WO2016078546A1 - Médicament prévenant la maladie d'alzheimer - Google Patents

Médicament prévenant la maladie d'alzheimer Download PDF

Info

Publication number
WO2016078546A1
WO2016078546A1 PCT/CN2015/094568 CN2015094568W WO2016078546A1 WO 2016078546 A1 WO2016078546 A1 WO 2016078546A1 CN 2015094568 W CN2015094568 W CN 2015094568W WO 2016078546 A1 WO2016078546 A1 WO 2016078546A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
amino acid
tat
ptpn1
sequence
Prior art date
Application number
PCT/CN2015/094568
Other languages
English (en)
Chinese (zh)
Inventor
朱铃强
刘丹
王雄
王玉田
黄和周
Original Assignee
华中科技大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 华中科技大学 filed Critical 华中科技大学
Publication of WO2016078546A1 publication Critical patent/WO2016078546A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the invention relates to the field of drug research and development, and particularly relates to a polypeptide and application thereof in preparing a medicament for treating or preventing senile dementia.
  • AD Alzheimer Disease
  • AD is a common neurodegenerative disease. Its main symptoms are memory loss, low cognitive ability and slow thinking, and progressive aggravation. Finally, life cannot take care of itself and die. ⁇ 10 years. With the acceleration of the aging process of society, the number of patients with AD has risen sharply, and it has become a serious disease that threatens the physical and mental health of the elderly, especially the elderly, and brings serious social, economic and family problems.
  • NFTs neurofibrillary tangles
  • a ⁇ ⁇ -amyloid
  • NFTs Since the discovery that the main component of NFTs is a double helix formed by the aggregation of abnormal, hyperphosphorylated Tau protein (Morris M, Maeda S, Vossel K, Mucke L. The many faces of tau. Neuron. 2011 May 12; 70(3) : 410-26), domestic and foreign scholars generally agree that the abnormality and hyperphosphorylation of the skeleton protein tau is a key step in the formation of NFTs. The accumulation of hyperphosphorylated tau protein is not only present in entangled form, but it is also a major component of dystrophic processes in neuropilic fibrils and senile plaques.
  • Alzheimer's disease There are currently no specific treatments and methods for the pathological features of Alzheimer's disease.
  • Commonly used drugs to alleviate Alzheimer's disease are: (1) Improve choline neurotransmitter: One of the main causes of Alzheimer's disease is memory loss due to choline deficiency.
  • acetylcholinesterase (AchE) inhibitors play an important role in the treatment of senile dementia by enhancing cholinergic effects;
  • drugs that improve cerebral blood circulation and brain cell metabolism these drugs can dilate cerebral blood vessels to improve senile dementia Patient's sugar, protein, nucleic acid, Metabolic disorders such as lipids;
  • calcium antagonists these drugs easily pass the blood-brain barrier, selectively dilate the cerebral blood vessels, thereby improving memory and cognitive function;
  • hormonal drugs mainly relieve the symptoms of female patients.
  • these drugs only slightly improve the symptoms of dementia in patients with advanced senile dementia or delay the progression of dementia. Therefore, the development of new drugs for the pathological features of Alzheimer's disease has become a global expectation.
  • TAT is a kind of cell penetrating peptides, which is an efficient transport carrier discovered in recent years. TAT can penetrate the cell membrane, nuclear membrane, carrying peptides, proteins and DNA molecules to enter the cytoplasm and nucleus through receptor transport to exert corresponding biological effects (Kilic E, Kilic U, Hermann DM.TAT fusion proteins against ischemic stroke: Current status and future perspectives. Front Biosci. 2006 May 1; 11:1716-21). Current research shows that HIV-TAT is able to cross all tissue cells without significant toxic side effects. TAT can bring the polypeptides connected to it into the cells within a few minutes, and can enter the neurons across the blood-brain barrier. The polypeptides brought into the cells retain their original biological activities and thus play a biological role.
  • PTPN1 also known as PTP1B, Tonks NK.Protein tyrosine
  • Phosphases--from housekeeping enzymes to masterregulators of signal transduction FEBS J. 2013 Jan; 280(2): 346-78.
  • Alzheimer's disease transgenic animal model By applying to the in vivo Alzheimer's disease transgenic animal model, it effectively exerts its biological function of blocking the binding of miR-124 to PTPN1 3'-UTR, promotes PTPN1 expression, and improves microtubule-associated protein in senile dementia model animals.
  • Tau hyperphosphorylation, amyloid deposition, and animal learning and memory disorders provide molecular targets for further development of clinical treatments for Alzheimer's disease.
  • polypeptide selected from the group consisting of:
  • polypeptide comprising a substitution, deletion and/or insertion of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide of the invention is TAT-siP-PTPN1, the sequence of which is set forth in SEQ ID NO: 1. That is, the TAT transmembrane peptide (GRKKRRQRRRPRQ) was ligated with siP-PTPN1 (a polypeptide that blocks the binding site of miR-124 and PTPN1, we named silencing peptide PTPN1, abbreviated as siP-PTPN1), of which siP-PTPN1 The sequence is PYGCRVIQRIGNYVIQHVASNNVEKIGGYVIRHVGGYVIRHVGNYVIQHVGNYVIQHVGCRVIQRIL.
  • the polypeptide of the present invention utilizes the transmembrane function of TAT to transport the siP-PTPN1 polypeptide into the bloodstream through the blood-brain barrier and is taken up by the brain nerve cells to exert its biological function.
  • a tag polypeptide such as HA (YPYDVPDYA)
  • HA YPYDVPDYA
  • a preferred polypeptide sequence of the invention is the sequence in which HA (YPYDVPDYA) is added at the end of SEQ ID NO: 1.
  • a preferred polypeptide of the invention is TAT-siP-PTPN1-HA (SEQ ID NO: 4).
  • Another object of the invention is also to provide a nucleotide sequence encoding a polypeptide of the invention.
  • the nucleotide sequence of the present invention is a nucleotide sequence encoding the small molecule polypeptide TAT-siP-PTPN1, preferably shown by SEQ ID NO: 2, but is not limited thereto. Due to the degeneracy of the codons, all nucleotide sequences encoding TAT-siP-PTPN1 polypeptides are within the scope of the invention.
  • the nucleotide sequence encoding the TAT-siP-PTPN1-HA polypeptide is SEQ ID NO:3.
  • the invention still further provides a nucleic acid construct comprising a nucleotide sequence of the invention operably linked to one or more controls that direct production of the polypeptide in a suitable host sequence.
  • the invention still further provides a recombinant expression vector comprising the nucleic acid construct.
  • Another object of the present invention is to provide a use of the polypeptide of the present invention for the preparation of a medicament for the treatment or prevention of Alzheimer's disease.
  • the present invention provides the use of a polypeptide TAT-siP-PTPN1 for the preparation of a medicament for treating or preventing Alzheimer's disease.
  • the polypeptide of the present invention is made into an intravenous injection, and after intravenous injection, it is found to be effective for improving hyperphosphorylation, amyloid deposition, animal learning and/or memory of the microtubule-associated protein tau exhibited by the senile dementia model animal. Obstacles provide molecular targets for further development of drugs for clinical treatment of Alzheimer's disease.
  • Another object of the present invention is to provide a medicament comprising the small molecule polypeptide of the present invention. Composition.
  • the technical measures adopted by the present invention are: Applicants have found that the 3'-UTR of m iR-124 and PTPN1 in the dementia model animal bind to each other and mediate the hyperphosphorylation of the downstream microtubule-associated protein tau, Amyloid deposition, as well as animal learning and memory disorders. Blocking the 3'-UTR of miR-124 and PTPN1 to each other can effectively improve the above symptoms.
  • the polypeptide enters the bloodstream and crosses the blood-brain barrier and is taken up by the brain's nerve cells to exert its biological function.
  • TAT-scramble-HA SEQ ID NO: 5
  • TAT-siP-PTPN1-HA SEQ ID NO: 4
  • This control sequence was obtained by artificially scrambling the siP-PTPN1 sequence.
  • the TAT-siP-PTPN1-HA polypeptide and the control TAT-scramble-HA were obtained by the applicant by prokaryotic expression, collection, and purification.
  • the invention proves that TAT-siP-PTPN1-HA has an exact therapeutic effect on the Alzheimer's model by applying TAT-siP-PTPN1-HA in an animal model of Alzheimer's disease.
  • the present invention has the following characteristics: (1) The small molecule polypeptide of the present invention is easy to induce expression purity, is completely soluble, is suitable for intravenous injection, and has no toxic side effects. (2) The polypeptide disclosed by the present invention is taken up by neurons through the blood-brain barrier, and can be transformed into a nervous system disease such as Alzheimer's disease, so that it has practical feasibility.
  • Coding sequence means a polynucleotide that directly specifies the amino acid sequence of a polypeptide.
  • the boundaries of the coding sequence are generally determined by an open reading frame that begins with a start codon (such as ATG, GTG or TTG) and ends with a stop codon (such as TAA, TAG or TGA).
  • the coding sequence can be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • control sequence means a nucleic acid sequence necessary for expression of a polynucleotide encoding a mature polypeptide of the invention.
  • Each control sequence may be native (ie, from the same gene) or foreign (ie, from a different gene) to the polynucleotide encoding the polypeptide, or be native or foreign to each other.
  • control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At least, the control sequence includes a promoter, And transcription and translation termination signals.
  • These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linkage of these control sequences to the coding region of a polynucleotide encoding a polypeptide.
  • expression includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • Expression vector means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to a control sequence providing for its expression.
  • host cell means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
  • host cell encompasses any progeny of a parent cell that is different from the parent cell due to mutations that occur during replication.
  • nucleic acid construct means a single or double stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a manner that is not inherent in nature. A segment containing a nucleic acid, or a synthetic nucleic acid molecule comprising one or more control sequences.
  • operably linked means a construct in which the control sequence is placed in position relative to the coding sequence of the polynucleotide such that the control sequence directs expression of the coding sequence.
  • Sequence identity The degree of association between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
  • EMBOSS European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.) 16:276-277
  • Needleman-Wunsch algorithm implemented in the Needle program (Needleman and Wunsch) (1), J. Mol. Biol. 48: 443-453) to determine sequence identity between two amino acid sequences.
  • These parameters used are gap open penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of the "longest identity" of the Nieder annotation (obtained using the -non-simplification option) is used as the percent identity and is calculated as follows: (consistent residue ⁇ 100) / (alignment length - total number of gaps in the alignment)
  • Niedel as in the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al, 2000, supra) (preferably version 5.0.0 or later)
  • the Nederman-Wengsch algorithm (Nedleman and Wunsch, 1970, supra) implemented in the program was used to determine sequence identity between two deoxyribonucleotide sequences. These parameters used are gap open penalty of 10, gap extension penalty of 0.5, and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of the "longest identity" of the Niedel label (obtained using the -non-simplification option) is used as the percent identity and is calculated as follows: (consistent deoxyribonucleotide ⁇ 100) / (alignment length - ratio The total number of vacancies in the pair)
  • the present invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide of the present invention has the polypeptide of the amino acid sequence of SEQ ID NO: 1.
  • the present invention also relates to a polypeptide comprising at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:1, which polypeptides are capable of For the treatment or prevention of senile dementia.
  • the invention relates to a polypeptide comprising a substitution, deletion and/or insertion of one or more (several) amino acid residues in the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide of the invention differs from the mature polypeptide of SEQ ID NO: 1 by no more than 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids.
  • amino acid changes can have subtle properties, ie, conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein.
  • conservative substitutions are within the scope of the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and day) Asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine) , threonine and methionine).
  • Amino acid substitutions that do not generally alter specific activity are known in the art and are for example, by H. Neurat and RL Hill, 1979, The Proteins, Academic Press (Academic Press) ), described in New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg , Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
  • amino acid changes have the property of altering the physicochemical properties of the polypeptide.
  • amino acid changes can increase the thermal stability of the polypeptide, alter substrate specificity, change the pH optimum, and the like.
  • Single or multiple amino acid substitutions, deletions and/or insertions can be made and tested using known methods of mutagenesis, recombination and/or shuffling, followed by relevant screening procedures, such as by Reidhaar (Reidhaar) -Olson) and Sauer, 1988, Science 241: 53-57; Bowie and Saar, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or those disclosed in WO 95/22625.
  • Other methods that may be used include error-prone PCR, phage display (e.g., Lowman et al, 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) and regional targeting Change (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
  • the mutagenesis/shuffling method can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896).
  • Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide.
  • Variants of these polypeptides may be based on a polynucleotide presented in the form of a mature polypeptide coding sequence (eg, a subsequence thereof), and/or by introduction of an amino acid sequence that does not alter the polypeptide, but corresponding to a host intended to produce the enzyme
  • the nucleotide substitution of the codon used by the organism, or by the introduction of nucleotide substitutions that may result in different amino acid sequences For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.
  • Mutations in the putative contact site amino acid can also be combined, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling. Physical analysis of the structure to determine the active site of the enzyme or other biological interactions. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899 - 904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. It is also possible to infer the identification of essential amino acids from the alignment with related polypeptides.
  • a tagged amino acid sequence such as HA, AviTag, Calmodulin-tag, Strep, Flag-tag, His, myc, Xpress, V5, TC, VSV, etc.
  • the detection does not affect the biological function of the peptide/protein.
  • a start codon such as ATG, is added, so a methionine (M) is added to the beginning of the translated polypeptide, which is required for expression of the polypeptide without affecting the polypeptide/protein.
  • M methionine
  • the invention also relates to nucleic acid constructs comprising a polynucleotide of the invention operably linked to one or more control sequences which, under conditions compatible with the control sequences, direct the coding sequence at the appropriate Expression in host cells.
  • Polynucleotides can be manipulated in a variety of ways to provide expression of the polypeptide. Depending on the expression vector, manipulation of the polynucleotide prior to its insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.
  • the control sequence may be a promoter, i.e., a polynucleotide recognized by a host cell to express a polynucleotide encoding a polypeptide of the present invention.
  • the promoter comprises transcriptional control sequences that mediate expression of the polypeptide.
  • the promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated, and heterozygous promoters, and may be extracellularly encoded by a homologous or heterologous source encoding the host cell. Or the gene acquisition of the intracellular polypeptide.
  • the invention also relates to recombinant expression vectors comprising the polynucleotides, promoters, and transcriptional and translational termination signals of the invention.
  • Different nucleotide and control sequences can be joined together to create a recombinant expression vector which can include one or more convenient restriction sites. Polynucleotides encoding the variant are allowed to be inserted or substituted at these sites.
  • the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
  • the coding sequence is located in the vector such that the coding sequence is operably linked to the appropriate control sequence for expression.
  • the invention also relates to recombinant host cells comprising a polynucleotide of the invention operably linked to one or more control sequences which direct the production of a polypeptide of the invention .
  • the construct or vector comprising the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier.
  • the host cell can be any cell useful for recombinant production of a polypeptide of the invention, such as a prokaryotic cell (bacterial cell) or a eukaryotic cell (e.g., a mammalian, insect, plant, fungal cell).
  • Figure 1 is a Coomassie blue staining of a small molecule polypeptide TAT-siP-PTPN1-HA.
  • Figure 2 is a diagram of immunoblot staining of a small molecule polypeptide TAT-siP-PTPN1-HA.
  • Figure 3 is a statistical diagram of the water maze results of TAT-siP-PTPN1-HA in improving learning and memory in Tg2576 mice.
  • Figure 4 is a graph showing the results of TAT-siP-PTPN1-HA improving the phosphorylation of tau protein in Tg2576 mice.
  • Figure 5 is a graph showing the improvement of A ⁇ deposition results in Tg2576 mice by TAT-siP-PTPN1-HA.
  • Figure 6 is a statistical diagram showing the results of Golgi staining of learning and memory in Tg2576 mice by TAT-siP-PTPN1-HA.
  • Figure 7 is a graph showing the results of long-term potentiation of learning and memory in Tg2576 mice by TAT-siP-PTPN1-HA.
  • the present invention can improve the senile plaque, tau hyperphosphorylation and senile senescence of senile dementia model animals by injecting TAT-siP-PTPN1-HA polypeptide into 12-month-old Tg2576 mice (purchased from The Jackson Laboratory). Learning and / or memory impairment.
  • polypeptides TAT-scramble-HA and TAT-siP-PTPN1-HA were directly dissolved in physiological saline to a final concentration of 10 mg/kg.
  • mice Male Tg2576 mice, SPF grade, weighing 25-32 grams, 20, were housed in a conventional environment.
  • Experimental group 1 control group (wild type control mouse Tg2576 mice); 2 model group (Tg2576 mice injected with TAT-scramble-HA); 3 treatment group (Tg2576 injected with TAT-siP-PTPN1-HA small) Rat); 10 per group.
  • Tg2576 mice 12-month-old Tg2576 mice were given a TAT-scramble-HA polypeptide by a single injection in the tail vein at a dose of 10 mg/kg.
  • Tg2576 mice 12-month-old Tg2576 mice were given TAT-siP-PTPN1-HA polypeptide by single injection in the tail vein at a dose of 10 mg/kg.
  • the nucleotide sequence of the peptide was synthesized by Wuhan Qingke Company, and it was constructed into the prokaryotic expression vector pGEX-5X-1 (purchased from GE Healthcare China) by molecular biology technology, and was competent in BL21 (purchased from Beijing full version). Transformation and amplification in the gold company).
  • pGEX-5X-1 purchased from GE Healthcare China
  • BL21 purchased from Beijing full version
  • Transformation and amplification in the gold company When the bacterial solution OD600 reached 0.6, 2 mM IPTG was added to induce 0.5 hour, 1 hour, and 2 hours, respectively, and finally, protein expression was observed by SDS-PAGE electrophoresis and Coomassie blue staining.
  • polypeptide After the polypeptide was induced to express, it was purified, and subjected to shearing using Factor Xa enzyme (purchased from NEB) to isolate the small molecule polypeptide of interest (about 8 kDa). Protein samples were purified and assayed by Western blotting using HA-specific antibodies (purchased from Abcam). Expression of TAT-siP-PTPN1-HA was detected by staining for specific antibodies recognizing HA, and the results confirmed the expression was successful.
  • Factor Xa enzyme purchased from NEB
  • mice's ability to learn and remember Morris water maze training and testing (see Figure 3)
  • the Morris water maze test system was used in the experiment, including a circular pool with a diameter of 120 cm and a height of 60 cm.
  • the cylindrical plexiglass platform has a diameter of 10 cm and a height of 40 cm.
  • the water level in the pool is about 45cm high, and the room temperature and water temperature are kept at 26 ⁇ 2°C.
  • the platform is placed at the center of a certain quadrant and is not about 2cm below the water surface.
  • the mice were placed in the water from the head of the 1/2 radians in either quadrant to the pool wall, and the incubation period (ie, the time from the entry point of the mouse to the platform) was measured.
  • the path was used to measure the learning and memory of the mice.
  • the indicator of test scores was used in the experiment, including a circular pool with a diameter of 120 cm and a height of 60 cm.
  • the cylindrical plexiglass platform has a diameter of 10 cm and a height of 40 cm.
  • the water level in the pool is about 45cm
  • the swimming time limit was 60 seconds. That is, the platform was not automatically found within 60 seconds.
  • the incubation period was recorded as 60 seconds. The tester guided him to the stage and rested. The next training will take place after 30 seconds.
  • the first training time was 6 days. On the 7th day, the time required for the mice to find the plateau was recorded, and the latency required for the mice to find the platform was recorded. Take a day off, go to the platform, calculate the dwell time of the mouse in the target quadrant within 1 minute, and analyze the number of crossings at the position of the platform to evaluate its memory ability.
  • mice in the treatment group had simple trajectory recovery and shorter latency; the mice in the model group had the shortest time and distance through the platform, and the number of times was the least, indicating that the mice in the model group Memory ability is impaired, the position of the platform is not remembered; the mice in the treatment group recovered to the time and distance of crossing the platform compared to the model group. Normally consistent, the number of crossing platforms increased accordingly, and its memory ability was significantly restored after TAT-siP-PTPN1-HA administration.
  • mice were sacrificed by decapitation of the cervical vertebrae, and the brain tissue was quickly removed and placed in a 0-4 ° C, 0.05 M Tris buffered saline solution (TB, pH 7.0) to rapidly separate the bilateral hippocampus.
  • Tris buffered saline solution TB, pH 7.0
  • the % protein homogenate was centrifuged at 1000 rpm for 5 minutes, the supernatant was taken, and the protein content was determined and used.
  • Sample preparation was performed with immunoblotting, and the antibody was washed with A ⁇ 42 antibody (purchased from A ⁇ eta GmBH, Germany), and the sample was added to develop color with tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB). And read at a wavelength of 450 nm on a spectrophotometer.
  • a ⁇ 42 has a high toxic effect.
  • the results showed that the release of A ⁇ 42 fragment in the model group was significantly increased.
  • TAT-siP-PTPN1-HA After treatment with TAT-siP-PTPN1-HA, the A ⁇ 42 in the treatment group returned to the normal group, further demonstrating that TAT-siP-PTPN1-HA can improve Alzheimer's disease.
  • mice were anesthetized and fixed on a stereotaxic instrument, and local craniotomy was performed.
  • the stimulation electrodes and recording electrodes were placed in the hippocampal CA3 and CA1 regions, respectively. Stimulate the field potential induced by the CA3 region. Select the appropriate stimulus and record electrode position. I/O curves were drawn, and 40% of the maximum amplitude of the induced fEPSP was selected as the stimulation intensity.
  • a series of high frequency stimulation 100 Hz, 1 second
  • Long-term time-course enhancement (LTP) recorded for 120 minutes.
  • the indicator of tau protein was related to Alzheimer's disease.
  • the A ⁇ content was also significantly reduced compared with the model group, indicating that TAT-siP-PTPN1-HA can alleviate the abnormal hyperphosphorylation of tau in the hippocampus of Tg2576 mice, reduce amyloid A ⁇ deposition and improve animal learning and/or memory impairment.
  • the polypeptide of the present invention can be used for the prevention and treatment of senile dementia.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un polypeptide de faible poids moléculaire, une séquence nucléotidique codant pour le polypeptide et l'utilisation du polypeptide dans la préparation d'un médicament pour le traitement et la prévention de la maladie d'Alzheimer.
PCT/CN2015/094568 2014-11-20 2015-11-13 Médicament prévenant la maladie d'alzheimer WO2016078546A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410668134.0A CN104479027B (zh) 2014-11-20 2014-11-20 一种防治老年痴呆的药物
CN201410668134.0 2014-11-20

Publications (1)

Publication Number Publication Date
WO2016078546A1 true WO2016078546A1 (fr) 2016-05-26

Family

ID=52753644

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/094568 WO2016078546A1 (fr) 2014-11-20 2015-11-13 Médicament prévenant la maladie d'alzheimer

Country Status (2)

Country Link
CN (1) CN104479027B (fr)
WO (1) WO2016078546A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104479027B (zh) * 2014-11-20 2017-06-16 华中科技大学 一种防治老年痴呆的药物
CN105237628B (zh) * 2015-11-17 2018-08-07 南开大学 一种用于治疗阿尔兹海默症的多肽
CN109553685B (zh) * 2017-09-25 2021-07-27 华中科技大学 一种小分子多肽及其在制备防治缺血性脑卒中药物中的应用
KR20210035825A (ko) * 2018-07-19 2021-04-01 제넨테크, 인크. 마커 분자를 기반으로 아밀로이드-양성 치매를 갖거나 발생 위험성이 있는 개체를 식별하는 방법 및 관련 용도
CN111253492B (zh) * 2018-11-30 2021-10-08 华中科技大学 一种透脑性多肽及其在制备防治老年痴呆药物中应用
CN110003342B (zh) * 2018-12-29 2020-09-08 华中科技大学 一种多肽及其在改善低社会等级对机体不良影响中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012032519A2 (fr) * 2010-09-07 2012-03-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Méthodes de diagnostic de la maladie de parkinson
CN104479027A (zh) * 2014-11-20 2015-04-01 华中科技大学 一种防治老年痴呆的药物

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009070805A2 (fr) * 2007-12-01 2009-06-04 Asuragen, Inc. Gènes régulés par le mir-124 et cheminements servant de cibles pour une intervention thérapeutique
CN101974089B (zh) * 2010-06-11 2012-09-26 华中科技大学 重组Trx-TAT-hMsrA融合蛋白及其在神经细胞保护方面的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012032519A2 (fr) * 2010-09-07 2012-03-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Méthodes de diagnostic de la maladie de parkinson
CN104479027A (zh) * 2014-11-20 2015-04-01 华中科技大学 一种防治老年痴呆的药物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, FANG ET AL.: "microRNA-124 and nervous system diseases", CHINESE JOURNAL OF NEUROLOGY, vol. 46, no. 1, 31 January 2013 (2013-01-31) *

Also Published As

Publication number Publication date
CN104479027B (zh) 2017-06-16
CN104479027A (zh) 2015-04-01

Similar Documents

Publication Publication Date Title
WO2016078546A1 (fr) Médicament prévenant la maladie d'alzheimer
Stetler et al. Heat shock proteins: cellular and molecular mechanisms in the central nervous system
Tofaris et al. Physiological and pathological properties of α-synuclein
Gomes et al. Aquaporins are multifunctional water and solute transporters highly divergent in living organisms
El-Agnaf et al. Formation and properties of amyloid-like fibrils derived from α-synuclein and related proteins
Ecroyd et al. Crystallin proteins and amyloid fibrils
JP6921006B2 (ja) 老化関連症状を治療するための方法および組成物
Cavallarin et al. The role of phosphorylation in synucleinopathies: focus on Parkinson's disease
JP2012176978A (ja) プロテインキナーゼｃのペプチドインヒビター
US10662419B2 (en) Cell-permeable (ICP) parkin recombinant protein and use thereof
JP2017524380A (ja) 腫瘍血管遮断剤であるポリペプチド、遺伝子、発現ベクター、及びその使用方法
CN110041420A (zh) 虎纹捕鸟蛛毒素-iv变体及其使用方法
KR20160064192A (ko) 프로톡신-ii 변이체 및 사용 방법
CN107325168B (zh) 一种经修饰的生长分化因子及其制备方法和应用
KR20170120703A (ko) 프로톡신-ii 변이체 및 사용 방법
CN107921089A (zh) 原毒素‑ii变体及使用方法
CN103097404B (zh) 具有镇痛作用并抑制asic通道的新型肽
CN110041408B (zh) 一种小分子多肽及其在制备防治帕金森综合症药物中的应用
Yamamoto et al. Effects of the Polyphenols Delphinidin and Rosmarinic Acid on the Inducible Intra-Cellular Aggregation of Alpha-Synuclein in Model Neuron Cells
WO2008120263A2 (fr) Antagonistes des récepteurs des prokinéticines, leurs dérivés et leurs utilisations
KR20140038396A (ko) 다중돌연변이체 타우 단백질 변이체 및 인간 타우 병변을 재현하기 위한 그의 용도
EP4046648A1 (fr) Utilisation de la molécule monomère de la neurotoxine post-synaptique de serpents de la famille des elapidae dans le traitement de la maladie d'alzheimer
JP2004508827A (ja) カルシウム結合タンパク質
CN112194730A (zh) 一种多肽ti-16及其用途
Galán-Acosta Recombinant Brichos Domains Delivered over the Blood-Brain Barrier: A Possible Way to Treat Alzheimer´ s Disease

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15860639

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15860639

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 15860639

Country of ref document: EP

Kind code of ref document: A1