Patent us20140205668 – surface modified proteinaceous spherical particles and uses thereof – google patents what is anoxic encephalopathy

Try the new google patents, with machine-classified google scholar results, and japanese and south korean patents. Publication number

US20140205668 A1

Publication type

Application

Application number

US 14/237,890

PCT number

PCT/IB2012/054037

Publication date

Jul 24, 2014

Filing date

Aug 8, 2012

Priority date

Aug 11, 2011

Also published as

US9504759, US20170088585, WO2013021353A1

Publication number

14237890, 237890, PCT/2012/54037, PCT/IB/12/054037, PCT/IB/12/54037, PCT/IB/2012/054037, PCT/IB/2012/54037, PCT/IB12/054037, PCT/IB12/54037, PCT/IB12054037, PCT/IB1254037, PCT/IB2012/054037, PCT/IB2012/54037, PCT/IB2012054037, PCT/IB201254037, US 2014/0205668 A1, US 2014/205668 A1, US 20140205668 A1, US 20140205668A1, US 2014205668 A1, US 2014205668A1, US-A1-20140205668, US-A1-2014205668, US2014/0205668A1, US2014/205668A1, US20140205668 A1, US20140205668A1, US2014205668 A1, US2014205668A1

what is anoxic encephalopathy


Inventors

Shai rahimipour, michal richman

Original assignee

Bar-ilan university

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Patent citations (2), referenced by (2), classifications (16), legal events (1)

External links: USPTO, USPTO assignment, espacenet

In order to attach the aβ binding element KLVFF to protein molecules which would form spherical protein particles, an amino acid having a thiol group, cysteine, was introduced on the C-terminal of the KLVFF peptide. Peptides having the sequence KLVFFC-NH 2 were synthesized using solid phase peptide synthesis. A rink amide MBHA (paramethylbenzhydrylamine) resin was used, with FMOC (fluorenylmethyloxycarbonyl) protection. Coupling was carried out in N-methyl-2-pyrrolidone (NMP), and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was used as the coupling agent.What is anoxic encephalopathy the peptides were cleaved from resin and deprotected by a mixture of trifluoroacetic acid (TFA):triisopropylsilane:H 2O (95:2.5:2.5) and purified to homogeneity by RP-HPLC (reversed phase high-performance liquid chromatography). The pure peptides were analyzed by mass spectrometry using MALDI-TOF/TOF (matrix-assisted laser desorption/ionization, time of flight) or ESI (electrospray ionization) mass spectrometer.

In addition, an indirect assay (tht [thioflavin T] assay) was performed as follows. In the tht assay, the tht dye binds to amyloid plaque formations and displays enhanced fluorescence. The fluorescence is measured and correlated to presence of amyloid plaque formations. Agents which bind and inhibit amyloid plaque formation decrease fluorescence of tht.What is anoxic encephalopathy the inhibitory activity of KLVFF-modified smops on aβ 40 aggregation was determined in black 96-well flat-bottom plates by incubating fresh monomerized (non-aggregated) solutions of aβ 40 (33 micromolar (μm), 15 μl) with different preparations of the smops (25 μl, table 1) in PBS. PBS was added to reach a volume of 50 μl, giving a final concentration of 10 μm for aβ 40. The inhibitory activity of the KLVFF-modified smops was compared to that of soluble KLVFF-NH 2 peptide alone or to the smops with the scrambled peptide, FVKLFC-NH 2 (SEQ ID NO 4). The plates were sealed with aluminum foil and incubated while shaking at 750 rpm at 37° C. For 72 hours to induce aggregation. Following the incubation, a solution of tht (24 μm, 150 μl) in glycine buffer (50 millimolar (mm), ph 8.5) was added to each well and the fluorescence of amyloid-bound tht was immediately measured using a plate reader (infinite M200, tecan, switzerland) at excitation and emission wavelengths of 430 and 492 nm, respectively.What is anoxic encephalopathy the tht solution was freshly prepared by diluting a tht stock solution (1 mm, in water) with glycine buffer (50 mm, ph 8.5). Experiments were performed in quadruplicate.

To induce optimal phagocytic activity, murine microglia (BV-2) cells were pre-stimulated with lipopolysaccharide (LPS) that generates inflammatory responses similar to those seen in AD patients. Phagocytotic activity was then studied by confocal microscopy as well as SEM. SMOPs were pre-loaded with nile-red as a fluorescent probe to allow their identification within the cells. To ensure that the smops localized to the intracellular space, the outer membrane and the nucleus of the live BV-2 cells were stained with alexa-red wheat-germ agglutinin (WGA) and hoechst dye, respectively.What is anoxic encephalopathy the stacked confocal images were then reconstructed to provide three-dimensional pictures of the cells. The images show that smops with a diameter of about 0.7-1 μm were better phagocytosed by the BV-2 cells than smops with larger diameter size. Further support for the phagocytosis of the smops by BV-2 cells was provided by SEM analyses, which demonstrated the formation of hole-like structures (2.5-5 μm) on the membrane of the cells stimulated with LPS and incubated with the smops. The holes most likely occurred on the cell membrane due to the disruption of the smops inside the cells following the sample preparation and conditions under which SEM analyses were performed.

To determine if the phagocytic pathway of the smops is distinct from that of aggregated aβ, BV-2 cells were incubated with pertussis toxin (PTX) and fucoidan as specific inhibitors of the G-protein receptor and the scavenger receptor, respectively, which have been shown to be essential for phagocytosis of aβ.What is anoxic encephalopathy previous studies have shown that inhibition of these receptors reduces the uptake of aβ by macrophages. LPS-treated cells were incubated with PTX and fucoidan and then exposed for 30 minutes to aggregated FITC-aβ or nile-red-loaded KLVFF-modified SMOP. The extent of phagocytosed FITC-aβ or KLVFF-modified SMOP was then compared by FACS for treated and non-treated cells. Treatment of BV-2 cells with either PTX or fucoidan reduced the uptake of the aggregated aβ, as expected. However, these agents had a minimal effect on the phagocytosis of smops, suggesting that the uptake mechanism of the smops is distinct from that of aβ, and may be effective in patients experiencing defective phagocytosis of aβ.What is anoxic encephalopathy

Example 2C PEG-modified smops•

Cyclic D,L-α-peptides were synthesized on 2-cl-trityl resin as follows. Fmoc-lys-odmab (0.78 g, 1 millimoles (mmol)) and DIEA (2 ml) in CH 2cl 2 (8 ml) were added to 2-cl-trityl chloride polystyrene resin (1 g, 1.6 mmol/gr) and the mixture was agitated for 4 hours. The resin was then washed with CH 2cl 2 (3×) and meoh and dried. The loading of the resin was then determined by quantification of fmoc release following treatment of the resin with 20% piperidine in DMF. Peptides were then synthesized on an aapptec automated peptide synthesizer (vantage) with HBTU/DIPEA mediated coupling reactions. Following completion of linear peptides, the resin was treated with 2% hydrazine hydrate in DMF to remove the dmab protecting group.What is anoxic encephalopathy the resin was then washed with 5% DIEA in DMF and the linear peptide was cyclized, while still on resin, using a NMP solution of pybop, HOBT and DIEA (5, 5 and 15 eq.)) for 6 hours. The peptides were cleaved from the resin by treatment with a mixture of 95% TFA: 2.5% triisopropylsilane and 2.5% water for 2 hours. Crude peptides were then purified to homogeneity (95% purity) by preparative RP-HPLC using a C 18 column, and identified by MALDI- or ESI-MS.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (i.E., about 60% identity, optionally about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.What is anoxic encephalopathy this definition also refers to the complement of a test sequence. The identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 75-100 amino acids or nucleotides in length, or, where not specified, across the entire sequence or a polynucleotide or polypeptide.

Example 4 administration of SMOP to humans for treatment of disease•

There is further provided in accordance with an embodiment of the invention, a particle comprising: a spherical protein layer comprising a protein, a polypeptide, a biopolymer or a polymer, and an amyloid-binding moiety conjugated to the protein layer. Optionally, the diameter of the particle is between about 0.2 and about 2 μm, between about 0.4 and about 1 μm, between about 0.7 to about 1 μm.What is anoxic encephalopathy optionally, the amyloid-binding moiety is a peptide which optionally binds aβ. Optionally, the peptide is derived from aβ. Optionally, the peptide comprises the amino acid sequence KLVFF. Optionally, the amyloid-binding moiety comprises a cyclic peptide having an even number of amino acids. Optionally, the cyclic peptide comprises 6 or 8 amino acids, optionally alternating L- and D-amino acids. Optionally, the cyclic peptide is composed of 6 amino acids, 3 of the amino acids in L-configuration and 3 of the amino acids in D-configuration. Optionally, the cyclic peptide is selected from the group consisting of cyclic peptides having the sequence: D-leucine-L-leucine-D-tryptophan-L-histidine-D-serine-L-lysine; D-leucine-L-leucine-D-tryptophan-L-histidine-D-serine-L-alanine; D-leucine-L-leucine-D-tryptophan-L-histidine-D-alanine-L-lysine; D-leucine-L-leucine-D-tryptophan-L-alanine-D-serine-L-lysine; D-leucine-L-leucine-D-alanine-L-histidine-D-serine-L-lysine; D-leucine-L-alanine-D-tryptophan-L-histidine-D-serine-L-lysine; D-alanine-L-leucine-D-tryptophan-L-histidine-D-serine-L-lysine; D-leucine-L-noreucine-D-tryptophan-L-histidine-D-serine-L-lysine and D-leucine-L-2-aminooctanoic acid-D-tryptophan-L-histidine-D-serine-L-lysine.What is anoxic encephalopathy optionally, the amyloid-binding moiety is bound to the protein through a disulfide bond. Optionally, the protein comprises an albumin protein. Optionally, the protein comprises human serum albumin or BSA. Optionally, the particle comprises an inner solvent layer, surrounded by the protein layer. Optionally, the solvent layer comprises an oil, optionally a pharmaceutically acceptable oil, optionally, canola oil. Optionally, the particle further comprising PEG attached to the surface of a particle. Optionally, the particle comprises a contrasting agent.