013, 8, 2483-2495]. Since no cysteine residue is present in the protein sequence, inventors replaced two alanine (at positions 14 and 45) with Cys and, after the complete assembly of the protein, the Cys residues were desulfurized to restore Ala [Wan, Q.; Danishefsky, S. J. Angew. Chem. Int. Ed. 2007, 46, 9248-9252].
[0070] The assembly of peptides was performed on Biotage Syro II peptide synthesizer [Sheppard, R. et al. J. Pept. Sci. 2003, 9, 545]. Fmoc (9-fluorenylmethyloxycarbonyI)- amino acids for synthesis of peptides were obtained from Iris-Biotech. All other protected amino acids and reagents for peptide synthesis were supplied by Sigma—Aldrich. The crude peptides were purified by RP-flash chromatography on Isolera Prime (Biotage) apparatus or preparative RP-HPLC. The purified fractions were characterized by HPLC-MS on Agilent 1200 equipped with Agilent 6130 MS.
[0071] Once the three peptides were purified through chromatographic techniques, the peptide hydrazide 1 was converted to thioester through NaNO.sub.2 oxidation and thiolysis in presence of peptide 2 and 4-mercaptophenylacetic acid (MPPA). After the ligation was completed, the product was purified and the ligation reaction, involving fragment (1-44) and Peptide 3, was repeated to obtain the complete protein SAA (1-76). The product was purified by preparative HPLC on Vydac C18 column (10 ×250 mm, 10 μ, 300 Å), flow rate 3.7 mL/min, gradient elution 25-55% B over 30 min. The purity and correct composition of the product was confirmed by HPLC-MS analysis
Preparation and characterization of Amyposomes®
[0072] Amyposomes® (Liposomes composed of Spingomyelin/cholesterol, functionalized with PA and with mApoE peptide (covalently linked to the outer surface) were synthesized as described [Balducci C. et al. J.Neurosci. 2014, 34: 14022-31, Bana et al., Nanomedecine 2013, doi:10.1016/j.nano.2013.12.001].
[0073] Briefly, Sphingomyelin and Cholesterol (1:1 molar ratio) were mixed with 2.5 molar % of mal-PEG-PE (also named, DSPE-mal, Sigma Aldrich 880126P-25MG) and with 5 molar% of phosphatidic acid (PA, Sigma Aldrich 830845P) in chloroform/methanol (2:1, v/v) and dried under a gentle stream of nitrogen followed by a vacuum pump for 3 h to remove traces of organic solvent. The resulting lipid film was rehydrated in phosphate-buffered saline containing 150 mM NaCI, pH 7.40 (PBS), vortexed and then extruded 10 times at 55° C. through a stack of two polycarbonate filters (100-nm pore size diameter) under 20 bar nitrogen pressure with an extruder. Liposomes were separated from possible unincorporated material by size-exclusion chromatography using PD-10 column and PBS as the eluent.
[0074] mApoE peptide was added to Liposomes in PBS to give a final peptide:mal-PEG-PE (or DSPE-PEG-MAL) molar ratio of 1.2:1 and incubated overnight at room temperature to form a thioether bond with mal-PEG-PE (or DSPE-PEG-MAL) since mApoE peptide reacts only with the portion of mal-PEG-PE present in the outer leaflet of the liposomes (50-60% of the total).
[0075] Liposomes composed of sphingomyelin/cholesterol, functionalized with PA and covalently linked with the peptide mApoE (Amyposomes®) were separated from unbound peptide using PD-10 column. The yield of coupling of the peptide to liposomes was assessed by measuring the tryptophan fluorescence intensity (λex =280 nm) of the incubation mixture and of Amyposomes® recovered from the PD-10 column. Spectra were recorded between 300 and 450 nm using a Cary Eclipse spectrofluorometer (Varian). The amount of peptide bound to liposomes was calculated from the Tryptophan (present in the peptide) fluorescence intensity of a known amount of the peptide dissolved in PBS, taken as the standard. Lipid recovery was measured by Stewart's assay [Stewart J.C., Anal. Biochem. 1980, 104, 10-14].
[0076] Preferred liposomes have the following composition
46,25 mol % cholesterol
46,25 mol % sphingomyelin
1.25-1,5% mol mal-PEG-PE linked to mApoE
1.25-1,0% mol mal-PEG-PE free
5 mol % phosphatidic acid
[0077] Wherein the sum of the % of mal-PEG-PE free and % mal-PEG-PE linked to mAPOE is 2,5%.
[0078] More preferred liposomes have the following composition: 46,25 mol% cholesterol 46,25 mol% sphingomyelin 2,5% mol mal-PEG-PE 5 mol% phosphatidic acid
[0079] These liposomes are functionalized on surface with 1,25 mol% of mAPOE. Liposomes size, Polidispersity Index (PDI) and ζ-potential were characterized as described previously [Gobbi M, et al. Biomaterials 2010;31:6519-29]. Liposomes without PA and mApoE (Plain) were used as controls.
Aggregation/Disaggregation Assay
[0080] Inhibition of protein aggregation or destabilization of preformed aggregates by Amyposomes was monitored essentially as described [ Bana L. et al., Nanomedicine. 2013 ,10:1583-90] by using the Thioflavin T (ThT) fluorescence assay, which identifies amyloid containing β-sheet structures. Fluorescence (λecc =450 nm; λem =480 nm) was measured with Wallac 1420 Victor2 spectrofluorometer (Perkin Elmer). Data were subtracted from fluorescence of Amyposomes alone.
[0081] ThT is a weakly fluorescent probe in water, but its fluorescence increases when it intercalates among the stacked β-sheets of aggregated amyloid proteins molecules. Therefore, the increase of ThT fluorescence during time in the presence of a protein, can be taken as a parameter related to the increased extent of protein aggregation.
[0082] On the other side, when ThT molecules entrapped within aggregates are released in water, as a result of disaggregation, then the fluorescence decreases. Therefore, the decrease of fluorescence over time can be taken as a parameter related to the decreased aggregation (or “increased disaggregation”).
[0083] To monitor the possible inhibiting effect of Amyposomes on protein aggregation, the non-aggregated form of proteins was added with 10 μM ThT and with different amounts of Amyposomes® directly in Costar 96-well black plates. The change in fluorescence was monitored continuously during time with Wallac 1420 Victor2 spectrofluorometer (Perkin Elmer). Alternatively, instead of continuously following the fluorescence, the non-aggregated form of proteins was added with different amounts of Amyposomes® and, at different times of incubation, an aliquot of the samples was withdrawn, added with 10 μM ThT, and the fluorescence measured as above described.
[0084] To monitor the possible destabilizing effect of Amyposomes on protein aggregates, the pre-aggregated form of proteins was added with 10 μM ThT and with different amounts of Amyposomes®, then the time course of fluorescent was followed as above described.
Atomic Force Microscopy
[0085] Aliquots of 10 μI amyloid fibrils obtained as described in Materials and Methods were allowed to adhere onto freshly cleaved mica for 10 min. The samples were washed 3 times with 200 μL Milli-Q water and air dried overnight. AFM was performed Tapping Mode in air using stiff silicon cantilevers (RTESP-Veeco, resonant frequencies ˜300 kHz, spring constant ˜40 N/m). AFM images were acquired at a scan rate of 1 Hz with a Nanowizard II (JPK Instruments, Berlin, Germany). Images were obtained by scanning the samples at a rate of 0.5-1.0 Hz, and 512 x 512 pixels were collected in each image and analysed using JPKs software. Samples were exhaustively examined to confirm their homogeneity.
Aggregation-Disaggregation Protocol for Aβ40
Preparation of Disaggregated Aβ40.
[0086] Lyophilized peptide was stored in sealed glass vials at −80 ° C. Prior to resuspension, each vial was allowed to equilibrate to room temperature for 30 min to avoid condensation upon opening the vial. Each vial of peptide was diluted in 100% HFIP to 1 mM using a glass gas-tight Hamilton syringe with a Teflon plunger and incubated under stirring for 30 min at RT. The HFIP was allowed to evaporate in the fume hood, and the resulting clear peptide films were dried under vacuum (6.7 mtorr) in a SpeedVac (Savant Instruments) and stored desiccated at −20° C. DMSO was added to solubilize the peptide film in order to obtain a solution disaggregated Aβ40 at 5 mM protein concentration. Then, the effect of Amyposomes® on protein aggregation was investigated by the ThT assay.
Preparation of Aggregated Aβ40.
[0087] Fibrils were prepared by diluting 5 mM Aβ40 in DMSO to 25 μM in PBS, immediately vortexing for 30 s, and incubating at 37° C. or 7 days.
[0088] Then the effect of Amyposomes® on protein fibrillary aggregates was investigated by the ThT assay (as described in methods above).
Aggregation-Disaggregation Protocol for TTR S52P
[0089] Recombinant S52P_TTR, at 35 μM concentration in 200 μl PBS (150 mM NaCI, pH 7.4) was incubated at 37° C. in Costar 96-well black plates in the presence of trypsin, at an enzyme:substrate ratio of 1:50, w/w to hydrolytically generate the amyloidogenic form of the protein. To monitor the influence of Amyposomes on protein aggregation, 1.5 mM of PMSF was added after 6h incubation, to inhibit the trypsin enzymatic activity. Under these conditions, the amyloidogenic form of the protein is generated but its aggregation into fibrils has not yet occurred. Afterwards, Amyposomes were added and the fibrillization process was followed by the ThT fluorescence assay, carried out as described above.
[0090] To monitor the effect of Amyposomes on disaggregation, S52P_TTR was incubated in the presence of trypsin, at an enzyme:substrate ratio of 1:50, w/w, to hydrolytically generate the amyloidogenic form of the protein. After 24h incubation, when formation of fibrils has already occurred, 1.5 mM of PMSF was added to inhibit the trypsin enzymatic activity. Afterwards, Amyposomes were added, and the disaggregation process was followed by ThT fluorescence assay, carried out as above described.
Aggregation-Disaggregation Protocol for β2 -Microglobulin (D76 N and ΔN6 variants)
[0091] The inhibition of β2-microglobulin aggregation by Amyposomes was investigated by the ThT assay, carrying out incubation of 40 μM recombinant β2-Microglobulin in 200 μl of PBS at 37° C. in the presence of different amounts of Amyposomes.
[0092] To monitor the effect of Amyposomes on protein disaggregation, 40 pM of recombinant β2-microglobulin in PBS was previously incubated at 37° C., under constant stirring to allow the fibril formation. After 24h incubation, ThT disaggregation assays were carried out in the presence of different amounts of Amyposomes.
Statistical analysis
[0093] Data of ThT assays were normalized to the ThT signal of reference samples containing all the reagents but lacking the amyloid protein.
EXAMPLES
Example 1: Characterization of Amyposomes®
[0094] The size of Amyposomes used within these experiments was 157 ±22.1 nm with a Polydispersity Index (PDI) of 0.072 (average of 15 different batches measured in triplicate).
Example 2: Aggregation-Disaggregation of A β40
[0095] As inferred from the values of the ThT fluorescence, immediately after that the disaggregated form Abeta40 was incubated in PBS, aggregation started and reached a maximum after 48 hours, then it remained constant.
[0096] In the presence of Amyposomes at different protein:Amyposomes ratio, a biphasic behaviour was observed: after an initial phase (24 h) during which an increase of aggregation was observed, then the aggregation decreased constantly over time and, in the case of 1:50 ratio, almost only disaggregated Abeta 40 was present after 4 days. The results are reported in
[0097] The influence of Amyposomes on disaggregation of Aβ40 was evaluated on preparations of previously aggregated Aβ40. The results are reported in
[0098] AFM imaging (
[0099] These results support the therapeutic effect of the liposomes of the present invention for the treatment and/or prevention of CAA.
Example 3: Aggregation-Disaggregation of TTR_S52P
[0100] Aggregation of TTR was triggered by addition of trypsin, catalyzing cleavage of a peptide fragment from the native protein [Mangione P.P. et al. J. Biol Chem. 2018, 293, 14192. Addition of a trypsin inhibitor after 5h (performed to prevent the possible hydrolysis of mApoE upon successive addition of Amyposomes) did not affect the aggregation, that started after 5h and reached a maximum 7 h later then remaining constant.
[0101] To study their influence on aggregation, Amyposomes were added to the protein immediately after the trypsin inhibitor and, in their presence, a reduction of the aggregation was observed. The reduction was higher at higher Amyposome amounts and, at a TTR: Amyposomes ratio of 1:50, the aggregation was 40% with respect to the protein in the absence of Amyposomes. The results are reported in
[0102] The influence of Amyposomes on disaggregation of TTR was evaluated on preparations of previously aggregated protein. The protein in the aggregated form was stable at least for 5 h.
[0103] In the presence of Amyposomes, the fibrils disaggregated. The higher the amount of Amyposomes, the higher was the disaggregation of the protein, so that in the case of TTR:Amyposomes 1:50 ratio, only a 5% residual of aggregates was observed after 5 h. The results are reported in
[0104] Imaged by AFM, TTR S52P produced morphologically typical mature amyloid fibrils, 4-7 nm in height emerging from a thick layer of short fibrils, geometrically ordered (
[0105] These results support the therapeutic effect of the liposomes of the present invention for the treatment and/or prevention of SSA and FAP.
Example 4: Aggregation-Disaggregation of β2 Microglobulin
[0106] Two variants of the protein (D76N and ΔN6) were investigated.
[0107] The amyloidogenic variant of β2-microglobulin, D76N, is associated with a familial form of the disease and is characterized by progressive bowel disfunction and extensive amyloid deposits in the spleen, liver, heart, salivary glands and nerves. ΔN6 is a ubiquitous constituent of β2-m amyloid deposits in patients affected by dialysis-related amyloidosis and, due to its capacity to act as a seed in the fibrillogenesis of full length β2-m, it could have a crucial role in dictating the clinical history of the disease.
[0108] The aggregation of D76N followed a biphasic behaviour: after 10 h incubation the aggregation started and increased slowly up to 19 h, then abruptly increased reaching a maximum at 21 h, then remained constant. On the other side, kinetics for ΔN6 variant were much faster, the aggregation starting immediately upon incubation, and reaching a maximum in 2 h, then remaining constant (
[0109] To study their influence on aggregation, Amyposomes were incubated with the protein, and a reduction of the aggregation was observed. The reduction increased on increasing the Amyposome amount and, at a D76N:Amyposomes ratio of 1:50, the final aggregation extent was 36% with respect to the protein alone. In the case of ΔN6, at a protein:Amyposomes ratio of 1:50, the aggregation extent was 20% with respect to the protein alone. The results are reported in
[0110] The influence of Amyposomes on disaggregation of β2 Microglobulin was evaluated on preparations of previously aggregated protein.
[0111] In the case of D76N, the protein in the aggregated form was stable at least for 3 days. ln the presence of Amyposomes, a decrease of fluorescence was observed starting at 24 h of incubation that decreased constantly up to 72 h. This effect was not evident at 1:2 protein: Amyposomes ratio, was minimal at 1:10 ratio, and was stronger, and comparable, at 1:30 and 1:50 ratios, leading to only a 15% residual aggregates after 3 days.
[0112] In the case of ΔN6, a decrease of fluorescence was observed starting immediately after incubation in the presence of Amyposomes, and the kinetics was much faster, the fluorescence reaching a minimum after 2 h. The effect increased on increasing the amount of Amyposomes. At 1:50 ratios, 50% residual aggregates were present. The results are reported in
[0113] These results support the therapeutic effect of the liposomes of the present invention for the treatment and/or prevention of all diseases associated with β2-microglobulin accumulation or deposit, comprising all dialysis-related amyloidosis, including familial form of dialysis-related amyloidosis, characterized by progressive bowel disfunction and extensive amyloid deposits in the spleen, liver, heart, salivary glands and nerves and sporadic forms of dialysis-related amyloidosis.
Example 5: Aggregation-Disaggregation of SAA(1-76)
[0114] SAA aggregation started after 48 hours incubation. Amyposomes were added to the protein and, in their presence, a strong reduction of the final aggregation extent was observed. The reduction increased on increasing the Amyposome amount and, at a TTR: Amyposomes ratio of 1:50, only disaggregated for was present. The results are reported in
[0115] The influence of Amyposomes on disaggregation of SAA(1-76) was evaluated on preparations of previously aggregated protein.
[0116] In the presence of Amyposomes, at 1:50 ratio the extent of aggregation decreased and after 72 h incubation 30% residual of aggregates was observed. The results are reported in
[0117] These results support the therapeutic effect of the liposomes of the present invention for the treatment and/or prevention of reactive amyloidosis, even associated to rheumatoid arthritis, atherosclerosis or for the treatment and/or prevention of end stage renal failure in patients with type 1 and 2 diabetes mellitus and diabetic kidney disease.
Example 6: Evaluation of the Binding of Amyposomes to Aggregates of Four Different Amyloidogenic proteins by Surface Plasmon Resonance (SPR)
[0118] The binding properties of the Amyposomes for the aggregates of four different amyloidogenic proteins: amyloid-β1-40 (Aβ1-40), transthyretin (TTR), and β2- microglobuline (β2M), in two mutated forms: β2MD76N and β2MΔN6 by SPR were evaluated.
METHOD
[0119] Amyposomes were flowed, at different concentrations (1.56, 3.125, 6.25, 12.5, 25 pM) of PA exposed on Amyposomes surface, over a chip surface coated with the protein aggregates (30 μg/mL, in acetate buffer, pH 4), following the same design previously used to demonstrate the binding of flowing liposomes to immobilized Aβ1-42 fibrils (Gobbi et al., 2010, Biomaterials 31: 6519). Bovine Serum Albumin was used as a negative control. The sensorgrams (time course of the SPR signal in Resonance Units, RU,
RESULTS
[0120] Amyposomes have no “specific” binding signal on BSA (i.e. the reference protein), even at the highest concentration tested. The estimated (see above) equilibrium dissociation constants (KD) were:
0.12 μM for Aβ1-40 fibrils;
1.75 μM on average for the two β2M aggregates;
14.2 μM for TTR aggregates.
[0121] These differences in the KD values were mainly due to differences in the dissociation rate constants, whereas the association rate contants were not markedly different.
[0122] The present SPR data (
Example 7: Effect of Treatment with Amyposomes on Vascular aβ in Cerebral Amyloid Angiopathy (CAA) Animal Models
MATERIALS
[0123] Common Reagents and reagents for immunohistochemistry were from Merck. Primary rabbit AβAntibodies (Catalog # 71-5800, INVITROGEN) and secondary anti-rabbit antibodies (BA-1000, VINCI-BIOCHEM) were from Thermo Fisher Scientific. Cerebral Amyloid angiopaty Mouse model was from Jackson Laboratories (Cat. # Stock :7027C57BL/6-Tg(Thyl-APPSwDutlowa)BWevn/Mmjax).
Mouse model utilized to study Cerebral Amyloid Angiopathy :Tg-SwDI mice.
[0124] The model used to evaluate the effect of Amyposomes is the triple transgenic C57 / 6-Tg (Thyl APPSwDutlowa) BWevn / Mmjax Hem izygous1,2 (Tg-SwDI mice). This mouse model has been primarily designed to study CAA (Jakel L. Animal Models of Cerebral Amyloid Angiopathy Clin. Sci. 2017 131:2469).
[0125] These mice express human APP770 containing the Swedish (K670N / M671L) ,Dutch (APP E693Q), Iowa (APP D694N) mutations under control of the mouse Thyl promoter. In this model, the fibrillary microvascular accumulations of Aβ begin at about 3 months of age. At 12 months 50% of the brain microvasculature has Aβ deposits increasing to 85-90% at the age of 24 months. Higher levels of Aβ.sub.40 compared with Aβ.sub.42 have been measured in isolated cerebral microvessels. Accumulation of Aβ in parenchima is diffuse. (Miao J. Am. J. Pathol. 2005).
[0126] These mice features mirror human Cerebral Amyloid Angiopathy. In fact, human brains affected by CAA show few parenchymal amyloid plaques while vascular Aβdeposits comprise predominantly Aβ40 (Suzuki,N et al. 1994 High tissue content of soluble Abeta1-40 is linked to cerebral amyloid angiopathy Am. J. Pathol . 145:452; Herzig MC, Nat Neurosci. 2004;7:954-60). Of note, parenchymal senile plaques in AD are composed principally of Aβ1- 42 (Dickson,D.W., et al. 1988 Alzheimer's disease. A double-labeling immunohistochemical study of senile plaques. Am. J. Pathol. 132, 86),
Evaluation of the effect of Amyposomes treatment on vascular Abeta in CAA mouse model Tg-SwDl.
[0127] SwDI mice (n=10, aged 6 months) were treated with 3 intraperitoneal injections /week (1 injection every other day) for 3 weeks. Each injection contained 2.6 mg of Amyposomes / 100μL of PBS. SwDI animals (n=10, aged 6 months) treated with PBS (100μL/injection) following the same scheme of treatment were used as control. The animals were sacrified and brain was post-fixed in paraformaldehyde (4%) for histology studies.
[0128] Vascular Aβ deposition was examined using rabbit Anti-Abeta antibodies. To this purpose brain cryostat sections (30 μm) were incubated for 1 h at room temperature with the primary antibody (rabbit Anti-Aβ, 1:200, Catalog # 71-5800, INVITROGEN). After incubation with the anti-rabbit biotinylated secondary antibody (1:200; 1 h at room temperature, Catalog # BA-1000, VINCI-BIOCHEM), immunostaining was developed using the avidin— biotin kit (PK4000, Vector Laboratories) and diaminobenzidine (D8001, Sigma, Italy).
[0129] As shown in
[0130] Amyposome-treated animals are reported in Fig . 10.