A polypeptide and uses thereof

Abstract

This application belongs to the field of biology, and specifically relates to a polypeptide and its applications. This application provides a polypeptide comprising an Alphabody and BDNF linked by a linker. The Alphabody has a structure shown in the general formula HRS1-L1-HRS2-L2-HRS3, wherein each of HRS1, HRS2, and HRS3 is independently an α-helix structure containing 2 to 4 consecutive heptapeptide repeating units, said heptapeptide repeating units being polypeptide fragments represented as "abcdefg" or "defgabc", wherein at least 50% of all a and d positions are occupied by isoleucine residues, and wherein each HRS begins with an aliphatic or aromatic amino acid residue located at the a or d position; L1 and L2 are each independently a linker fragment. The polypeptide of this application can increase the exposure of BDNF in plasma and prolong its half-life.

Description

Technical Field

[0001] This application belongs to the field of biology, and specifically relates to a polypeptide and its applications. Background Technology

[0002] Brain-derived neurotrophic factor (BDNF) is a protein with neurotrophic effects. BDNF is the most abundant neurotrophic factor in the body and is widely expressed in the nervous system. It not only has regulatory functions on the growth, development, induced differentiation and synaptic connection of embryonic neurons, but also participates in activity-dependent neuronal plasticity, including post-injury regeneration, repair and protection, especially the protection of cognitive function-related areas such as the prefrontal cortex and hippocampus.

[0003] BDNF is widely distributed in tissues such as the central nervous system, peripheral nervous system, endocrine system, bone, and cartilage. It plays a regulatory role in the growth, development, induced differentiation, and synaptic connections of embryonic neurons, and is crucial in the formation of neural networks. Simultaneously, it participates in activity-dependent neuronal plasticity, including post-injury regeneration, repair, and protection, particularly in the protection of cognitively relevant areas such as the prefrontal cortex and hippocampus. BDNF can act on certain neurons in the central and peripheral nervous systems, helping the survival of existing neurons and promoting the growth and differentiation of new neurons and synapses.

[0004] In the endoplasmic reticulum, BDNF exists in the form of pre-pro-BDNF. After translocating to the Golgi apparatus, the signal sequence in the pre-region is cleaved, forming pro-BDNF, the precursor BDNF protein. Subsequently, pro-BDNF is further cleaved to form mature BDNF (mBDNF). After binding to the TrkB receptor, mBDNF can activate three intracellular signaling pathways: the extracellular regulated protein kinase (MEK-ERK) signaling pathway, the phosphatidylinositol kinase (PI3K) pathway, and the phospholipase CPLC-γ signaling pathway. These pathways help maintain neurogenesis, promote neuronal differentiation and survival, and play an important role in maintaining synaptic plasticity in adults and in the structure and function of brain neurons.

[0005] Existing research indicates that using BDNF to treat central nervous system diseases requires a carrier or peptide formulation. For example, WO2016208696A1 discloses a fusion protein of human transferrin receptor antibody and BDNF. WO2023055155A1 discloses a peptide composed of a brain endothelial cell surface protein-binding peptide linked to a cell-permeable peptide, which can be linked to brain-derived neurotrophic factor (BDNF). CN109157530A discloses a PEG-PLGA nanosphere carrying a BDNF gene plasmid, which, after transfection into neural stem cells, can highly express the functional protein of brain-derived neurotrophic factor (BDNF). CN106188308A discloses the preparation of a brain-targeting protein transporter by activating the carboxyl group of DHA and undergoing an acid-amine condensation reaction with the amino group on the side chain of BDNF amino acid residues. Domestic researchers have constructed a TAT-BDNF prokaryotic expression plasmid, the product of which can be rapidly transduced into cerebellar granule cells and has neuroprotective effects (Chinese Journal of Clinical Anatomy, 2006, Vol. 3). CN101008012A discloses a novel expression plasmid for recombinant human glial cell-derived neurotrophic factor BDNF, which is constructed by fusing an 11-amino acid polypeptide containing 9 lysine residues to the N-terminus of mature human BDNF.

[0006] Although many technologies already exist, there is still much room for exploration in developing new and more effective peptides for BDNF. Summary of the Invention

[0007] The technical problem to be solved by this application is to provide a polypeptide that can increase the exposure of BDNF in plasma and prolong its half-life.

[0008] The purpose of this application is to provide a polypeptide and its application.

[0009] This application provides a polypeptide comprising an Alphabody and BDNF linked by a linker. The Alphabody has a structure represented by the general formula HRS1-L1-HRS2-L2-HRS3, wherein each of HRS1, HRS2, and HRS3 is independently an α-helix structure comprising 2 to 4 consecutive heptapeptide repeating units, the heptapeptide repeating units being polypeptide fragments represented as "abcdefg" or "defgabc", wherein at least 50% of all a and d positions are occupied by isoleucine residues, and each HRS begins with an aliphatic or aromatic amino acid residue located at the a or d position; L1 and L2 are each independently a linker fragment. The polypeptide provided by this application can increase the exposure of BDNF in plasma and prolong its half-life.

[0010] An "Alphabody" can generally be defined as a self-folding, single-chain triplet, in which HRS1, HRS2, and HRS3 together form a triplet α-helical coiled structure.

[0011] The terms “heptapeptide,” “heptapeptide unit,” or “heptapeptide repeating unit” are used interchangeably herein and will be interpreted as a (poly)peptide fragment having 7 residues, repeated 2 or more times within each heptapeptide repeating sequence of the alphabody, polypeptide, or composition contemplated herein, and denoted as “abcdefg” or “defgabc,” where the symbols “a” through “g” denote conventional heptapeptide positions. Heptapeptide repeating units are preferably determined based on optimal overlap of (HppHppp)n or (HpppHpp)n sequence patterns having actual amino acid sequences, where “H” and “p” denote hydrophobic and polar residues, respectively, and where “n” is a number equal to or greater than 2. The start and end points of each heptapeptide repeating unit are then taken as the first and last hydrophobic residues at the a or d positions, respectively. The conventional H-residues are preferably selected from aspartic acid, arginine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, histidine, glutamine, threonine, serine, and alanine, more preferably from aspartic acid, arginine, and isoleucine, and most preferably from isoleucine. The conventional p-residues are preferably selected from glycine, alanine, cysteine, serine, threonine, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, and arginine. In cases where this simple method cannot definitively assign amino acid residues to seven-residue repeat sequences, more specialized analytical methods can be used, such as using specialized software like the COILS method by Lupas et al. (Science 1991, 252:1162-1164; http: / / www.russell.embl-heidelberg.de / cgi-bin / coils-svr.pl). However, it should be noted that the heptapeptide motifs present in the Alphabody (or polypeptides or compositions containing such Alphabody) as envisioned herein are not strictly limited to the above-mentioned representatives (i.e., “abcdefg”, “HPPHPPP”, “defgabc”, and “HPPPHPP”).

[0012] Each HRS begins with an aliphatic or aromatic amino acid residue at the a or d position. This means that when the heptapeptide repeat unit is represented as "abcdefg", it begins with the amino acid at the a position; and when the heptapeptide repeat unit is represented as "defgabc", it begins with the amino acid at the d position. The aliphatic amino acid is selected from leucine, isoleucine, valine, methionine, etc. The aromatic amino acid is selected from phenylalanine, aspartic acid, arginine, etc. In a preferred embodiment, the amino acid at the a or d position is selected from aspartic acid, arginine, and isoleucine.

[0013] In a preferred embodiment of this application, the sequence of HRS1 includes the sequence shown in SEQ ID NO:13 or SEQ ID NO:14, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:13 or SEQ ID NO:14. Specifically, it can be:

[0014] DAAIQKQISAIQKQIASLPPE(SEQ ID NO:13); or

[0015] IAAIQKQISAIQKQIASLPPE (SEQ ID NO: 14).

[0016] In a preferred embodiment of this application, the sequence of HRS2 includes the sequence shown in SEQ ID NO:15 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:15. Specifically, it can be:

[0017] VQQIQKQISAIQKQIASPNLS (SEQ ID NO: 15).

[0018] In a preferred embodiment of this application, the sequence of HRS3 includes the sequence shown in SEQ ID NO:16 or SEQ ID NO:17, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:16 or SEQ ID NO:17. Specifically, it can be:

[0019] RQQIQLQIAKIQAIIELLKAG(SEQ ID NO:16); or

[0020] IQQIQLQIAKIQAIIELLKAG (SEQ ID NO: 17).

[0021] The linker fragments in the alphabody structure are preferably conformationally flexible to ensure relaxed (unhindered) association of the three heptapeptide repeat sequences as an α-helical coil-and-coil structure. Further, in the context of an alphabody, 'L1' should represent linker fragment one, i.e., the linker between HRS1 and HRS2, while 'L2' should represent linker fragment two, i.e., the linker between HRS2 and HRS3. Suitable linkers for the polypeptides contemplated herein will be apparent to those skilled in the art and can generally be any linker in the art for connecting amino acid sequences, provided that the linker fragments are structurally flexible (in the sense that they do not affect the characteristic three-dimensional coil-and-coil structure of the alphabody). The two linkers L1 and L2 in a particular alphabody structure may be the same or different. Based on the further disclosure herein, those skilled in the art will be able to determine the optimal linker, optionally after performing a limited number of routine experiments. In a particular embodiment, linkers L1 and L2 are sequences consisting of 2 to 30 amino acid residues. Based on the other disclosures herein, those skilled in the art will be able to determine the optimal linker fragment for a particular alphabody of the invention (optionally after performing a limited number of routine experiments). In a specific, non-limiting embodiment, the amino acid of the linker fragment is selected from glycine, serine, threonine, alanine, proline, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, and arginine, etc.

[0022] In a preferred embodiment of this application, the sequence of L1 includes the sequence shown in SEQ ID NO:18 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:18. Specifically, it can be: IRARP (SEQ ID NO:18).

[0023] In a preferred embodiment of this application, the sequence of L2 includes the sequence shown in SEQ ID NO:19 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:19. Specifically, it can be:

[0024] AAD (SEQ ID NO:19).

[0025] In addition to the above description, Alphabody also includes N-terminal and C-terminal flanking regions. Those skilled in the art should understand that the excess residues at the N-terminus of HRS1 are N-terminal flanking regions, and the excess residues at the C-terminus of HRS3 are C-terminal flanking regions.

[0026] In some embodiments, the N-terminal flanking region includes a sequence as shown in SEQ ID NO:20 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:20. Specifically, it can be:

[0027] GD (SEQ ID NO:20).

[0028] In some embodiments, the C-terminal flanking region includes a sequence as shown in SEQ ID NO:21 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:21. Specifically, it can be:

[0029] ITSPYYFELINKARTVEGVNALKHDILAAHAT (SEQ ID NO: 21).

[0030] The "half-life" of an alphabody or peptide is generally defined as the time required for its serum or plasma concentration in vivo to decrease to 50%. The in vivo half-life of an alphabody or peptide can be determined in any manner known to those skilled in the art, such as by pharmacokinetic analysis. As will be apparent to those skilled in the art, half-life can be expressed using parameters such as t1 / 2-α, t1 / 2-β, and area under the curve (AUC). Typically, an increased in vivo half-life is characterized by an increase in one or more, preferably all three, of the parameters t1 / 2-α, t1 / 2-β, and AUC.

[0031] In a specific embodiment, the linker is a flexible spacer sequence comprising glycine and serine; preferably, it also includes proline and alanine. In a particular embodiment, the linker contains 3 to 20 repeats of the "glycine / serine / proline / alanine-rich" sequence.

[0032] In some preferred embodiments, the linker sequence includes the sequence shown in SEQ ID NO:3 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:3. Specifically, SEQ ID NO:3 is: GPASPSAPASPSAPASG.

[0033] In some preferred embodiments, the sequence of BDNF includes the sequence shown in SEQ ID NO:12, specifically:

[0034] HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYE TKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLT IKRGR.

[0035] In some preferred embodiments, the C-terminal of the Alphabody is connected to the N-terminal of the BDNF via a linker, forming an Alphabody-linker-BDNF connection sequence. In some preferred embodiments, another connection method may also be used, for example, the C-terminal of the BDNF is connected to the N-terminal of the Alphabody via a linker, forming a BDNF-linker-Alphabody connection sequence.

[0036] In some preferred embodiments, the sequence of the alphabody includes the sequence shown in SEQ ID NO:1 or SEQ ID NO:2, or the sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0037] Specifically, the sequence of the alphabody can be ABshL12d_ALB_C:

[0038] GDDAAIQKQISAIQKQIASLPPEIRARPPVQQIQKQISAIQKQIASPNLSAADRQQIQL QIAKIQAIIELLKAGITSPYYFELINKARTVEGVNALKHDILAAHAT (SEQ ID NO: 1).

[0039] The alphabody sequence can also be ABshL12d_AC1al_ALB_C:

[0040] GDIAAIQKQISAIQKQIASLPPEIRARPPVQQIQKQISAIQKQIASPNLSAADIQQIQLQI AKIQAIIELLKAGITSPYYFELINKARTVEGVNALKHDILAAHAT (SEQ ID NO: 2).

[0041] In one specific embodiment, the structure of the polypeptide may be represented as a sequence including the sequence shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

[0042] In one specific embodiment, the structure of the polypeptide can be represented as BDNF-linker-ABshL12d_ALB_C, and its sequence is shown in SEQ ID NO:8:

[0043] HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGRGPASPSAP ASPSAPASGGDDAAIQKQISAIQKQIASLPPEIRARPPVQQIQKQISAIQKQIASPNLSAADRQQIQLQIAKIQAIIELLKAGITSPYYFELINKARTVEGVNALKHDILAAHAT (621F without label).

[0044] In one specific embodiment, the structure of the polypeptide can be represented as BDNF-linker-ABshL12d_AC1al_ALB_C, and its sequence is shown in SEQ ID NO:9:

[0045] HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGRGPASPSAP ASPSAPASGGDIAAIQKQISAIQKQIASLPPEIRARPPVQQIQKQISAIQKQIASPNLSAADIQQIQLQIAKIQAIIELLKAGITSPYYFELINKARTVEGVNALKHDILAAHAT (621G without label).

[0046] In one specific embodiment, the structure of the polypeptide can be represented as ABshL12d_ALB_C-linker-BDNF, and its sequence is shown in SEQ ID NO:10:

[0047] GDDAAIQKQISAIQKQIASLPPEIRARPPVQQIQKQISAIQKQIASPNLSAADRQQIQLQIAKIQAIIELLKAGITSPYYFELINKARTVEGVNALKHDILAAHATGPASPSAPASPSAPASGHSDP ARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR (unlabeled 621I).

[0048] In one specific embodiment, the structure of the polypeptide can be represented as ABshL12d_AC1al_ALB_C-linker-BDNF, and its sequence is shown in SEQ ID NO:11:

[0049] GDIAAIQKQISAIQKQIASLPPEIRARPPVQQIQKQISAIQKQIASPNLSAADIQQIQLQIAKIQAIIELLKAGITSPYYFELINKARTVEGVNALKHDILAAHATGPASPSAPASPSAPASGHSDP ARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR (621J without label).

[0050] In some preferred embodiments, the N-terminus and / or C-terminus of the peptide includes tags that assist in expression or purification. In specific embodiments of this application, the tags include one or more of the following: c-Myc tag, HA tag, VSV-G tag, FLAG tag, V5 tag, or HIS tag. Preferably, the tags are both HIS and V5 tags. More preferably, the N-terminus of the peptide is an HIS tag, and the C-terminus is a V5 tag.

[0051] In one specific embodiment, the sequence includes the sequence shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7 or the sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0052] In one specific embodiment, the sequence of the polypeptide is shown in SEQ ID NO:4:

[0053] HHHHHHHHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGRGPASPSAPAS PSAPASGGDDAAIQKQISAIQKQIASLPPEIRARPPVQQIQKQISAIQKQIASPNLSAADRQQIQLQIAKIQAIIELLKAGITSPYYFELINKARTVEGVNALKHDILAAHATGSGKPIPNPLLGLDST(621F).

[0054] In one specific embodiment, the sequence of the polypeptide is shown in SEQ ID NO:5:

[0055] HHHHHHHHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGRGPASPSAPAS PSAPASGGDIAAIQKQISAIQKQIASLPPEIRARPPVQQIQKQISAIQKQIASPNLSAADIQQIQLQIAKIQAIIELLKAGITSPYYFELINKARTVEGVNALKHDILAAHATGSGKPIPNPLLGLDST(621G).

[0056] In one specific embodiment, the sequence of the polypeptide is shown in SEQ ID NO:6:

[0057] HHHHHHHHGDDAAIQKQISAIQKQIASLPPEIRARPPVQQIQKQISAIQKQIASPNLSAADRQQIQLQIAKIQAIIELLKAGITSPYYFELINKARTVEGVNALKHDILAAHATGPASPSAPASPSAPASGHSDPA RRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGRGSGKPIPNPLLGLDST(621I).

[0058] In one specific embodiment, the sequence of the polypeptide is shown in SEQ ID NO:7:

[0059] HHHHHHHHGDIAAIQKQISAIQKQIASLPPEIRARPPVQQIQKQISAIQKQIASPNLSAADIQQIQLQIAKIQAIIELLKAGITSPYYFELINKARTVEGVNALKHDILAAHATGPASPSAPASPSAPASGHSDPA RRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGRGSGKPIPNPLLGLDST(621J).

[0060] A second aspect of this application provides an isolated nucleic acid molecule that encodes the aforementioned polypeptide.

[0061] In some embodiments, the nucleic acid molecules of this application can typically be obtained using PCR amplification, recombination, or artificial synthesis. One feasible method is to synthesize the relevant sequences artificially, especially when the fragment length is short. Generally, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0062] A third aspect of this application provides a vector comprising the aforementioned nucleic acid molecule. "Vector" refers to a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. The vector in this invention generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0063] A fourth aspect of this application provides a host cell comprising the aforementioned nucleic acid molecule or the aforementioned vector. "Host cell" can refer to any cell suitable for expression via an expression vector, allowing the expression of the nucleic acid molecule of this invention to be permitted when a promoter is present in the host cell. Preferably, the nucleic acid molecule is carried in a vector for nucleic acid replication and expression. Cells compatible with the vector are selected; for example, the host cell can be a prokaryotic cell, such as a bacterial cell (Escherichia coli); or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as mammalian cells (CHO, COS, HEK, HeLa, and 3T3 cells). The introduction of the nucleic acid molecule into the host cell can be performed using conventional methods well known to those skilled in the art, including but not limited to calcium phosphate transfection, DEAE-glucan-mediated transfection, cationic lipid-mediated transfection, electroporation, translocation, microinjection, transduction, scratch loading, and bombardment.

[0064] A fifth aspect of this application provides a method for generating the aforementioned polypeptide, comprising culturing the aforementioned host cells to generate and isolate the polypeptide. The culturing can be carried out in a conventional nutrient fermentation medium. Suitable media can be selected based on the choice of host cells and / or the regulatory requirements of the expression vector. Such media are known to those skilled in the art. If desired, the media may contain additional components that are more favorable to the transformed host than to other potentially contaminating microorganisms, such as antibiotics, specifically ampicillin.

[0065] The sixth aspect of this application provides a pharmaceutical composition comprising the aforementioned polypeptide and a pharmaceutically acceptable carrier.

[0066] The seventh aspect of this application provides the use of the aforementioned polypeptide, the aforementioned nucleic acid molecule, the aforementioned carrier, the aforementioned host cell, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating or preventing BDNF-mediated diseases.

[0067] In some implementations, BNDF-mediated diseases include cardiovascular and cerebrovascular diseases. Preferably, cardiovascular and cerebrovascular diseases include one or more of cardiovascular diseases, brain diseases, and brain tumors.

[0068] In some implementations, cardiovascular diseases include one or more of the following: acute coronary syndrome, aneurysm, angina pectoris, atherosclerosis, aortic stenosis, myocardial fatty degeneration, hypertensive heart disease, pulmonary hypertension, myocardial ischemia-reperfusion injury, cardiomyopathy, coronary artery disease, heart failure, ischemic heart disease, lipogenic cardiomyopathy, myocardial infarction, and pericardial disease.

[0069] In some implementations, brain diseases include one or more of the following: encephalitis, Parkinson's disease, epilepsy, Huntington's disease, Alzheimer's disease, Luger's disease, Pick's disease, Kreutzfeldt-Jacob's disease, progressive supranuclear palsy, spinocerebellar degeneration, cerebellar atrophy, multiple sclerosis, stroke, depression, and post-traumatic stress disorder.

[0070] In some embodiments, brain tumors include one or more of the following: glioma, glioblastoma, meningioma, astrocytoma, acoustic neuroma, chondroma, medulloblastoma, gangliocytoma, schwannoma, neurofibroma, neuroblastoma, epidural, intramedullary, and intrasheath tumors.

[0071] The eighth aspect of this application provides a method for treating or preventing BDNF-mediated diseases, comprising administering to a subject a therapeutically effective dose of a polypeptide, nucleic acid molecule, carrier, host cell, or pharmaceutical composition.

[0072] By way of example, treatment methods may be used for subjects with the following symptoms: acute coronary syndrome, aneurysm, angina pectoris, atherosclerosis, aortic stenosis, myocardial fatty degeneration, hypertensive heart disease, pulmonary hypertension, myocardial ischemia-reperfusion injury, cardiomyopathy, coronary artery disease, heart failure, ischemic heart disease, lipogenic cardiomyopathy, myocardial infarction, pericardial disease, encephalitis, Parkinson's disease, epilepsy, Huntington's disease, Alzheimer's disease, Luger's disease, Pick's disease, Kreutzfeldt-Jacob's disease, progressive supranuclear palsy, spinocerebellar degeneration, cerebellar atrophy, multiple sclerosis, stroke, depression, post-traumatic stress disorder, glioma, glioblastoma, meningioma, astrocytoma, acoustic neuroma, chondroma, medulloblastoma, gangliocytoma, schwannoma, neurofibroma, neuroblastoma, and one or more combinations of epidural, intramedullary, and intrasheath tumors.

[0073] In some embodiments, the subject is a mammal or mammalian cells. Mammals include, for example, rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. Primates include, for example, monkeys, apes, or Homo sapiens. Mammalian cells include, for example, human embryonic kidney cells, and more specifically, HEK293 cells.

[0074] Detailed application instructions

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any discrepancy, the definitions provided in this application shall prevail. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference.

[0076] The terms “comprising,” “including,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other elements or method steps not listed. Those skilled in the art will understand that the foregoing term “comprising” encompasses the meaning of “consisting of.”

[0077] The term "one or more species" or similar expression "at least one species" can mean, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more species.

[0078] When the lower and upper limits of a numerical range are disclosed, any numerical value falling within that range and any included range are specifically disclosed. In particular, each range of values ​​disclosed herein should be understood as representing each numerical value and range encompassed within a wider range.

[0079] The term "pharmaceutically acceptable" refers to a substance that, within the bounds of normal medical judgment, is suitable for contact with a patient's tissues without causing undue toxicity, irritation, allergic reactions, etc., has a reasonable benefit-risk ratio, and is effective for its intended use.

[0080] The term "pharmaceutical composition" refers to a composition containing one or more polypeptides of this application, a physiologically / pharmaceuticalally acceptable carrier, and other components such as physiologically / pharmaceuticalally acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0081] The term "pharmaceutically acceptable carrier" refers to substances that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. "Pharmaceutically acceptable carriers" include, but are not limited to, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, disintegrants, stabilizers, solvents, or emulsifiers.

[0082] The terms “administration” or “giving” refer to methods that enable the delivery of a compound or composition to a desired biological site of action. These methods include, but are not limited to, oral or parenteral administration (including intraventricular, intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular injection, infusion, intrathecal administration, or transforaminal administration), local administration, and rectal administration.

[0083] As used herein, the term "treatment" includes relieving, reducing, or improving a disease or symptom; preventing other symptoms; improving or preventing underlying metabolic factors of symptoms; inhibiting a disease or symptom, for example, preventing the development of a disease or symptom; reducing a disease or symptom; promoting the remission of a disease or symptom; or causing the symptom of a disease or symptom to cease; and extends to include prevention. "Treatment" also includes achieving therapeutic and / or preventive benefits. A therapeutic benefit refers to the eradication or improvement of the condition being treated. Furthermore, a therapeutic benefit is achieved by eradicating or improving one or more physical symptoms associated with an underlying disease, and an improvement in the patient's condition can be observed even though the patient may still have the underlying disease. A preventive benefit refers to the use of a composition by a patient to prevent the risk of a certain disease, or the use by a patient when experiencing one or more physical symptoms of a disease, even though the disease has not yet been diagnosed.

[0084] The terms “active ingredient,” “therapeutic agent,” “active substance,” or “active agent” refer to a chemical entity that can effectively treat or prevent a target disorder, disease, or symptom.

[0085] For the purposes of pharmaceuticals, pharmaceutical units, or active ingredients, the terms "effective amount," "therapeutic effective amount," or "preventive effective amount" refer to a sufficient quantity of a drug or agent that provides acceptable side effects while achieving the desired therapeutic effect. The determination of the effective amount varies from person to person, depending on the individual's age and general condition, as well as the specific active substance. The appropriate effective amount in a given case can be determined by a person skilled in the art based on routine testing.

[0086] As used herein, “individual” includes both human and non-human animals. Exemplary human individuals include individuals suffering from a disease (such as the disease described herein) (referred to as patients) or healthy individuals. In this application, “non-human animal” includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0087] The term "room temperature" refers to a temperature ranging from 10°C to 40°C. In some embodiments, "room temperature" refers to a temperature ranging from 15°C to 30°C; in other embodiments, "room temperature" refers to a temperature ranging from 18°C ​​to 25°C.

[0088] The term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be conveniently performed using, for example, computer programs such as the Align program (DNAstar, Inc.). The algorithm integrated into the ALIGN program can also be used to determine the percentage identity between two amino acid sequences using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the Needleman and Wunsch algorithms in the GAP program integrated into the GCG software package can be used to determine the percentage identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix, along with gap weights and length weights.

[0089] The following detailed description of the application is intended to illustrate non-limiting embodiments, enabling other skilled in the art to more fully understand the technical solutions, principles, and practical applications of this application, so that other skilled in the art can modify and implement this application in many forms to best suit the requirements of a particular purpose.

[0090] Beneficial effects

[0091] This application discloses for the first time a novel polypeptide form containing BDNF, which can increase the exposure of BDNF in plasma and prolong its half-life, thus helping to retain BDNF in the body. Attached Figure Description

[0092] Figure 1 The results of NFAT luciferase reporter assay in HEK293 cells overexpressing TrkB in Example 4 are shown. 1A represents the results of treatment with 5 nM recombinant protein sample for 7 h, 1B represents the results of treatment with 2 nM recombinant protein sample for 4 h, and 1C represents the results of treatment with 0.5 nM recombinant protein sample for 4 h.

[0093] Figure 2 The results of the in vivo PK experiment of the recombinant protein in Example 5 are shown. Detailed Implementation

[0094] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

[0095] Example

[0096] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Unless otherwise specified, all proportions or percentages used herein are by weight.

[0097] The main instruments used in the following embodiments are shown below:

[0098]

[0099] Example 1

[0100] Design the sequence structure of the recombinant BDNF fusion protein

[0101] 621D: Wild-type BDNF has an N-terminus linked to an uncut His-tag and a C-terminus linked to a V5-tag; the specific sequence is shown below:

[0102] HHHHHH HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQ

[0103] YFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGRGS GKPIPNPLLGLDST (SEQ ID NO:14, underscore indicates tag).

[0104] 621F: Wild BDNF is linked to an optimized Alphabody, ABshL12d_ALB_C, with the N-end linked to an uncut His-tag and the C-end linked to a V5-tag; the specific sequence is shown below:

[0105]

[0106] (SEQ ID NO:4, underline indicates tag, bold indicates ABshL12d_ALB_C, italic indicates linker).

[0107] 621G: Wild BDNF is linked to another optimized Alphabody, ABshL12d_AC1aI_ALB_C. The N-terminus is linked to an uncut His-tag, and the C-terminus is linked to a V5-tag. The specific sequence is as follows:

[0108]

[0109] (SEQ ID NO:5, underline indicates tag, bold indicates ABshL12d_AC1aI_ALB_C, italic indicates linker).

[0110] 621I: ABshL12d_ALB_C is linked to the wild BDNF, the N end is linked to the uncut His-tag, and the C end is linked to the V5-tag. The specific sequence is as follows:

[0111]

[0112] (SEQ ID NO:6, underline indicates tag, bold indicates ABshL12d_ALB_C, italic indicates linker).

[0113] 621J: ABshL12d_AC1aI_ALB_C is linked to the wild BDNF, the N end is linked to the uncut His-tag, and the C end is linked to the V5-tag. The specific sequence is as follows:

[0114]

[0115] (SEQ ID NO:7, underline indicates tag, bold indicates ABshL12d_AC1aI_ALB_C, italic indicates linker).

[0116] All constructs have a C-terminal V5 tag for subsequent detection with an anti-V5 antibody, which can be used for albumin affinity assays (see below) and PK analysis.

[0117] Example 2

[0118] Expression and purification of recombinant proteins

[0119] The sequence of the recombinant protein constructed in Example 1 was cloned into the pET-16b vector through the NcoI and XhoI restriction sites to obtain the construct. The construct was then transformed into Escherichia coli pLysS strain, and the recombinant strain was cultured to obtain the recombinant protein.

[0120] The specific culture conditions were as follows: overnight pre-culture with an OD600 of 0.1 was inoculated into 3L of LB medium (containing ampicillin) and grown at 37°C. When the OD600 reached 0.5-0.6, recombinant protein production was induced with 1mM IPTG and grown at 37°C for 4 hours. The precipitate was collected by centrifugation, resuspended in 30ml of buffer (containing 50mM HEPES, 500mM NaCl, pH=7.2), and then cell lysis was performed.

[0121] The specific method for cell lysis was as follows: 10 μg / mL DNase, 5 mM MgCl2, and 1 mM AEBSF were added to the resuspended solution. The mixture was incubated in a vortex mixer for 15 minutes, sonicated on ice for 5 minutes (70% power, 0.5 min circulation), then placed in a vortex mixer for another 15 minutes. The mixture was then centrifuged at 2000 rpm for 20 min at 4°C, and the supernatant and precipitate were collected separately. SDS-PAGE of the collected supernatant and precipitate samples showed that most of the protein was expressed in inclusion bodies.

[0122] The inclusion body sample was then refolded and purified: An appropriate amount of refolding buffer (50 mM MEPES pH 7.2 / 4 M GuHCl) was added to the precipitate, and the mixture was stirred at room temperature until the precipitate dissolved. The solution was centrifuged at 14,000 rpm for 15 min at 4 °C, and the supernatant was collected. An appropriate amount of Ni-NTA resin was added to the supernatant, and the mixture was incubated at 4 °C for 4 h. The resin was then transferred to a chromatography column, washed with refolding buffers containing different concentrations of imidazole, and subsequently eluted with a refolding buffer containing 300 mM imidazole. Finally, the eluted purified sample was concentrated by dialyzing (1XPBS pH 9.0, 10% glycerol).

[0123] Example 3

[0124] Affinity assay for recombinant proteins

[0125] 1. Experimental objective: To test the affinity of recombinant proteins and establish a solution-based ELISA method for detecting albumin affinity.

[0126] 2. Experimental sample: The purified recombinant protein obtained in Example 2 was prepared in-house.

[0127] 3. Test Procedure:

[0128] Methods for detecting albumin affinity using ELISA:

[0129] The first step (calibrating the ELISA) is to obtain a dose-dependent signal by binding the V5-tagged recombinant protein to biotin-tagged human serum albumin (HSA) captured on a neutral avidin plate.

[0130] The second step (solution ELISA) involves selecting a fixed concentration of the fusion construct from the linear region of the resulting S-shaped binding curve for subsequent ELISA steps. In the solution ELISA step, a fixed concentration of recombinant protein is co-incubated with diluted albumin; the resulting signal disappears and can be converted into a binding curve in solution.

[0131] In this way, soluble Kd was obtained.

[0132] The specific steps are as follows:

[0133] Coat the ELISA plate with neutral avidin (10 μg / ml) in PBS solution (2 h, room temperature); block (2% PBS solution, 1 h, room temperature), wash 4 times; add pre-incubated biotinylated human albumin biotin-HSA (10 μg / ml) to the ELISA plate and incubate for 60 min, wash 4 times; add recombinant protein sample, incubate for 1 h, wash 4 times; stain with V5-HRP for 0.5 h, wash 4 times; develop with developing solution (until the blue is sufficiently strong); stop the reaction with 0.5 M H2SO4 after 6 min.

[0134] 4. Experimental Results:

[0135] The Kd values ​​of human albumin were obtained (see Table 1). No binding of unfused BDNF (621D) to human albumin was observed, while 621I and 621J showed high affinity for human albumin.

[0136] Table 1 Albumin affinity of recombinant proteins

[0137]

[0138] Example 4

[0139] In vitro cell experiments of recombinant proteins

[0140] 1. Experimental objective: To verify the recombinant protein in vitro in cells and to detect the NFAT-dependent transcription of BDNF-activated receptor TrkB in HEK293 cells expressing TrkB using a luciferase dual reporter gene system.

[0141] 2. Experimental sample: The purified recombinant protein obtained in Example 2 was prepared in-house.

[0142] 3. Test Procedure:

[0143] In this embodiment, the Renida luciferase was carried on the pRL-SV40 vector, and the firefly luciferase and NFAT were both located on the pGL4-NFAT(F) vector.

[0144] The specific experimental steps are as follows:

[0145] First, HEK293 cells in good condition with a density of about 70-80% were seeded and passaged in medium containing 10% FBS.

[0146] Then, pRL-SV40 and pGL4-NFAT(F) were co-transfected with TrkB. The transfection reagent mixture (Fugene, DNA and OptiMEM) was incubated at room temperature for about 20 min. After removing the culture medium, 200 μl of the transfection reagent mixture was added. After transfection, the purified recombinant protein sample prepared in Example 2 was added. Fluorescence detection values ​​were obtained after treating at different concentrations for different times.

[0147] 4. Experimental Results:

[0148] The results are as follows Figure 1 As shown, 621D, 621F, 621I, and 621J all demonstrated the ability to activate the receptor. Among the fusion peptides (621F, 621I, and 621J), 621J exhibited the best activation ability at the lowest concentration of 0.5 nM.

[0149] Example 5

[0150] In vivo PK experiment of recombinant protein

[0151] 1. Experimental objective: To administer the recombinant protein of this application to mice via intravenous injection, determine the blood concentration of the recombinant protein in mice, calculate PK parameters, and evaluate the pharmacokinetic properties of the recombinant protein of this application.

[0152] 2. Experimental materials:

[0153] (1) Experimental sample: The purified recombinant protein obtained in Example 2 was prepared in-house.

[0154] (2) Experimental animals: ICR mice, SPF grade, male.

[0155] 3. Test Procedure:

[0156] (1) Dosage information:

[0157] Drug preparation: Calculate the preparation volume based on the weight of the weighed drugs. The concentration of the 621D stock solution is 0.85 mg / mL, and the concentration of the 621J stock solution is 0.75 mg / mL. Prepare a working solution of 0.6 mg / mL using PBS (pH=9) containing 10% glycerol for later use.

[0158] Route of administration: Intravenous injection, dose: 3 mg / kg.

[0159] Dosage frequency and duration: Single dose.

[0160] (2) Experimental methods:

[0161] ICR mice were stratified by body weight and randomly divided into groups of 6 mice each. The mice were fasted overnight before the experiment. After intravenous administration of the drug, 50 μL of blood was collected from the fundus vein at 0, 0.083, 0.25, 0.5, 1, 2, and 4 hours and placed in sample tubes containing the anticoagulant sodium heparin. The tubes were then incubated on wet ice at 4000 rpm. -1 Centrifuge for 10 min to separate the plasma, and freeze it in a -80℃ freezer until analysis.

[0162] (3) Detection method:

[0163] A standard curve was prepared using 621D and 621J, and the protein content in the plasma sample was detected using ELISA (refer to the steps for affinity detection in Example 3).

[0164] 4. Experimental Results:

[0165] The results are as follows Figure 2 As shown, 621D and 621J can be detected in mouse plasma, and 621J has higher plasma exposure and a longer half-life compared to 621D.

Claims

1. A polypeptide comprising an alphabody and BDNF linked by a linker, characterized in that, The Alphabody has a structure represented by the general formula HRS1-L1-HRS2-L2-HRS3, wherein each of HRS1, HRS2, and HRS3 is independently an α-helix structure comprising 2 to 4 consecutive heptapeptide repeating units, which are polypeptide fragments represented as "abcdefg" or "defgabc", wherein at least 50% of all a and d positions are occupied by isoleucine residues, and wherein each HRS begins with an aliphatic or aromatic amino acid residue located at the a or d position; L1 and L2 are each independently a linker fragment.

2. The polypeptide according to claim 1, characterized in that, The linker sequence includes the sequence shown in SEQ ID NO:3 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:3; And / or, the sequence of the BDNF includes the sequence shown in SEQ ID NO:12 or the sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:12; And / or, the sequence of HRS1 includes the sequence shown in SEQ ID NO:13 or SEQ ID NO:14 or the sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:13 or SEQ ID NO:14; And / or, the sequence of HRS2 includes the sequence shown in SEQ ID NO:15 or the sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:15; And / or, the sequence of HRS3 includes the sequence shown in SEQ ID NO:16 or SEQ ID NO:17 or the sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:16 or SEQ ID NO:17; And / or, the sequence of L1 includes the sequence shown in SEQ ID NO:18 or the sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:18; And / or, the sequence of L2 includes the sequence shown in SEQ ID NO:19 or the sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:

19.

3. The polypeptide according to claim 2, characterized in that, The sequence of the Alphabody includes the sequence shown in SEQ ID NO:1 or SEQ ID NO:2, or the sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:1 or SEQ ID NO:

2.

4. The polypeptide according to claim 1, characterized in that, The N-terminus and / or C-terminus of the polypeptide contains tags that assist in expression or purification.

5. The polypeptide according to claim 4, characterized in that, The tag includes one or more of the following: c-Myc tag, HA tag, VSV-G tag, FLAG tag, V5 tag, or HIS tag; preferably, the tag is an HIS tag and a V5 tag; more preferably, the peptide contains an HIS tag at the N-terminus and a V5 tag at the C-terminus.

6. The polypeptide according to claim 1, characterized in that, The sequence of the polypeptide includes sequences such as SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, or sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:

11.

7. An isolated nucleic acid molecule encoding a polypeptide as described in any one of claims 1-6.

8. A vector comprising the nucleic acid molecule as described in claim 7.

9. A host cell comprising the nucleic acid molecule as claimed in claim 7 or the vector as claimed in claim 8.

10. A method for producing a polypeptide as claimed in any one of claims 1-6, the method comprising culturing a host cell as claimed in claim 9 to produce and isolate the polypeptide.

11. A pharmaceutical composition comprising the polypeptide as described in any one of claims 1-6, and further comprising a pharmaceutically acceptable carrier.

12. Use of the polypeptide of any one of claims 1-6, the nucleic acid molecule of claim 7, the carrier of claim 8, the host cell of claim 9, or the pharmaceutical composition of claim 11 in the preparation of a medicament for the treatment or prevention of BDNF-mediated diseases.

13. The use as described in claim 12, characterized in that, The BDNF-mediated diseases include cardiovascular and cerebrovascular diseases; preferably, the cardiovascular and cerebrovascular diseases include one or more of cardiovascular diseases, brain diseases, and brain tumors.

14. The use as described in claim 13, characterized in that, The cardiovascular diseases mentioned include one or more of the following: acute coronary syndrome, aneurysm, angina pectoris, atherosclerosis, aortic stenosis, myocardial fatty degeneration, hypertensive heart disease, pulmonary hypertension, myocardial ischemia-reperfusion injury, cardiomyopathy, coronary heart disease, heart failure, ischemic heart disease, lipotoxic cardiomyopathy, myocardial infarction, and pericardial disease.

15. The use as described in claim 13, characterized in that, The brain diseases mentioned include one or more of the following: encephalitis, Parkinson's disease, epilepsy, Huntington's disease, Alzheimer's disease, Luger's disease, Pick's disease, Kreutzfeldt-Jacob's disease, progressive supranuclear palsy, spinocerebellar degeneration, cerebellar atrophy, multiple sclerosis, stroke, depression, and post-traumatic stress disorder.

16. The use as described in claim 13, characterized in that, The brain tumors include one or more of the following: glioma, glioblastoma, meningioma, astrocytoma, acoustic neuroma, chondroma, medulloblastoma, gangliocytoma, schwannoma, neurofibroma, neuroblastoma, epidural, intramedullary, and intrasheath tumors.

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