WO2023045662A1 - 多肽的抗抑郁、抗焦虑用途及复合体 - Google Patents

多肽的抗抑郁、抗焦虑用途及复合体 Download PDF

Info

Publication number
WO2023045662A1
WO2023045662A1 PCT/CN2022/113892 CN2022113892W WO2023045662A1 WO 2023045662 A1 WO2023045662 A1 WO 2023045662A1 CN 2022113892 W CN2022113892 W CN 2022113892W WO 2023045662 A1 WO2023045662 A1 WO 2023045662A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
ht2ar
tat
mice
anxiety
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2022/113892
Other languages
English (en)
French (fr)
Inventor
谭震
余治健
李书鹏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Chenyang Biological Technology Co Ltd
Original Assignee
Shenzhen Chenyang Biological Technology Co Ltd
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 Shenzhen Chenyang Biological Technology Co Ltd filed Critical Shenzhen Chenyang Biological Technology Co Ltd
Priority to JP2024518969A priority Critical patent/JP2024535104A/ja
Priority to EP22871715.3A priority patent/EP4406547A4/en
Priority to KR1020247011564A priority patent/KR20240058153A/ko
Priority to US18/695,187 priority patent/US20240390459A1/en
Publication of WO2023045662A1 publication Critical patent/WO2023045662A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1787—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Definitions

  • the present invention relates to the fields of antidepression and anti-anxiety, in particular to the use of polypeptides in anti-depression and anti-anxiety.
  • Depression is a mental disorder with depression as the main clinical symptom caused by various reasons. It has the characteristics of high morbidity, disability and suicide. The pathogenesis of depression has not been fully elucidated, and most of the commonly used antidepressants in clinical practice are developed based on the "monoamine neurotransmitter hypothesis".
  • depression is related to the lack of monoamine neurotransmitters such as dopamine (Dopamine, DA), 5-hydroxytryptamine (5-hydroxytryptamine, 5-HT), norepinephrine (NA) in the brain; antidepressant Drugs block the reuptake of monoamine neurotransmitters by inhibiting the function of 5-HT and NA transporters, increasing their concentration in the synaptic cleft, thereby improving depressive symptoms.
  • Dopamine dopamine
  • 5-HT 5-hydroxytryptamine
  • NA norepinephrine
  • first-generation antidepressant drugs monoamine oxidase inhibitors (MAOI) and tricyclic antidepressant drugs
  • second-generation antidepressant drugs First-generation antidepressants: selective serotonin reuptake inhibitors (SSRIs); third-generation antidepressants: dual serotonin and norepinephrine reuptake inhibitors, norepinephrine reuptake inhibitors (SNRIs) And norepinephrine and serotonergic antidepressants.
  • NMDA receptors body, CRF1 receptor, ⁇ receptor, ⁇ receptor, GABAB receptor, M choline receptor, IDO, CysLT1R, PDE4, PPAR ⁇ , PPAR ⁇ , NOS, etc.
  • NMDA receptors body, CRF1 receptor, ⁇ receptor, ⁇ receptor, GABAB receptor, M choline receptor, IDO, CysLT1R, PDE4, PPAR ⁇ , PPAR ⁇ , NOS, etc.
  • the inventors conducted a lot of research based on the molecular changes of depression models, and found a new mechanism of depression pathogenesis, thus completing the present invention.
  • 5-HT receptors are a group of G protein-coupled receptors and ligand-gated ion channels that appear centrally in the central nervous system and peripherally in the peripheral nervous system.
  • 5HT receptors can be divided into seven subfamilies 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7.
  • 5-HT2 receptors have A, B, C Three subtypes, namely, three receptor proteins of 5-HT2A, 5-HT2B and 5-HT2C.
  • 5-HT2A receptor 5-HT2AR
  • the DA receptor is an in vivo receptor that acts through its corresponding membrane receptor.
  • DA receptors can be divided into five types: D1, D2, D3, D4 and D5.
  • D1 receptor (D1R) is widely expressed in the brain.
  • 5-HT2AR regulates neurotransmitters not only in a single form but also in a complex form, such as 5-HT1AR/5-HT2AR and 5-HT2AR/oxytocin receptor (OXTR).
  • the present invention provides a polypeptide having an amino acid sequence (SKDNSDGVNEKVSCV) as shown in SEQ ID NO:1.
  • SKDNSDGVNEKVSCV amino acid sequence having an amino acid sequence as shown in SEQ ID NO:1.
  • the use of the polypeptide of the present invention in the preparation of medicines for treating and/or preventing depression or anxiety is also provided.
  • the length of the polypeptide of the present invention may be 15 to 90 aa, especially 15 to 50 aa, 15 to 30 aa, 15 to 20 aa.
  • polypeptides of the invention may be derived from 5-HT2AR.
  • the 5-HT2AR derived from the polypeptide can be queried through biological information banks (such as Genbank, EMI, DDBJ, etc.), and the polypeptide derived from 5-HT2AR can be confirmed on this basis.
  • biological information banks such as Genbank, EMI, DDBJ, etc.
  • the polypeptide in addition to containing the sequence shown in SEQ ID NO: 1 (ie, the 457th to 471st positions of the amino acid sequence in NP_001365853), the polypeptide can also be in One or more amino acid residues are included in the N-terminal direction.
  • polypeptide of the present invention consists of positions 442 to 471 of the amino acid sequence corresponding to NP_001365853.
  • polypeptide of the present invention consists of positions 385 to 471 of the amino acid sequence corresponding to NP_001365853.
  • the 5-HT2AR is derived from a primate.
  • the 5-HT2AR is of human origin.
  • treating means causing a desired or beneficial effect in a patient, which may include reducing the frequency or severity of one or more symptoms of a disease, or suppressing or Inhibiting the further development of a disease, condition or disorder.
  • prevention refers to preventing or delaying the onset of a disease, or preventing the appearance of clinical or subclinical symptoms thereof.
  • the polypeptide of the present invention plays an antidepressant role through a new mechanism (5-HT2AR/D1R complex) in the pathology of depression, has the characteristics of fast onset, good activity and small side effects, and has good clinical development value .
  • the polypeptide of the present invention can also achieve anxiolytic effect.
  • polypeptides of the present invention disrupt the interaction of the 5-HT2AR/D1R complex, but have no effect on 5-HT2AR or D1R expression was not significantly affected.
  • the polypeptides of the invention act by specifically disrupting the interaction of the 5-HT2AR/D1R complex.
  • the invention provides a nucleic acid molecule encoding a polypeptide of the invention.
  • the use of the nucleic acid molecule of the present invention in the preparation of a medicament for treating and/or preventing depression or anxiety is also provided.
  • the nucleic acid molecule of the present invention is used to produce the antigenic peptide of the present invention, and those skilled in the art can appropriately adjust the sequence of the nucleic acid molecule according to the expression system used.
  • the nucleic acid molecule has a sequence as shown in SEQ ID NO:2.
  • the invention provides an expression vector comprising a nucleic acid molecule of the invention.
  • the use of the expression vector of the present invention in the preparation of medicines for treating and/or preventing depression or anxiety is also provided.
  • Nucleic acid sequences of the present invention can be inserted into expression vectors using various known methods.
  • nucleic acid molecules can be inserted into appropriate restriction endonuclease sites.
  • Standard techniques for cloning, isolation, amplification and purification, as well as enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonuclease, etc., and various separation techniques in operation are known to those skilled in the art and commonly used techniques.
  • the present invention provides a host cell comprising a nucleic acid molecule or expression vector of the present invention.
  • a host cell comprising a nucleic acid molecule or expression vector of the present invention.
  • the use of the host cell of the present invention in the preparation of medicines for treating and/or preventing depression or anxiety is also provided.
  • Various expression systems such as expression vectors and host cells in prokaryotic expression systems and eukaryotic expression systems, can be used to produce the polypeptides of the present invention.
  • Host cells can include COS-7 cell line of monkey kidney fibroblasts and other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, Hela and BHK cells Tie.
  • Mammalian expression vectors should contain an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking non- Transcribed sequence.
  • DNA sequences derived from, for example, the SV40 splice and polyadenylation sites can be used to provide the desired non-transcribed genetic elements.
  • Expression vectors can be introduced into host cells by a variety of methods familiar to those skilled in the art, including but not limited to, for example, calcium phosphate transfection, DEAE-dextran-mediated transfection, or electroporation.
  • the present invention provides a complex comprising the polypeptide of the present invention and a (transport) carrier linked thereto for permeating the blood-brain barrier.
  • the carrier for permeating the blood-brain barrier may be: HIV-1 Tat protein, insulin, cationized albumin, anti-rat transferrin receptor monoclonal antibody (OX26), human insulin receptor One or more of human murine monoclonal antibody (HIRMAb), Penetratin, transduction domain of Tat protein, Pep-1 peptide, S4 13 -PV, Magainin 2 and Buforin 2.
  • the TAT transduction domain can be transduced into cells across the membrane, and its amino acid is YGRKKRRQRRR (as shown in SEQ ID NO: 3).
  • the polypeptide of the present invention can be linked with a carrier for permeating the blood-brain barrier through appropriate linking techniques.
  • Exemplary linking techniques may be avidin-biotin technology, polyethylene glycol (PEG) based spacer technology, fusion protein technology, and the like.
  • PEG polyethylene glycol
  • the polypeptide of the present invention can be directly linked to the transduction domain of Tat protein by fusion protein technology.
  • a linker can also be used to link the polypeptide of the present invention with a carrier for permeating the blood-brain barrier.
  • exemplary linkers may be flexible linkers with glycine, such as G, GSG, GSGGSG, GSGGSGG, GSGGSGGG, GGGGSGGG, GGGGS, and SGG, among others.
  • the present invention provides a method of treating depression in a subject suffering from or at risk of depression comprising:
  • An effective amount of a polypeptide or complex of the invention is administered to the subject.
  • the polypeptide or complex reduces 5-HT2AR/D1R interaction, thereby treating depression in the subject.
  • a method of treating an anxiety disorder in a subject, the subject having or at risk of having an anxiety disorder comprising:
  • An effective amount of a polypeptide or complex of the invention is administered to the subject.
  • the polypeptide or complex reduces 5-HT2AR/D1R interaction, thereby treating anxiety in the subject.
  • an effective amount refers to an amount of an active agent sufficient to induce a desired biological result.
  • the result can be alleviation of a sign, symptom or cause of a disease, or any other desired change in a biological system.
  • therapeutically effective amount is used herein to mean any amount of a formulation which, when applied repeatedly to an affected area over a period of time, results in a substantial improvement in the condition of the disease. The amount will vary with the condition being treated, the stage of progression of the condition and the type and concentration of formulation employed. Those skilled in the art can determine the appropriate amount by routine experimentation.
  • Subject refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, rodents, monkeys, humans, farm animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vitro or cultured in vitro.
  • the invention provides a polypeptide or complex of the invention for use as a medicament.
  • use as a medicament refers to use in the treatment or prevention of depression or anxiety.
  • Figure 1 shows the immunoblot results of the co-immunoprecipitation of the hippocampal tissue of mice
  • Figure 2 shows the immunoblot results of co-immunoprecipitation carried out in CRS, CMS and CSDS mouse models
  • Fig. 3 shows the position of the fragment involved in embodiment 3 on 5-HT2AR
  • Figure 4 shows the Western blot results of GST pulldown analysis of mouse hippocampal tissue using the fragments in Example 3;
  • Figure 5 shows the immunoblot results of co-immunoprecipitation of hippocampal tissues of mice treated with polypeptides
  • Figure 6 shows the results of immunoblotting of the hippocampus of mice treated with polypeptides
  • FIG. 7 shows the results of OFT, FST and TST analysis on mice treated with polypeptides, wherein, *: P ⁇ 0.05; **: P ⁇ 0.01;
  • Figure 8 shows the results of FST, TST and SPT analysis of CRS, CMS and CSDS mice treated with polypeptides, wherein, *: P ⁇ 0.05; **: P ⁇ 0.01; ***: P ⁇ 0.001; ****: P ⁇ 0.0001;
  • Figure 9 shows the immunoblot results of co-immunoprecipitation of hippocampal tissues of CRS, CMS and CSDS mice treated with polypeptides
  • Figure 10 shows the result of immunoblotting of the hippocampal tissue of CRS mice treated with polypeptides
  • FIG 11 shows the results of OFT (for central zone residence time) analysis of mice treated with polypeptides, wherein **: P ⁇ 0.01;
  • Figure 12 shows the results of EPM analysis on mice treated with polypeptides, where *: P ⁇ 0.05; **: P ⁇ 0.01.
  • mice Unless otherwise specified, the experimental animals used in the examples were adult C57BL/6J male mice (12-14 weeks), which were housed at 18-22° C. under 12 hours of light/12 hours of darkness. Food and drink and tap water were freely available throughout.
  • Co-immunoprecipitation was performed using protein samples (100-500 ⁇ g of protein) from mouse hippocampal tissue. Precipitation was performed using an anti-5-HT2AR mouse antibody (SantaCruz Biotechnology, sc-166775) and 25 ⁇ l protein A/G+agarose bead slurry (santaCruz Biotechnology, sc-2001).
  • the anti-5-HT2AR antibody can precipitate D1R, but not D2R, which indicates that 5-HT2AR can form a complex with D1R in vivo, but cannot form a complex with D2R.
  • CRS chronic restraint stress stress
  • CMS chronic moderate stress stress
  • CSDS chronic social stress stress
  • mice were fixed horizontally for 6 hours (10:00 to 16:00) every day in an acrylic cylindrical flat-bottomed restraint (25 ⁇ 90 mm) for 2 weeks.
  • the filter has several slots to securely restrain mice according to the size of each mouse and inhibit physical movement of limbs without causing pain. After being restrained, mice were immediately returned to their cages. Unrestrained mice (control) remained in their home cages when the CRS procedure was not performed, and neither control nor CRS mice had access to food and water during the CRS exposure period.
  • mice were subjected to various stresses, including: restraint (4 hours), cage tilting (45 times, 12 hours each), light-dark cycle reversal (once), flashing light (12 hours) and dirty Cage (2 times, 14 hours each time). The program lasted 6 weeks. Control mice received no corresponding stress.
  • mice were exposed to different CD1-aggressive mice (target mice) for 10 minutes a day for a total of 10 days. After 10 min of exposure, the experimental mice were separated by a plastic divider containing holes. In addition, experimental mice were exposed to chronic stress for 24 hours through a separator placed in the middle of the cage. Control mice were subjected to a similar procedure (non-social stress), but using mice of the same breed instead of aggressive mice.
  • the 5-HT2AR full-length cDNA clone (Genbank accession number: NM_001378924) was first amplified to obtain the CT region (K385-V471) of 5-HT2AR and the third cDNA fragment of the intracellular loop (IL3) region (F255-V324). These fragments were subcloned into the BamH1/EcoR1 or BamH1/Xho1 sites of the pGEX-4T-3 plasmid (Youbo no: VT1255). Initiating methionine residues and stop codons were incorporated where appropriate.
  • CT region of 5-HT2AR can pulldown D1R.
  • the GST fusion protein (GST-5HT2AR-KV) containing the KV region of 5-HT2AR, the GST fusion protein (GST-5HT2AR-NN) containing the NN region of 5-HT2AR, and the GST fusion protein (GST-5HT2AR-NN) containing 5-HT2AR were used.
  • the GST fusion protein in the DV region of HT2AR (GST-5HT2AR-DV) was subjected to pulldown analysis, and the western blot results are shown in Figure 4B.
  • DV region DA (D442-A456) region of 5-HT2AR and SV (D442-V471) region of 5-HT2AR, and the specific positions of these regions on 5-HT2AR are shown in Figure 3 .
  • the GST fusion protein containing the DA region of 5-HT2AR (GST-5HT2AR-DA) and the GST fusion protein containing the SV region of 5-HT2AR (GST-5HT2AR-SV) were used for pulldown analysis,
  • the results of western blot are shown in C in Figure 4.
  • the 5HT2AR-SV polypeptide (S457-V471) can bind D1R from mouse hippocampal tissue, indicating that 5-HT2AR can interact with D1R through the carboxyl tail.
  • the C-terminus of the SV region (S457-V471) of 5-HT2AR was combined with the transduction domain of HIV-1 Tat protein (such as SEQ ID NO: 3, hereinafter referred to as TAT) N-terminal fusion, to obtain a fusion protein that can penetrate the blood-brain barrier, named TAT-5HT2AR-SV.
  • the DV region (D442-V471) of 5-HT2AR was fused to TAT to obtain TAT-5HT2AR-DV; the CT region (K385-V471) of 5-HT2AR was fused to TAT to obtain TAT-5HT2AR-CT.
  • mice were treated with TAT-5HT2AR-SV, TAT-5HT2AR-DV, and TAT-5HT2AR-CT, and only TAT-treated mice were used as controls (both treated with a single intraperitoneal administration, 3nmol/g). After 1 hour of treatment, the used mice were analyzed according to the co-immunoprecipitation method in Example 1, and the results are shown in FIG. 5 .
  • TAT-5HT2AR-DV and TAT-5HT2AR-CT also successfully disrupted the 5-HT2AR/D1R interaction, and did not cause significant changes in the expressions of 5-HT2AR and D1R.
  • mice treated with TAT-5HT2AR-SV, TAT-5HT2AR-DV, and TAT-5HT2AR-CT were used in Open field test (OFT), forced swimming test (FST) and tail suspension test (TST) were performed 1 hour after treatment, and only Tat-treated mice were used as controls.
  • OFT Open field test
  • FST forced swimming test
  • TST tail suspension test
  • OFT Mice were acclimated to the experimental environment for 1 hour and placed in a 45 x 45 x 30 cm chamber. Record a 5 min video of the mouse's locomotor activity. The total distance covered by the mice was measured and analyzed and expressed in millimeters.
  • mice were placed in a plexiglass cylinder (height 70 cm, diameter 30 cm) filled with water (water temperature 23 ⁇ 1° C.), and the height of the water was more than 30 cm. Mice were videotaped for 5 minutes and analyzed, recording periods of inactivity. Mice were defined as immobile when they floated motionless in the water or with their noses above the water surface; when they moved horizontally across the cylinder, they were defined as swimming; and when they moved vertically against the cylinder wall, they were defined as climbing.
  • TST Mice were individually tape-suspended 40 cm above the floor in rectangular compartments (length 55 cm x width 20 cm x depth 11.5 cm). Record the video for a total of 5 minutes, and record the duration of stillness. EthoVisionXT software was used for recording and analysis.
  • CRS, CMS and CSDS mouse models were respectively constructed according to the method described in Example 2; TAT-5HT2AR-CT or TAT were treated respectively (both single intraperitoneal administration, 3nmol/g); then FST and TST were performed on CRS mice, and FST, TST and sucrose preference test were performed on CMS and CSDS mice respectively ( SPT).
  • mice were habituated to 1% sucrose solution for 3 days and randomly divided into groups. To assess their individual sucrose intake, mice were deprived of water and food for 24 h over 3 days. On the second day, each mouse had free access to two bottles containing sucrose and water. After 2.5 hours the positions of the water and sucrose-containing bottles were changed for a total test time of 5 hours. At last, record the volume of the consumed water and sucrose solution, and calculate according to formula (1):
  • mice treated with TAT-5HT2AR-SV all significantly reduced the increase in rest time induced by stress compared with the mice treated with TAT (A to C , FST and TST), and significantly enhanced sucrose preference (A to C, SPT).
  • the TAT-5HT2AR-DV treatment group and the TAT-5HT2AR-CT treatment group obtained similar results to the TAT-5HT2AR-SV treatment group.
  • Example 6 According to the co-immunoprecipitation method in Example 1, the processed CRS, CMS and CSDS mice (TAT-5HT2AR-SV, TAT-5HT2AR-DV, TAT-5HT2AR-CT and TAT) in Example 6 were analyzed. The blot results are shown in Figure 9, respectively.
  • the hippocampal tissues of the treated CRS mice were directly subjected to Western blot analysis to investigate the expression levels of D1R and 5-HT2AR, and the results are shown in FIG. 10 .
  • the levels of D1R and 5-HT2AR did not have any significant changes between TAT-5HT2A-SV and TAT treated groups, which indicated that the polypeptide of the present invention is specific for the coupling of D1R and 5-HT2AR.
  • mice treated with TAT-5HT2AR-DV or TAT-5HT2AR-CT were tested separately, and the results showed that the TAT-5HT2AR-DV treatment group and TAT-5HT2AR-CT treatment group significantly reduced stress
  • mice treated with TAT-5HT2AR-SV, TAT-5HT2AR-DV, and TAT-5HT2AR-CT were used in Open field test (OFT) and elevated plus maze test (EPM) for testing central zone residence time were performed 1 hour after treatment, and TAT-only treated mice served as controls.
  • OFT Open field test
  • EPM elevated plus maze test
  • OFT used to test the residence time in the central area: Divide the bottom surface of the open space into 25 equal-area grids, and the middle 9 grid areas are the central area. After receiving the polypeptide treatment for 1 hour, the mice were placed in the central area to record for 5 minutes, and the time the mice stayed in the central area was recorded.
  • EPM consists of two open arms (25cm ⁇ 8cm) and two closed arms (25cm ⁇ 8cm), the intersection is the central area (8cm ⁇ 8cm), 40cm high from the ground.
  • TAT-5HT2A-SR polypeptide can significantly increase the residence time of mice in the central region; similarly, TAT-5HT2AR-DV polypeptide and TAT-5HT2AR-CT also significantly increased the mice in The residence time in the central area (P ⁇ 0.01) indicated that it effectively reduced anxiety.
  • TAT-5HT2A-SR polypeptide significantly increased the open arm residence time (A) of mice, decreased the closed arm residence time (B), without affecting the total exercise capacity (C); similarly , TAT-5HT2AR-DV polypeptide and TAT-5HT2AR-CT also significantly increased the open arm residence time (P ⁇ 0.01) and decreased the closed arm residence time (P ⁇ 0.05) of mice without affecting the total exercise capacity. It shows that the polypeptide of the present invention effectively reduces anxiety.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Neurosurgery (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Psychiatry (AREA)
  • Pain & Pain Management (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Endocrinology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)

Abstract

本发明涉及多肽的抗抑郁、抗焦虑用途，提供了具有如SEQ ID NO:1所示序列的多肽在制备用于治疗和/或预防抑郁症或焦虑症的药物中的用途。本发明还涉及含有多肽的复合体。

Description

多肽的抗抑郁、抗焦虑用途及复合体 技术领域
本发明涉及抗抑郁、抗焦虑领域，具体涉及多肽在抗抑郁、抗焦虑中的用途。
背景技术
抑郁症是由各种原因引起的以心境低落为主要临床症状的精神障碍疾病，具有发病率高、致残率高和自杀率高等特点。抑郁症的发病机制尚未完全阐明，目前临床上常用的抗抑郁药物大多是基于“单胺神经递质假说”而研发的。该假说认为，抑郁症的发生与脑内多巴胺(Dopamine,DA)、5-羟色胺(5-hydroxytryptamine,5-HT)、去甲肾上腺素(NA)等单胺类神经递质缺乏有关；抗抑郁药物通过抑制5-HT和NA转运体功能阻断单胺类神经递质再摄取，增加其在突触间隙的浓度，从而改善抑郁症状。
根据抗抑郁药物的作用机制及被开发时间，临床上将常用的抗抑郁药物分为如下几类，即第一代抗抑郁药物：单胺氧化酶抑制剂(MAOI)和三环类抗抑郁药物；第二代抗抑郁药物：选择性5-羟色胺再摄取抑制剂(SSRI)；第三代抗抑郁药物：5-羟色胺和去甲肾上腺素双重再摄取抑制剂、去甲肾上腺素再摄取抑制剂(SNRI)及去甲肾上腺素和5-羟色胺能抗抑郁剂等。
这类依据“单胺神经递质假说”所开发的药物虽有明确疗效，但有效率仅为60％，并且其抗抑郁疗效显现较慢(通常在服药后2～3周才显现)，此外，这类药物还存在长期应用时不良反应大和停药后易复发等缺点。
除抑郁症经典的“单胺神经递质假说”外，近年来随着研究者对抑郁症发病机制的深入研究，发现了一些基于非单胺递质的新型抗抑郁药物分子靶标，如NMDA受体、CRF1受体、δ受体，κ受体、GABAB受体、M胆碱受体、IDO、CysLT1R、PDE4、PPARγ、PPARδ、NOS等。围绕这些新靶标发现了一些具有抗抑郁活性的物质，部分物质已进入临床研究阶段。
为了提供更为丰富、更佳的治疗策略并更好地阐明发病机制，在抑郁症领域仍存在着探索与发现更重要的发病机制和治疗靶点的强烈需求。
发明内容
为达到上述目的，发明人基于抑郁症模型的分子变化进行了大量研究，并发现了抑郁症致病的一种新机制，从而完成了本发明。
具体而言，5-HT受体是一群于中枢神经系统中央处和末梢神经系统周边出现的G蛋白偶联受体及配体门控离子通道。5HT受体可分为七个亚科5-HT1、5-HT2、5-HT3、5-HT4、5-HT5、5-HT6和5-HT7。5-HT2受体又有A、B、C三种亚型，即，5-HT2A、5-HT2B和5-HT2C三种受体蛋白。其中，5-HT2A受体(5-HT2AR)作为一种兴奋性5-HT受体，在哺乳动物的大脑中广泛表达。
DA受体是通过其相应的膜受体发挥作用的一种位于生物体内的受体。DA受体可分为五种：D1、D2、D3、D4和D5。D1受体(D1R)在脑内表达广泛。
发明人认识到，5-HT2AR除单独形式外，也通过复合物的形式，如5-HT1AR/5-HT2AR和5-HT2AR/催产素受体(OXTR)，对神经递质进行调节。
目前，不存在报道显示5-HT2AR在体内会与D1R形成蛋白复合物，更未有任何报道揭示5-HT2AR/D1R复合物与抑郁症发生之间的关联。而本发明人意外地发现5-HT2AR通过其羧基端与D1R形成蛋白复合物，并且该复合物具有抑郁症致病性，随后通过进一步研究发现一种多肽能降低5-HT2AR/D1R的相互作用并因此减轻抑郁症状和焦虑症状。
据此，在第一方面，本发明提供了一种多肽，其具有如SEQ ID NO:1所示的氨基酸序列(SKDNSDGVNEKVSCV)。此外，还提供了本发明的多肽在制备用于治疗和/或预防抑郁症或焦虑症的药物中的用途。
在一些实施方式中，本发明的多肽长度可为15至90aa，尤其15至50aa、15至30aa、15至20aa。例如，16aa、17aa、18aa、19aa、20aa、21aa、22aa、23aa、24aa、25aa、26aa、27aa、28aa、29aa、30aa、31aa、32aa、33aa、34aa、35aa、36aa、37aa、38aa、39aa、40aa、41aa、42aa、43aa、44aa、45aa、46aa、47aa、48aa或49aa。
在一些实施方式中，本发明的多肽可源自5-HT2AR。可通过生物信息库(如Genbank、EMI、DDBJ等)查询多肽所源自的5-HT2AR，并在此基础上确认源自5-HT2AR的多肽。例如，在多肽源自5-HT2AR(Genbank登录号：NP_001365853)的情况下，除含有SEQ ID NO:1所示的序列(即NP_001365853中氨基酸序列的第457至471位)外，多肽还可以在N端方向上包括一个或更多个氨基酸残基。
在一个示例性的实施方式中，本发明的多肽由NP_001365853对应的氨基酸序列的第442至471位组成。
在一个示例性的实施方式中，本发明的多肽由NP_001365853对应的氨基酸序列的第385至471位组成。
在一些实施方式中，5-HT2AR源自灵长目动物。
在优选的实施方式中，5-HT2AR源自人。
如本文所使用的，术语“治疗”是指在患者中引起希望的或有益的作用，所述希望的或有益的作用可包括减少疾病一个或更多个症状的频率或严重性，或者阻抑或抑制疾病、病症或失调的进一步发展。
如本文所使用的，术语“预防”是指防止或延缓疾病的发生，或者防止其临床或亚临床症状的出现。
本发明的多肽通过抑郁症病理中一种的新的机制(5-HT2AR/D1R复合物)起到抗抑郁作用，具有起效快、活性好且副作用小的特点，具有较好的临床开发价值。此外，本发明的多肽亦可实现抗焦虑的作用。
需要说明的是，如下文中实施例4和7，及图5、6、9和10中所证实的，本发明的多肽破坏5-HT2AR/D1R复合物的相互作用，但对5-HT2AR或D1R的表达并没有显著影响。换句话说，本发明的多肽通过特异性破坏5-HT2AR/D1R复合物的相互作用起作用。
在第二方面，本发明提供了一种核酸分子，其编码本发明的多肽。此外，还提供了本发明的核酸分子在制备用于治疗和/或预防抑郁症或焦虑症的药物中的用途。
本发明的核酸分子用于生产本发明的抗原肽，根据所采用的表达系统，本领域技术人员能够适当地对核酸分子的序列进行调整。
在示例性的实施方式中，所述核酸分子具有如SEQ ID NO:2所示的序列。
在第三方面，本发明提供了一种表达载体，其包含本发明的核酸分子。此外，还提供了本发明的表达载体在制备用于治疗和/或预防抑郁症或焦虑症的药物中的用途。
可以利用多种已知方法将本发明的核酸序列插入到表达载体中。例如，可将核酸分子插入适当的限制性内切核酸酶位点。用于克隆、分离、扩增及纯化的标准技术，及操作中涉及DNA连接酶、DNA聚合酶、限制性内切核酸酶等的酶学反应以及各种分离技术，属于本领域技术人员已知并常用的技术。
在第四方面，本发明提供了一种宿主细胞，其包含本发明的核酸分子或表达载体。此外，还提供了本发明的宿主细胞在制备用于治疗和/或预防抑郁症或焦虑症的药物中的用途。
可以使用多种表达系统，如原核表达系统和真核表达系统中的表达载体和宿主细胞来生产本发明的多肽。接下来以哺乳动物表达系统为例进行说明，宿主细胞可包括猴肾成纤维细胞的COS-7细胞系以及能够表达相容性载体的其它细胞系，如C127、3T3、CHO、Hela和BHK细胞系。哺乳动物表达载体应包含复制起点、合适的启动子和增强子以及任何必需的核糖体结合位点、聚腺苷酸化位点、剪接供体和受体位点、转录终止序列以及5’侧翼非转录序列。源自例如SV40剪接和聚腺苷酸化位点的DNA序列可以用于提供所需的非转录性遗传元件。可以通过本领域技术人员熟悉的多种方法，包括但不限于例如磷酸钙转染、DEAE-葡聚糖介导的转染或电穿孔，将表达载体引入宿主细胞中。
在第五方面，本发明提供了一种复合体，其包括本发明的多肽及连接于其上的用于通透血脑屏障的(转运)载体。
在示例性的实施方式中，用于通透血脑屏障的载体可以是：HIV-1Tat蛋白、胰岛素、阳离子化白蛋白、抗大鼠转铁蛋白受体的单抗(OX26)、人胰岛素受体鼠源性单抗(HIRMAb)、Penetratin、Tat蛋白的转导结构域、Pep-1肽、S4 13-PV、Magainin 2和Buforin 2中的一种或更多种。例如，TAT转导结构域能跨膜转导至胞内，其氨基酸为YGRKKRRQRRR(如SEQ ID NO:3所示)。
本发明的多肽可通过适当的连接技术与用于通透血脑屏障的载体连接。示例性的连接技术可以是亲和素-生物素技术、基于聚乙二醇(PEG)的空间臂技术、融合蛋白技术等。例如，在使用HIV-1Tat蛋白的转导结构域作为通透血脑屏障的载体的情况下，可以通过融合蛋白技术，直接将本发明的多肽与Tat蛋白的转导结构域相连。
在一些实施方式中，使用融合蛋白技术的情况下，也可以使用连接子(Linker)将本发明的多肽与用于通透血脑屏障的载体相连。示例性的连接子可以是带有甘氨酸的柔性连接子，如G、GSG、GSGGSG、GSGGSGG、GSGGSGGG、GGGGSGGG、GGGGS和SGG等。
在第六方面，本发明提供了一种治疗受试者的抑郁症的方法，所述受试者患有抑郁症或有患抑郁症的风险，包括：
向所述受试者施用有效量的本发明的多肽或复合体。
所述多肽或复合体降低5-HT2AR/D1R的相互作用，从而治疗所述受试者的抑郁症。
另外，还提供了一种治疗受试者的焦虑症的方法，所述受试者患有焦虑症或有患焦虑症的风险，包括：
向所述受试者施用有效量的本发明的多肽或复合体。
所述多肽或复合体降低5-HT2AR/D1R的相互作用，从而治疗所述受试者的焦虑症。
术语“有效量”或“治疗有效量”是指足以诱导所需生物学结果的活性剂的量。该结果可以是疾病的体征、症状或原因的减轻，或生物系统的任何其他期望的改变。术语“治疗有效量”在本文中用于表示当在一段时间内重复施用于受影响区域时引起疾病状况实质性改善的制剂的任何量。该量将随所治疗的病症、病症的进展阶段以及所用制剂的类型和浓度而变化。本领域技术人员能够通过常规实验确定适当的量。
“受试者”、“个体”或“患者”在本文中可互换使用，其是指脊椎动物，优选哺乳动物，更优选人。哺乳动物包括但不限于鼠类、猴子、人类，农场动物，和宠物。还包括体外获得的或体外培养的生物实体的组织、细胞及其后代。
在第七方面，本发明提供了本发明的多肽或复合体，其用作药物。
在一些实施方式中，用作药物是指用于治疗或预防抑郁症或焦虑症。
附图说明
图1示出了对小鼠的海马组织进行免疫共沉淀的免疫印迹结果；
图2示出了CRS、CMS和CSDS小鼠模型进行的免疫共沉淀的免疫印迹结果；
图3示出了实施例3中所涉及的片段在5-HT2AR上的位置；
图4示出了使用实施例3中的片段对小鼠海马组织进行GST pulldown分析的免疫印迹结果；
图5示出了对经多肽处理的小鼠的海马组织进行免疫共沉淀的免疫印迹结果；
图6示出了对经多肽处理的小鼠的海马组织进行免疫印迹的结果；
图7示出了对经多肽处理的小鼠进行OFT、FST和TST分析的结果，其中，*：P<0.05；**：P<0.01；
图8示出了对经多肽处理的CRS、CMS和CSDS小鼠进行FST、TST和SPT分析的结果，其中，*：P<0.05；**：P<0.01；***：P<0.001；****：P<0.0001；
图9示出了对经多肽处理的CRS、CMS和CSDS小鼠的海马组织进行免疫共沉淀的免疫印迹结果；
图10示出了对经多肽处理的CRS小鼠的海马组织进行免疫印迹的结果；
图11示出了对经多肽处理的小鼠进行OFT(用于中央区停留时间)分析的结果，其中，**：P<0.01；
图12示出了示出了对经多肽处理的小鼠进行EPM分析的结果，其中，*：P<0.05；**：P<0.01。
具体实施方式
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
下面将结合实施例对本发明的实施方式进行详细描述，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
实施例中未注明具体条件的，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产产商的，为可以通过商购获得的常规产品。
除另有说明外，实施例中所使用的实验动物为成年C57BL/6J雄性小鼠(12-14周)，其饲养在18-22℃下，12小时光照/12小时黑暗。在整个过程中可以自由获得饮食和自来水。
实施例中所涉及的统计学分析采用单因素方差(anova)分析，然后进行后比较来评估平均值之间的差异。除非另有说明，使用独立样本t检验来比较任何给定的两组之间的差异。
实施例1 体内形成5-HT2AR/D1R复合物的确认
使用来自小鼠海马组织的蛋白质样本(100-500μg的蛋白质)进行免疫共沉淀。使用抗5-HT2AR的小鼠抗体(SantaCruz生物技术，sc-166775)与25μl蛋白A/G+琼脂糖珠浆(santaCruz生物技术，sc-2001)进行沉淀。
沉淀后进行免疫印迹分析：经变性的蛋白质在8％SDS-PAGE凝胶上分离，然后转移到硝酸纤维素膜，用TBST封闭膜；接下来用抗D1R(SantaCruz生物技术，sc-33660)、抗D2R(SantaCruz生物技术，sc-5303)的小鼠抗体4℃孵育过夜；最后采用HRP偶联的二抗处理1小时并使用ECL超级信号化学发光试剂盒检测信号，使用图像实验室软件对条带进行密度分析，结果如图1所示。
由图1可知，抗5-HT2AR抗体能沉淀D1R，但不沉淀D2R，这表明5-HT2AR在体内可以与D1R形成复合物，而不能与D2R形成复合物。
实施例2 复合物病理意义的验证
为了探索该复合物的病理意义，我们构建了不同的抑郁症模型，包括：慢性束缚应力应激(CRS)、慢性中度压力应激(CMS)和慢性社会压力应激(CSDS)。各模型的构建方法如下：
CRS小鼠模型的构建：小鼠每天水平固定6小时(10点到16点)在丙烯酸圆柱形平底头约束器(25×90mm)中，持续2周。过滤器有几个插槽，可以根据每只老鼠的大小牢固地束缚小鼠，并抑制肢体的身体运动而不会引起疼痛。在被限制后，老鼠立即被放回自己的笼子里。非约束小鼠(对照)在没有进行CRS程序时仍留在自家的笼子里，对照小鼠和CRS小鼠在CRS暴露期间都无法获得食物和水。
CMS小鼠模型的构建：小鼠受到各种压力，包括：约束(4小时)、笼倾斜(45次，每次12小时)、光-暗周期逆转(一次)、闪光(12小时)和脏笼(2次，每次14小时)。该方案持续6周。对照小鼠没有受到相应压力。
CSDS小鼠模型的构建：小鼠暴露于不同的CD1攻击性小鼠(目标小鼠)，每天10min，共10天。接触10min后，实验小鼠被一个包含孔的塑料分隔器分开。此外，实验小鼠通过被放置在笼子中间的分离器暴露于慢性压力下24小时。对照组小鼠也采用了类似的程序(非社会压力)，但使用的是同一品种小鼠，而非攻击性小鼠。
分别使用所构建的CRS小鼠模型、CMS小鼠模型和CSDS小鼠模型，按照实施例1中的免疫共沉淀和免疫印迹方法，研究这些模型中D1R和5-HT2AR的相互作用，结果如图2所示。
由图2可知，在CRS、CMS和CSDS模型中均发现5-HT2AR/D1R复合物的显著增加，证实了该复合物在抑郁症中的病因作用。
实施例3 复合物相互结合位点的确认
为了确认5-HT2AR/D1R复合物的相互结合位点，首先扩增5-HT2AR全长cDNA克隆(Genbank登录号：NM_001378924)得到5-HT2AR的CT区域(K385-V471)和5-HT2AR第三胞内环(IL3)区域(F255-V324)的cDNA片段。将这些片段亚克隆到pGEX-4T-3质粒(优宝no：VT1255)的BamH1/EcoR1或BamH1/Xho1位点。适当时地合并起始蛋氨酸残基和终止密码子。对所有构建物都进行了重测序以确认适当地实现了剪接融合。使用E.coli BL21活性细胞(康体健康生命技术公司，no：KTSM104L)进行表达，并从细菌裂解液中纯化得到含有5-HT2AR的第3个胞内环(IL3)的GST融合蛋白(GST-5HT2AR-IL3)、含有将5-HT2AR的CT区域的GST融合蛋白(GST-5HT2AR-CT)。其中，IL3区域和CT区域的编码序列在5-HT2AR上的具体位置 如图3所示。
将溶解的小鼠海马组织提取物500μg用1×PBS/1％Triton X-100稀释，随后与20μl蛋白GST树脂饱和GST蛋白或15μg GST融合蛋白在4℃孵育过夜。珠子用1×PBS/1％Triton X-100洗涤1-8次。结合蛋白用2×上样缓冲液洗脱，SDS-PAGE分离，用各自的抗体进行免疫印迹，结果如图4中A所示。
由图4中A可知，5-HT2AR的CT区域可以pulldown D1R。
接下来，为了进一步挖掘5-HT2AR与D1R之间正确的相互作用序列/位点，我们将该CT区域分割为：5-HT2AR的KV(K385-V411)区域、5-HT2AR的NN(N412-N441)区域和5-HT2AR的DV(D442-V471)区域，这些区域在5-HT2AR上的具体位置如图3所示。
按照本实施例中的方法，使用含有5-HT2AR的KV区域的GST融合蛋白(GST-5HT2AR-KV)、含有5-HT2AR的NN区域的GST融合蛋白(GST-5HT2AR-NN)、含有5-HT2AR的DV区域的GST融合蛋白(GST-5HT2AR-DV)进行pulldown分析，免疫印迹结果如图4中B所示。
由图4中B可知，5-HT2AR的DV区域可以pulldown D1R。
进一步地，我们将该DV区域分割为：5-HT2AR的DA(D442-A456)区域和5-HT2AR的SV(D442-V471)区域，这些区域在5-HT2AR上的具体位置如图3所示。
按照本实施例中的方法，使用含有5-HT2AR的DA区域的GST融合蛋白(GST-5HT2AR-DA)、含有5-HT2AR的SV区域的GST融合蛋白(GST-5HT2AR-SV)进行pulldown分析，免疫印迹结果如图4中C所示。
由图4中C可知，5HT2AR-SV多肽(S457-V471)可以从小鼠海马组织中亲和D1R，表明5-HT2AR可以通过羧基尾部与D1R相互作用。
实施例4 多肽在体内对复合物的破坏作用的确认
为了验证5-HT2AR羧基端多肽在体内对5-HT2AR/D1R复合物的作用，将5-HT2AR的SV区域(S457-V471)C端与HIV-1型Tat蛋白的转导结构域(如SEQ ID NO:3所示，下文称为TAT)的N端融合，得到可以通透血脑屏障的融合蛋白，命名为TAT-5HT2AR-SV。类似地，将5-HT2AR的DV区域(D442-V471)与TAT融合，得到TAT-5HT2AR-DV；将5-HT2AR的CT区域(K385-V471)与TAT融合，得到TAT-5HT2AR-CT。
使用TAT-5HT2AR-SV、TAT-5HT2AR-DV、TAT-5HT2AR-CT对小鼠进行处理，仅TAT处理的小鼠用作对照(处理方式均为单次腹腔给药，3nmol/g)。处理1小时后，使用的小鼠，按照实施例1中免疫共沉淀方法进行分析，结果如图5所示。
由图5可知，5HT2AR-SV在小鼠海马组织中成功破坏了5-HT2AR/D1R的相互作用。
此外，取TAT-5HT2AR-SV和TAT处理的小鼠的海马组织直接进行免疫印迹分析。由图6可知，经处理后5-HT2AR和D1R的表达均没有显著变化。
另一方面，TAT-5HT2AR-DV和TAT-5HT2AR-CT同样成功破坏了5-HT2AR/D1R的相互作用，并没有引起5-HT2AR和D1R的表达均没有显著变化。
实施例5 多肽的抗抑郁样作用的评估
为了评价本发明的多肽抗抑郁样作用，使用经TAT-5HT2AR-SV、TAT-5HT2AR-DV、TAT-5HT2AR-CT处理的小鼠(处理方式均为单次腹腔给药，3nmol/g)在处理1小时后进行旷场试验(OFT)、强制游泳试验(FST)和悬尾试验(TST)，仅Tat处理的小鼠作为对照。试验方法如下：
OFT：小鼠适应实验环境1小时，并放置在45×45×30cm的腔室中。记录一段5分钟的视频，观察小鼠的运动活动。测量和分析小鼠覆盖的总距离，并以毫米表示。
FST：小鼠被放置在一个充满水(水温23±1℃)的有机玻璃圆柱体中(高度70厘米，直径30厘米)，水的高度为30厘米以上。对小鼠录像5分钟并进行分析，记录静止的时长。小鼠一动不动地漂浮在水中或使鼻子高于水面时被定义为静止；在整个圆柱体上的水平运动时被定义为游泳；而对着圆柱体壁的垂直运动时被定义为攀爬。
TST：小鼠分别以胶带悬挂在地板上方40厘米处，矩形隔间(长度55厘米×宽度20厘米×深度11.5厘米)内。记录视频共5分钟，记录静止的时长。采用EthoVisionXT软件进行记录和分析。
部分结果示于图7中，由左侧OFT结果可知可知，TAT-5HT2AR-SV处理的小鼠的总运动距离与TAT处理组无显著差异，表明多肽对小鼠的运动能力无影响；此外，由中间的FST结果及右侧的TST结果可知，与对照组(仅Tat处理)相比，TAT-5HT2AR-SV处理组的静止时间显著减少。TAT-5HT2AR-DV处理组和TAT-5HT2AR-CT处理组获得了与TAT-5HT2AR-SV处理组相似的结果。以上证实了本发明的抗抑郁作用。
实施例6 使用药物评价模型评估多肽的抗抑郁能力
为进一步评估多肽作为抗抑郁药物的能力，按照实施例2所描述的方法分别构建CRS、CMS和CSDS小鼠模型；构建1小时后分别使用10μM的TAT-5HT2AR-SV、TAT-5HT2AR-DV、TAT-5HT2AR-CT或TAT分别处理(处理方式均为单次腹腔给药，3nmol/g)；随后对CRS进行FST和TST，并对CMS和CSDS小鼠分别进行FST、TST和蔗糖偏好试验(SPT)。
其中，FST和TST操作按照实施例6中的描述进行；SPT具体操作如下：
SPT使用两瓶自由选择范式进行。小鼠用1％的蔗糖溶液习惯3天，并随机分组。为了评估它们个体的蔗糖摄入量，小鼠在3天内剥夺24小时的水和食物。第二天，每只老鼠都可以自由接触两瓶含有蔗糖和水的瓶子。2.5小时后改变水和含蔗糖瓶的位置，总试验时间为5小时。最后，记录所消耗的水和蔗糖溶液的体积，并按下式(I)计算：
Figure PCTCN2022113892-appb-000001
由图8中分析结果可知，对全部三种药物评价模型，TAT-5HT2AR-SV处理的小鼠相较于TAT处理的小鼠均显著地减少了应激诱导的静止时间增加量(A至C，FST和TST)，并且显著地增强了蔗糖偏好(A至C，SPT)。TAT-5HT2AR-DV处理组和TAT-5HT2AR-CT处理组获得了与TAT-5HT2AR-SV处理组相似的结果。
实施例7 在动物模型中验证多肽破坏复合物相互作用的有效性
按照实施例1中免疫共沉淀方法，对实施例6中经处理的CRS、CMS和CSDS小鼠(TAT-5HT2AR-SV、TAT-5HT2AR-DV、TAT-5HT2AR-CT和TAT)进行分析，免疫印迹结果分别如图9所示。
与预期一致，在所有三种模型中，5-HT2AR/D1R偶联水平均显著增加；与TAT处理组相比，TAT-5HT2AR-SV处理组显著降低了应激小鼠海马组织中5-HT2AR与D1R的相互作用(图9中A至C)。
进一步地，取经处理的CRS小鼠的海马组织直接进行了免疫印迹分析，以考察D1R和5-HT2AR表达水平，结果如图10所示。有趣的是，D1R和5-HT2AR的水平在TAT-5HT2A-SV与TAT处理组之间均没有任何显著变化，这表明本发明的多肽对D1R 和5-HT2AR的偶联具有特异性。
此外，对经TAT-5HT2AR-DV或TAT-5HT2AR-CT处理的CRS、CMS和CSDS小鼠分别进行了测试，结果表明TAT-5HT2AR-DV处理组和TAT-5HT2AR-CT处理组显著降低了应激小鼠海马组织中5-HT2AR与D1R的相互作用，但未引起D1R和5-HT2AR水平的显著变化。
实施例8 多肽的抗焦虑样作用的评估
为了评价本发明的多肽抗焦虑样作用，使用经TAT-5HT2AR-SV、TAT-5HT2AR-DV、TAT-5HT2AR-CT处理的小鼠(处理方式均为单次腹腔给药，3nmol/g)在处理1小时后进行用于测试中央区停留时间的旷场试验(OFT)和高架十字迷宫试验(EPM)，仅TAT处理的小鼠作为对照。试验方法如下：
OFT(用于测试中央区停留时间)：将旷底面等分成25个等面积的格子，中间9个格子区域为中央区。小鼠接受多肽处理1小时后放置于中央区记录5min，记录小鼠在中央区停留的时间。
EPM：由两个开放臂(25cm×8cm)和两个闭合臂(25cm×8cm)组成，交叉处为中央区(8cm×8cm)，离地40cm高。接受多肽处理1小时后，将小鼠面向开放臂放置于中央区，记录其自由活动5min。每只动物检测完成后，用70％酒精擦拭装置。记录小鼠在开放臂停留的时间、在闭合臂停留时间和小鼠总运动距离。
部分OFT结果示于图11中，TAT-5HT2A-SR多肽能显著增加了小鼠在中央区的停留时间；类似地，TAT-5HT2AR-DV多肽和TAT-5HT2AR-CT也显著增加了小鼠在中央区的停留时间(P<0.01)，表明其有效地降低了焦虑情绪。
部分EPM结果示于图12中，TAT-5HT2A-SR多肽显著增加了小鼠的开放臂停留时间(A)，降低闭合臂停留时间(B)，而不影响总运动能力(C)；类似地，TAT-5HT2AR-DV多肽和TAT-5HT2AR-CT也显著增加了小鼠的开放臂停留时间(P<0.01)，降低闭合臂停留时间(P<0.05)，而不影响总运动能力。表明本发明的多肽有效地降低了焦虑情绪。

Claims (10)

  1. 多肽在制备用于治疗和/或预防抑郁症或焦虑症的药物中的用途，其中，所述多肽具有如SEQ ID NO:1所示的氨基酸序列。
  2. 根据权利要求1所述的用途，其中，所述多肽长度为15至50aa。
  3. 根据权利要求1所述的用途，其中，所述多肽长度为15至30aa。
  4. 根据权利要求1至3中任一项所述的用途，其中，所述多肽源自5-HT2A受体。
  5. 根据权利要求4所述的用途，其中，所述5-HT2A受体源自灵长目动物，优选人。
  6. 核酸分子在制备用于治疗和/或预防抑郁症或焦虑症的药物中的用途，其中，所述核酸分子编码权利要求1至5中任一项所述的多肽。
  7. 表达载体在制备用于治疗和/或预防抑郁症或焦虑症的药物中的用途，其中，所述表达载体包含如权利要求6所述的核酸分子。
  8. 宿主细胞在制备用于治疗和/或预防抑郁症或焦虑症的药物中的用途，其中，所述宿主细胞包含如权利要求6所述的核酸分子或权利要求7所述的表达载体。
  9. 一种复合体，其包括权利要求1至5中任一项所述的多肽及连接于其上的用于通透血脑屏障的载体。
  10. 根据权利要求9所述的复合体，其中，所述用于通透血脑屏障的载体选自由以下项组成的组中的一种或更多种：HIV-1 Tat蛋白、胰岛素、阳离子化白蛋白、抗大鼠转铁蛋白受体的单抗、人胰岛素受体鼠源性单抗、Penetratin、Tat蛋白的转导结构域、Pep-1肽、S4 13-PV、Magainin 2和Buforin 2。
PCT/CN2022/113892 2021-09-26 2022-08-22 多肽的抗抑郁、抗焦虑用途及复合体 Ceased WO2023045662A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2024518969A JP2024535104A (ja) 2021-09-26 2022-08-22 ポリペプチドおよび複合体の抗うつ用途および抗不安用途
EP22871715.3A EP4406547A4 (en) 2021-09-26 2022-08-22 APPLICATION AGAINST DEPRESSION AND ANXIETY OF A POLYPEPTIDE AND A COMPLEX
KR1020247011564A KR20240058153A (ko) 2021-09-26 2022-08-22 폴리펩타이드 및 복합체의 항-우울증 및 항-불안증 적용
US18/695,187 US20240390459A1 (en) 2021-09-26 2022-08-22 Anti-depression and anti-anxiety application of polypeptide and complex

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111128332.4 2021-09-26
CN202111128332.4A CN113855784B (zh) 2021-09-26 2021-09-26 多肽的抗抑郁、抗焦虑用途及复合体

Publications (1)

Publication Number Publication Date
WO2023045662A1 true WO2023045662A1 (zh) 2023-03-30

Family

ID=78994512

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/113892 Ceased WO2023045662A1 (zh) 2021-09-26 2022-08-22 多肽的抗抑郁、抗焦虑用途及复合体

Country Status (6)

Country Link
US (1) US20240390459A1 (zh)
EP (1) EP4406547A4 (zh)
JP (1) JP2024535104A (zh)
KR (1) KR20240058153A (zh)
CN (1) CN113855784B (zh)
WO (1) WO2023045662A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113855784B (zh) * 2021-09-26 2025-04-15 深圳辰扬生物科技有限公司 多肽的抗抑郁、抗焦虑用途及复合体
CN114767832B (zh) * 2022-04-21 2025-05-16 深圳辰扬生物科技有限公司 多肽及其用途

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1920033A (zh) * 2006-03-29 2007-02-28 山东中医药大学 猕猴5羟色胺2a受体的部分基因及其应用
CN101160127A (zh) * 2005-01-19 2008-04-09 艾尼纳制药公司 用于预防或治疗进行性多灶性白质脑病而作为5-ht2a血清素受体调节剂的二芳基和芳基杂芳基脲衍生物
CN101875961A (zh) * 2003-07-22 2010-11-03 艾尼纳制药公司 用于预防和治疗相关病症而作为5-ht2a血清素受体调节剂的二芳基和芳基杂芳基脲衍生物
US20180263976A1 (en) * 2012-07-12 2018-09-20 Glytech Llc Composition and method for treatment of depression and psychosis in humans
WO2018229744A1 (en) * 2017-06-12 2018-12-20 Glytech Llc. Treatment of depression with nmda antagonists and d2/5ht2a or selective 5ht2a antagonists
CN113855784A (zh) * 2021-09-26 2021-12-31 深圳辰扬生物科技有限公司 多肽的抗抑郁、抗焦虑用途及复合体

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050070577A1 (en) * 2003-07-03 2005-03-31 Pfizer Inc. Compositions containing a serotonin selective reuptake inhibitor and a 5-HT2A receptor antagonist
WO2007090094A2 (en) * 2006-01-27 2007-08-09 The University Of Mississippi Medical Center Thermally-targeted delivery of medicaments, including doxorubicin
US20120321609A1 (en) * 2011-04-11 2012-12-20 Stephen Ferguson Method of treating psychological disorders
EP3613433B1 (en) * 2014-05-30 2022-01-05 Shanghai Henlius Biotech, Inc. Anti-epidermal growth factor receptor (egfr) antibodies
JP6655074B2 (ja) * 2014-06-20 2020-02-26 ジェネンテック， インコーポレイテッド シャガシンに基づく足場組成物、方法及び使用
EP3831844B1 (en) * 2015-06-26 2024-03-13 Prindex S.r.l. Diagnosis and therapy of multiple sclerosis
CN108992661B (zh) * 2018-09-18 2019-09-20 北京大学深圳研究生院 一种多肽在制备静脉、腹腔或鼻腔给药药物中的应用
CN109106942B (zh) * 2018-09-18 2019-11-22 深圳瑞健生物科技有限公司 一种可通过血脑屏障的多肽在制备药物中的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101875961A (zh) * 2003-07-22 2010-11-03 艾尼纳制药公司 用于预防和治疗相关病症而作为5-ht2a血清素受体调节剂的二芳基和芳基杂芳基脲衍生物
CN101160127A (zh) * 2005-01-19 2008-04-09 艾尼纳制药公司 用于预防或治疗进行性多灶性白质脑病而作为5-ht2a血清素受体调节剂的二芳基和芳基杂芳基脲衍生物
CN1920033A (zh) * 2006-03-29 2007-02-28 山东中医药大学 猕猴5羟色胺2a受体的部分基因及其应用
US20180263976A1 (en) * 2012-07-12 2018-09-20 Glytech Llc Composition and method for treatment of depression and psychosis in humans
WO2018229744A1 (en) * 2017-06-12 2018-12-20 Glytech Llc. Treatment of depression with nmda antagonists and d2/5ht2a or selective 5ht2a antagonists
CN113855784A (zh) * 2021-09-26 2021-12-31 深圳辰扬生物科技有限公司 多肽的抗抑郁、抗焦虑用途及复合体

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. NP_001365853
CAI BOYAN , XIE HAIFANG , YU WENWEN: "Research progress on the relationship between depression and neurotransmitters", JOURNAL OF LIAONING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE, vol. 9, no. 5, 5 September 2007 (2007-09-05), pages 51 - 52, XP093052387, ISSN: 1673-842X, DOI: 10.13194/j.jlunivtcm.2007.05.53.caiby.120 *
LIN JIAN-YANG, JIANG MING-YAN, KAN ZHOU-MI, CHU YANG: "Influence of 5-HTR2A genetic polymorphisms on the efficacy of antidepressants in the treatment of major depressive disorder: A meta-analysis", JOURNAL OF AFFECTIVE DISORDERS, vol. 168, 15 October 2014 (2014-10-15), NL , pages 430 - 438, XP093052385, ISSN: 0165-0327, DOI: 10.1016/j.jad.2014.06.012 *
See also references of EP4406547A4

Also Published As

Publication number Publication date
EP4406547A1 (en) 2024-07-31
KR20240058153A (ko) 2024-05-03
EP4406547A4 (en) 2025-10-08
JP2024535104A (ja) 2024-09-26
US20240390459A1 (en) 2024-11-28
CN113855784A (zh) 2021-12-31
CN113855784B (zh) 2025-04-15

Similar Documents

Publication Publication Date Title
JP7663513B2 (ja) 調節された合成遺伝子発現システム
JP2018513384A (ja) Ｒｏｒ１−ｒｏｒ２結合のモジュレーター
US20220257782A1 (en) Peptide therapeutics for autoimmune diseases and inflammatory diseases
KR101833971B1 (ko) 중간엽 줄기세포 또는 xcl1을 포함하는 근육질환의 예방 또는 치료용 약학적 조성물
WO2023045662A1 (zh) 多肽的抗抑郁、抗焦虑用途及复合体
US8779093B2 (en) Neuronal viability factor and use thereof
US20170233444A1 (en) Rtef-1 variants and uses thereof
US8101564B2 (en) Methods for regulating osteoclast differentiation and bone resorption using LRRc17
US8193153B2 (en) Trophic factor for the treatment of retinal degenerative diseases
WO2019062325A1 (zh) 一种衍生自rps23rg1的多肽及其应用
WO2023202663A1 (zh) 多肽及其用途
AU2003288434A1 (en) Peptides, antibodies thereto, and their use in the treatment of central nervous system damage
US11629186B2 (en) Anti-CCL8 antibodies and uses thereof
WO2025167587A1 (zh) 多肽偶联物及其在射血分数保留的心衰中的应用
WO2017055370A1 (en) Treatment of bone growth disorders
Remez et al. The progressive rod–cone degeneration (PRCD) protein is secreted through the conventional ER/Golgi-dependent pathway
HK40121458A (zh) 多肽及其用途
US20090036380A1 (en) Composition And Method For Brain Tumor Therapy
CN119161448B (zh) 一种mdga2靶向多肽及其应用
Klitchyk Cell Death Signaling Mediated by Dependence Receptors EphA4 and EphB3 Following Induced Stress
EP1445315A1 (en) Human and mammalian stem cell-derived neuronal survival polypeptides
Mandati The role of the C-terminus Merlin in its tumor suppressor function
KR20170121449A (ko) Ｃｒｉｆ１을 유효성분으로 포함하는 자가면역질환의 예방 또는 치료용 조성물

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: 22871715

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 18695187

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2024518969

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20247011564

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2022871715

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022871715

Country of ref document: EP

Effective date: 20240426