WO2023174116A1 - Régulateur wnt5a et son utilisation - Google Patents

Régulateur wnt5a et son utilisation Download PDF

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WO2023174116A1
WO2023174116A1 PCT/CN2023/080207 CN2023080207W WO2023174116A1 WO 2023174116 A1 WO2023174116 A1 WO 2023174116A1 CN 2023080207 W CN2023080207 W CN 2023080207W WO 2023174116 A1 WO2023174116 A1 WO 2023174116A1
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wnt5a
polypeptide
sequence
amino acid
seq
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PCT/CN2023/080207
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Chinese (zh)
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徐贞仲
杨帆
谢亚凯
陈晓莹
杨巍
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浙江大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to the field of biomedicine, and in particular to Wnt5a regulators and their applications.
  • DNP diabetic neuropathic pain
  • the main clinical symptoms of DNP are allodynia, spontaneous pain, and paresthesia (tingling, pinprick, or electric shock sensation), which affects 25% to 30% of diabetic patients. Since the pathogenesis of DNP is still unclear and effective treatments are lacking, it is crucial to elucidate the pathogenesis of DNP and explore new DNP treatment targets.
  • DRG dorsal root ganglion
  • DRG neurons are in direct contact with metabolites in the circulatory system and are more susceptible to damage by toxic metabolites caused by hyperglycemia. Functional abnormality of DRG neurons is considered to be a direct factor mediating the occurrence and maintenance of DNP. Larger-diameter type A neurons in the DRG emit low-threshold, myelinated nerve fibers (type A ⁇ , some type A ⁇ ) that mediate the perception of light touch. Multiple studies have confirmed that in neuropathic pain, class A DRG neurons play a key role in the occurrence of tactile allodynia.
  • a DRG neurons play an important role in the occurrence of diabetic neuralgia. Damage to A ⁇ nerve fibers can be observed in animal models of type I and type II diabetes and in diabetic patients with large fiber neuropathy. In diabetic rat models, spontaneous discharge and ectopic discharge of A ⁇ nerve fibers are considered to be key factors in the occurrence of tactile allodynia. However, the molecular basis of the involvement of class A DRG neurons in the occurrence of diabetic neuralgia remains to be further studied. Therefore, clarifying the mechanism by which class A DRG neurons are involved in the occurrence of diabetic neuralgia and developing new DNP therapeutic targets centered on this mechanism may become a promising treatment for diabetes. An effective method for treating neuropathic pain such as neuralgia.
  • the object of the present invention is to provide a Wnt5a regulator.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned Wnt5a modulator.
  • Another object of the present invention is to provide the application of the above-mentioned Wnt5a modulator.
  • Another object of the present invention is to provide a method for preventing and/or treating neuropathic pain.
  • the first aspect of the present invention provides the use of a Wnt5a modulator for the preparation of a drug or a pharmaceutical composition, the drug or the pharmaceutical composition is used for one selected from the following group or multiple uses:
  • the Wnt5a modulator is selected from: antibodies, polypeptides, shRNA, dsRNA, miRNA, antisense oligonucleotides, compounds, or combinations thereof.
  • the Wnt5a modulator is selected from the group consisting of Wnt5a neutralizing antibodies and Wnt5a inhibitors.
  • the Wnt5a inhibitor is a polypeptide or Box5, wherein the sequence of the polypeptide has at least one amino acid residue that can specifically bind to Wnt5a.
  • At least part of the sequence of the polypeptide is identical to part of the amino acid sequence of the TRPV1 channel.
  • the TRPV1 channel has a region that specifically binds to Wnt5a;
  • the homology between at least part of the sequence of the polypeptide and part of the region specifically binding to Wnt5a is greater than 70%.
  • the amino acid sequence of the TRPV1 channel and the Wnt5a specific binding region are located in the extracellular S5-S6 loop of the TRPV1 channel;
  • the amino acid sequence of the TRPV1 channel and the Wnt5a specific binding region are located in the extracellular S3-S4 loop of the TRPV1 channel.
  • the sequence of the polypeptide includes N292, E611, S293, Q286, N606, S289, N605, R202, S612, E203, S210, K283, E652, E649, K208, D547, R281, R284 , at least one amino acid residue among K808, D647, R290, N288 and N653.
  • the sequence of the polypeptide includes at least one amino acid residue selected from N605, L461, K604, N606, V610, E611, P614, K616, R618, D647, L648, E649, E652 and D655, Preferably there are at least two types, more preferably at least three types, more preferably at least five types, more preferably at least seven types.
  • the sequence of the polypeptide includes at least one amino acid residue selected from the group consisting of E611, N606, K604, V610, E611, P614, K616 and R618.
  • sequence of the polypeptide includes at least one amino acid residue selected from D647, L648, E649, E652 and D655.
  • the polypeptide has a polypeptide selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. At least one amino acid sequence shown, fragments thereof and modified forms thereof or mutants thereof.
  • a second aspect of the present invention provides a Wnt5a modulator, the Wnt5a modulator comprising at least one polypeptide, the sequence of the polypeptide having at least one amino acid residue that can specifically bind to Wnt5a; and
  • At least part of the sequence of the polypeptide is identical to part of the amino acid sequence of the TRPV1 channel.
  • the TRPV1 channel has a region that specifically binds to Wnt5a;
  • At least a portion of the sequence of the polypeptide is identical to a portion of the region that specifically binds to Wnt5a; or, at least a portion of the sequence of the polypeptide is homologous to a portion of the region that specifically binds to Wnt5a. sex is greater than 70%.
  • sequence of the polypeptide includes at least one group of amino acid residues selected from the following:
  • the polypeptide has the properties shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 At least one amino acid sequence, fragments thereof and modified forms thereof or mutants thereof.
  • a third aspect of the present invention provides a pharmaceutical composition comprising the Wnt5a modulator described in the second aspect of the present invention and its pharmaceutically acceptable excipients.
  • a fourth aspect of the present invention provides a method for preventing and/or treating neuropathic pain and its complications, the method comprising the steps of:
  • the Wnt5a modulator according to the second aspect of the present invention or the pharmaceutical composition according to the third aspect of the present invention is administered to the subject.
  • the present invention has at least the following advantages:
  • the present invention develops a modulator based on Wnt5a, a new analgesic target, which can effectively relieve neuropathic pain.
  • Figure 1 is a schematic diagram of the expression levels of different Wnt5a isoforms in DRG after STZ treatment according to an embodiment of the present invention
  • Figure 2 is a schematic diagram of co-localization detection of Wnt5a mRNA and NF200 in DRG according to an embodiment of the present invention
  • Figure 3 is a statistical diagram of the area distribution of Wnt5a+ and NF200+ neurons in DRG according to an embodiment of the present invention
  • Figure 4 is a schematic diagram of the proportions of Wnt5a + , Wnt5a + /NF200 + , and Wnt5a + /IB4 + neurons before and after STZ treatment according to the embodiment of the present invention
  • Figure 5 is a schematic diagram of the effect of STZ treatment on the expression abundance of Wnt5a in Wnt5a + neurons according to an embodiment of the present invention
  • Figure 6 is a schematic diagram of Wnt5a expression detected by q-PCR in wild type, STZ treatment, and Gpr177 knockout according to the embodiment of the present invention
  • Figure 7 is a schematic diagram showing the content of Wnt5a in the DCR of wild-type, STZ-treated, and Gpr177-knocked-out Wnt5a detected by Western blot according to the embodiment of the present invention
  • Figure 8 is a schematic diagram showing the content of wild-type, STZ-treated, and Gpr177-knockout Wnt5a in cerebrospinal fluid detected by Western blot according to the embodiment of the present invention
  • Figure 9 is a schematic diagram of the expression and secretion of Wnt5a in primary cultured DRG neurons detected by Western blot according to the embodiment of the present invention.
  • Figure 10 is a quantitative statistical diagram showing the secretion and expression of Wnt5a by primary cultured DRG neurons in an embodiment of the present invention
  • FIG 11 shows the effect of intrathecal injection of the Wnt5a-specific antagonist Box5 on the foot withdrawal threshold of mice according to the Von Frey test in the embodiment of the present invention
  • Figure 12 is the effect of intrathecal injection of the neutralizing antibody Anti-Wnt5a IgG on the paw withdrawal threshold of mice according to the Von Frey test in the embodiment of the present invention
  • Figure 13 is a schematic diagram of the action potential of Trpv1+DRG neurons in current clamp recording according to an embodiment of the present invention
  • Figure 14 is a quantitative statistical diagram of the action potential firing frequency of Trpv1+DRG neurons in current clamp recording according to an embodiment of the present invention.
  • Figure 15 is a diagram showing Trpv1+DRG neurons induced by Box5 perfusion administration according to the embodiment of the present invention. Schematic diagram of the influence of current threshold on action potential firing;
  • Figure 16 is a schematic diagram of the experimental scheme in Example 2(F) of the present invention.
  • Figure 17 is a schematic diagram of the foot withdrawal threshold of wild-type and Wnt5a CKO mice tested for mechanical allodynia according to the embodiment of the present invention.
  • Figure 18 is a schematic diagram illustrating the impact of Wnt5a knockout on aversive escape time in mice according to the Real-time PEA experiment in the embodiment of the present invention
  • Figure 19 is a schematic diagram of the expression levels of Cre and Wnt5a in the DRG and lumbar enlarged spinal cord according to an embodiment of the present invention.
  • Figure 20 is a quantitative statistical diagram of the expression levels of Cre and Wnt5a in the DRG and lumbar enlarged spinal cord according to an embodiment of the present invention
  • Figure 21 is a schematic diagram of satellite glial cell marker protein GS staining according to an embodiment of the present invention (scale bar is 50 ⁇ m);
  • Figure 22 is a schematic diagram of macrophage marker protein IBA1 staining according to an embodiment of the present invention (scale bar is 50 ⁇ m);
  • Figure 23 is a schematic diagram of the co-localization of satellite glial cell marker protein GS and macrophage marker protein IBA1 according to an embodiment of the present invention (scale bar is 50 ⁇ m);
  • Figure 24 is a schematic diagram showing the distribution of nerve fiber endings in the epidermis in the hairless area of the sole after PGP9.5 immunostaining after knocking out Wnt5a according to an embodiment of the present invention
  • Figure 25 is a quantitative statistical diagram showing the distribution of nerve fiber endings in the epidermis in the hairless area of the sole after Wnt5a is knocked out according to an embodiment of the present invention.
  • Figure 26 is a schematic diagram of the foot withdrawal threshold of mechanical allodynia in mice caused by intrathecal injection of gradient doses of Wnt5a in the Von Frey test according to the embodiment of the present invention
  • Figure 27 is a schematic diagram of the effect of intrathecal injection of Real-time PEA on the aversive escape response of mice to mechanical stimulation according to the embodiment of the present invention
  • Figure 28 is a schematic diagram of Wnt5a-induced iCa2+ activity in primary cultured DRG neurons according to an embodiment of the present invention.
  • Figure 29 is a real-time iCa2+ signal diagram of representative neurons in Ca2+ activity recording according to an embodiment of the present invention.
  • Figure 30 is an analysis showing neurons responding to Wnt5a+ and Cap+ according to the embodiment of the present invention. Schematic diagram of overlap and proportion of neurons producing iCa2+ activity in response to stimulation with Wnt5a at different concentrations;
  • Figure 31 is a schematic diagram of DRG small neurons firing action potentials induced by Wnt5a in an embodiment of the present invention.
  • Figure 32 is a schematic diagram of inward current induced by Wnt5a and Cap according to an embodiment of the present invention.
  • Figure 33 is a diagram showing the results of the preparation of spinal cord slices and patch clamp recording experiments according to the embodiment of the present invention.
  • Figure 34 is a diagram showing the influence of Wnt5a on the frequency and amplitude of sEPSCs firing according to an embodiment of the present invention.
  • Figure 35 is a schematic diagram of DRG small neuron action potential firing in current clamp recording according to an embodiment of the present invention.
  • Figure 36 is a quantitative statistical diagram of the action potential firing frequency of DRG small neurons according to an embodiment of the present invention.
  • Figure 37 is a schematic diagram of detecting Wnt5a secretion levels in WT and db/db mice by western blotting according to the embodiment of the present invention.
  • Figure 38 is a schematic diagram of the foot withdrawal threshold of mice after intrathecal injection of 5 ⁇ g Box5 in WT and db/db mice according to the embodiment of the present invention.
  • Figure 39 is a schematic diagram of the foot withdrawal threshold of mice after intrathecal injection of 4 ⁇ g anti-Wnt5a antibody in WT and db/db mice according to the embodiment of the present invention.
  • Figure 40 is a schematic diagram of Wnt5a-induced Ca2+ activity in HEK293T cells according to an embodiment of the present invention.
  • Figure 41 is a real-time Ca2+ signal recording chart of representative HEK293T cells in Ca2+ imaging in an embodiment of the present invention
  • Figure 42 is a graph showing the percentage of cells with positive responses to Wnt5a-induced Ca2+ activity when transfected with different types of TRP channels in an embodiment of the present invention
  • Figure 43 is a schematic diagram of Wnt5a-induced inward current according to an embodiment of the present invention.
  • Figure 44 is a percentage graph of the inward current amplitude induced by Wnt5a (10ng/ml) and the current amplitude induced by the corresponding positive control reagent in an embodiment of the present invention
  • Figure 45 shows the single-channel Outside-out patch clamp after administration of Wnt5a according to the embodiment of the present invention. record chart;
  • Figure 46 is a schematic diagram of Wnt5a-induced single-channel current according to an embodiment of the present invention.
  • Figure 47 is a model diagram of the stable binding state of Wnt5a-TRPV1 protein according to an embodiment of the present invention.
  • Figure 48 is a schematic diagram of the binding energy of amino acid residues in the TRPV1 channel protein that may bind to Wnt5a according to an embodiment of the present invention
  • Figure 49 is a schematic diagram of the influence of the Von Frey test antagonist peptide AA601-25 on the foot withdrawal threshold of mechanical allodynia in mice according to the embodiment of the present invention.
  • Figure 50 is a schematic diagram of the influence of the Von Frey test antagonist peptides AA453-72, AA644-56 and control peptides on the foot withdrawal threshold of mechanical allodynia in mice according to the embodiment of the present invention
  • Figure 51 is a schematic diagram of iCa2+ activity induced by Wnt5a (1ng/ml) or Cap (1 ⁇ M) according to an embodiment of the present invention
  • Figure 52 is a schematic diagram of the influence of the Von Frey test antagonist peptide AA601-25 on the foot withdrawal threshold of mechanical allodynia in mice intraperitoneally injected with STZ according to the embodiment of the present invention
  • Figure 53 is a schematic diagram of the effect of knocking out Trpv1 on the foot withdrawal threshold of mechanical allodynia in mice with intrathecal injection of Wnt5a according to the Von frey test in the embodiment of the present invention
  • Figure 54 is a schematic diagram of the effect of knocking out Trpv1 on the foot withdrawal threshold of mechanical allodynia in STZ mice intraperitoneally injected according to the Von frey test in the embodiment of the present invention
  • Figure 55 is a schematic diagram of the expression of GPR177 and WNT5A mRNA in human DRG tissue sections detected by in situ hybridization experiments according to the embodiment of the present invention.
  • Figure 56 is a diagram showing the diameter distribution pattern of GPR177+ neurons and all DRG neurons according to an embodiment of the present invention.
  • Figure 57 is a schematic diagram of the proportion of WNT5A+ and NF200+ neurons among GPR177+ neurons according to an embodiment of the present invention.
  • Figure 58 is a schematic diagram of Wnt5a secretion levels in the cerebrospinal fluid of diabetic patients according to an embodiment of the present invention.
  • Figure 59 is a schematic diagram of the correlation between Wnt5a secretion level and NRS score in the cerebrospinal fluid of diabetic patients according to an embodiment of the present invention.
  • Figure 60 is a diagram of HEK293T cells transfected with human Trpv1-EGFP plasmid according to the embodiment of the present invention.
  • Granule schematic diagram of the inward current generated by Wnt5a activation in HE293T cells.
  • the inventors confirmed through animal experiments that specific antagonists of Wnt5a or neutralizing antibodies of Wnt5a can improve STZ-induced allodynia.
  • the inventors further designed an antagonistic polypeptide to block the binding and activation of TRPV1 by Wnt5a, and confirmed through in-vivo experiments that the antagonistic polypeptide can effectively inhibit Wnt5a and diabetes-induced neuropathic pain, providing a basis for clinical treatment of neuropathic pain. Pain provides new analgesic targets and intervention strategies.
  • Some embodiments of the present invention provide the use of a Wnt5a modulator for the preparation of a medicament or a pharmaceutical composition for one or more uses selected from the group consisting of:
  • Wnt5a modulators described in the present invention include, but are not limited to, small molecules, peptides, organic molecules, cyclic molecules, heterocyclic molecules, lipids, charged lipids, glycolipids, polar lipids, and non-polar lipids. and aptamer; in some preferred embodiments, the Wnt5a modulator is a polypeptide, for example: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and any of the sequences shown in SEQ ID NO. 6 or their mutants.
  • the Wnt5a modulators can form complexes with each other, and the polypeptides in these complexes can be the same, different, or a mixture of the same polypeptide and different polypeptides.
  • the composition may comprise other compounds, either separated from the polypeptide or covalently or non-covalently associated with the polypeptide, wherein non-limiting examples of such compounds include detectable moieties, e.g.
  • the Wnt5a modulator is selected from: antibodies, polypeptides, shRNA, dsRNA, miRNA, antisense oligonucleotides, compounds, or combinations thereof.
  • the Wnt5a modulator is selected from the group consisting of Wnt5a neutralizing antibodies and Wnt5a inhibitors.
  • the Wnt5a inhibitor is a polypeptide, wherein the sequence of the polypeptide has at least one amino acid residue that can specifically bind to Wnt5a.
  • the Wnt5a inhibitor is Box5.
  • At least part of the sequence of the polypeptide is identical to part of the amino acid sequence of the TRPV1 channel.
  • the TRPV1 channel has a region that specifically binds to Wnt5a;
  • At least part of the sequence of the polypeptide is identical to part of the region that specifically binds to Wnt5a; or,
  • the homology between at least part of the sequence of the polypeptide and part of the region specifically binding to Wnt5a is greater than 70%.
  • the specific binding region to Wnt5a is located in the extracellular S5-S6 loop of the TRPV1 channel, or the specific binding region to Wnt5a is located in the extracellular S3-S4 loop of the TRPV1 channel.
  • the sequence of the polypeptide includes N292, E611, S293, Q286, N606, S289, N605, R202, S612, E203, S210, K283, E652, E649, K208, D547, R281, R284 , K808, D647, R290, N288 and N653 of at least one amino acid residue.
  • the sequence of the polypeptide includes at least one amino acid residue selected from N605, L461, K604, N606, V610, E611, P614, K616, R618, D647, L648, E649, E652 and D655, Preferably there are at least two types, more preferably at least three types, more preferably at least five types, more preferably at least seven types.
  • the sequence of the polypeptide includes at least one amino acid residue selected from the group consisting of E611, N606, K604, V610, E611, P614, K616 and R618.
  • sequence of the polypeptide includes at least one amino acid residue selected from D647, L648, E649, E652 and D655.
  • the polypeptide has the amino acid sequence shown in SEQ ID NO. 1, fragments thereof and modified forms thereof, or mutants thereof.
  • the polypeptide has the amino acid sequence shown in SEQ ID NO.2, its fragments and modified forms or mutants thereof; more preferably, the polypeptide is as shown in SEQ ID NO.2 A fragment of an amino acid sequence, such as a polypeptide with an amino acid sequence shown in SEQ ID NO.3, or a polypeptide with an amino acid sequence shown in SEQ ID NO.6.
  • the polypeptide has the amino acid sequence shown in SEQ ID NO. 3, fragments thereof and modified forms thereof or mutants thereof.
  • the polypeptide has the amino acid sequence shown in SEQ ID NO.4, its fragments and modified forms or mutants thereof; more preferably, the polypeptide is as shown in SEQ ID NO.5 A fragment of an amino acid sequence, such as a polypeptide with an amino acid sequence shown in SEQ ID NO.3, or a polypeptide with an amino acid sequence shown in SEQ ID NO.6.
  • the polypeptide has the amino acid sequence shown in SEQ ID NO. 5, fragments thereof and modified forms thereof or mutants thereof.
  • the polypeptide has the amino acid sequence shown in SEQ ID NO. 6, fragments thereof and modified forms thereof or mutants thereof.
  • the inventor found through experiments that the antagonistic effect of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO.3 is better than SEQ ID NO.1 and SEQ ID NO.6.
  • Some embodiments of the invention provide a Wnt5a modulator, the Wnt5a modulator comprising at least one polypeptide having a sequence having at least one amino acid residue that can specifically bind to Wnt5a; and
  • At least part of the sequence of the polypeptide is identical to part of the amino acid sequence of the TRPV1 channel.
  • the TRPV1 channel has a region that specifically binds to Wnt5a;
  • At least a portion of the sequence of the polypeptide is identical to a portion of the region that specifically binds to Wnt5a; or, at least a portion of the sequence of the polypeptide is homologous to a portion of the region that specifically binds to Wnt5a. sex is greater than 70%.
  • sequence of the polypeptide includes at least one group of amino acid residues selected from the following:
  • sequence of the polypeptide includes E611, N606 and N605.
  • sequence of the polypeptide includes S612 and N606.
  • sequence of the polypeptide includes E652 and E649.
  • sequence of the polypeptide includes D647 and E649.
  • a pharmaceutical composition which pharmaceutical composition includes the Wnt5a modulator described in the second aspect of the present invention and its pharmaceutically acceptable excipients.
  • nucleic acid encoding said polypeptide sequence is provided.
  • a vector comprising a nucleic acid according to the second aspect of the invention is provided.
  • the vector is at least one of bacteria, yeast, mammalian, viral, expression, shuttle or plasmid.
  • the vector may further comprise control elements such that the modified protein is expressed constitutively or under the control of an inducible promoter.
  • host cells containing the vectors described above are also provided.
  • the host cell can be a prokaryotic or eukaryotic cell.
  • the host cells or vectors are administered or implanted into a mammal, such as a rodent or a human, for therapeutic purposes.
  • a method for preventing and/or treating neuropathic pain comprising the steps of:
  • a GPR177 inhibitor is administered to the subject.
  • any concentration range, percentage range, ratio range or integer range shall be understood to include the value of any integer within the stated range and, where appropriate, fractions thereof (such as tenths and hundredths of an integer). one part). It will be understood that the terms “a” and “an” as used herein refer to “one or more” of the listed components, unless otherwise stated. The use of alternatives (eg, "or”) should be understood to mean one, both, or any combination of the alternatives. As used herein, the terms “include” and “include” are used synonymously.
  • polypeptides comprising various combinations of components (eg, domains or regions) and substituents described herein to the same extent as if each polypeptide were set forth individually. Therefore, the selection of specific components of individual polypeptides is within the scope of the present disclosure.
  • the term "about” and its grammatical equivalents with respect to a reference value may include a range of the value plus or minus 10% from the value, such as plus or minus 10%, 9%, 8%, 7% from the value ,6%, Range of values of 5%, 4%, 3%, 2% or 1%.
  • the quantity "about 10" includes quantities from 9 to 11.
  • Wnt5a is an atypical Wnt isoform that exerts its functions primarily through the atypical WNT/PCP or WNT/Ca2+ signaling pathways.
  • Wnt is a protein involved in the Wnt signaling system, including, for example, Wnt5a, Wnt11, and Wnt3a.
  • the term "antagonist” refers to a substance that binds to a receptor and blocks the agonist-mediated effects of that receptor.
  • a Wnt5a antagonist binds to Wnt5a and blocks its binding to the TRPV1 channel. .
  • inhibitor refers to a substance that blocks or reduces the rate of a chemical reaction.
  • a Wnt5a inhibitor herein blocks or reduces the activity of Wnt5a.
  • binding domain or “binding region” or “binding domain” refers to a domain, region, part or site of a protein, polypeptide, oligopeptide or peptide or antibody or a binding domain derived from an antibody , which has the ability to specifically recognize and bind to target molecules (such as antigens, ligands, receptors, substrates or inhibitors).
  • target molecules such as antigens, ligands, receptors, substrates or inhibitors.
  • specific binding refers to the binding domain with an affinity equal to or greater than 105M-1 or Ka (i.e., the equilibrium association constant of a specific binding interaction in units of 1/M) binds to the target.
  • peptide refers to a molecule comprising a sequence of amino acids bound by peptide bonds, regardless of length, post-translational modification or function.
  • the term "vector” refers to a delivery vehicle for a polynucleotide.
  • the vector in genetic engineering recombinant technology, includes a polynucleotide sequence encoding a specific protein that is operably inserted to achieve expression of the protein.
  • Vectors are used to transform, transduce or transfect host cells, and the genetic material elements delivered by the vector can be expressed in the host cells.
  • a "vector” in this disclosure may be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (nucleic acid sequences including a series of appropriate expression control elements).
  • polypeptide refers to a protein that occurs naturally or is produced or altered by chemical or other means by recombination, which can essentially be conceived as a three-dimensional structure of a protein that has been processed post-translationally in the same manner as the native protein.
  • the polypeptides described herein can be prepared by any method known in the art, such as by recombinant DNA methods or by chemical synthesis methods.
  • the term "mutant" of a polypeptide refers to a polypeptide of said polypeptide that has one or more mutations relative to the starting polypeptide sequence, e.g., one or more amino acid residues are Another amino acid residue is substituted, or it has one or more amino acid residues inserted or deleted. Such changes must have less than 100% sequence identity or similarity to the starting polypeptide. In one embodiment, the variant will have an amino acid sequence that is about 60% to less than 100% amino acid sequence identity or similarity to the amino acid sequence of the starting polypeptide.
  • the variant will have an amino acid sequence from about 75% to less than 100%, from about 80% to less than 100%, from about 85% to less than 100%, from about 90% to less than 100%, from the amino acid sequence of the starting polypeptide. Amino acid sequences that have about 95% to less than 100% amino acid sequence identity or similarity.
  • TRPV1 channel and “capsaicin receptor” are used interchangeably and are a cation channel that refers to one of the TRPV channel submembers expressed in nociceptive primary sensory neurons, the TRPV channel Sub-members also include TRPV1, TRPV2, TRPV3 and TRPV4, etc.
  • TRPV1 channel extracellular S5-S6 loop refers to a cyclic peptide chain outside the RPV1 channel, and its specific amino acid sequence (mouse source) is shown in SEQ ID NO.2,
  • SEQ ID NO.2 (the humanized sequence) is SEQ ID NO.4:
  • TRPV1 channel extracellular S3-S4 loop is another cyclic peptide chain outside the RPV1 channel, and its specific amino acid sequence (mouse source) is shown in SEQ ID NO.1,
  • SEQ ID NO.1 AYYRPVEGLPPYKLNNTVGD.
  • the term "pharmaceutically acceptable” refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions are considered “pharmaceutically acceptable” when approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopeia for use in animals, and more particularly in humans.
  • the term "pharmaceutically acceptable excipient” refers to an excipient whose administration can be tolerated by the patient to whom it is administered.
  • Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, Sweeteners, preservatives, isotonic agents and combinations thereof. Selection of appropriate excipients Selection and use are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed.
  • the formulation may further include one or more carriers, diluents, preservatives, solubilizers, buffers, albumin to prevent protein loss from the vial surface, and the like.
  • treatment refers to therapeutic treatment or prophylactic/preventive treatment.
  • a treatment is therapeutic if the symptoms of at least one disease improve in the individual receiving the treatment, or the treatment delays the progression of the progressive disease in the individual, or prevents the onset of an additional related disease.
  • neuropathic pain refers to pain caused by pathological damage to any part of the nervous system, such as neuropathic pain caused by chemotherapy, neuropathic pain caused by injury and inflammation, etc., such as sciatica, intercostal nerves, etc. Pain, diabetic neuralgia, etc.
  • Example 1 Wnt5a secreted by DRG neurons is a necessary condition for the occurrence of DNP.
  • mice After the mice were deeply anesthetized with isoflurane, their heads were quickly decapitated and the blood was discarded.
  • the mouse L3-L6 DRG tissue was isolated and used for total RNA extraction.
  • the specific operation method of RNA extraction was carried out according to the instructions of the RNA extraction kit, and then the RNA was eluted with 40 ⁇ l RNase-free H 2 O. After measuring the RNA concentration, Take 300ng of total RNA for cDNA synthesis.
  • the cDNA synthesis operation was performed according to the instructions of the HiScript II Q RT SuperMix kit.
  • the synthesized cDNA was used for real-time fluorescence quantitative PCR (qRT-PCR) to detect the expression changes of the corresponding genes.
  • the reaction mixture is: 10 ⁇ l 2X Premix Ex Taq TM II, 1 ⁇ l 10 ⁇ M Primer F, 1 ⁇ l 10 ⁇ M Primer R, 7 ⁇ l ddH 2 O, 1 ⁇ l cDNA, total volume 20 ⁇ l.
  • the reaction conditions are: 95°C for 2min; 95°C for 10s, 60°C for 50s, read the plate, 40cycles; 65°C ⁇ 95°C gradient temperature, 0.5°C/cycle, read the melt curve.
  • Gapdh was used as an internal reference gene, and the relative expression changes of the target genes were calculated using the 2 - ⁇ Ct method. The experimental results obtained are shown in Figure 1.
  • the in situ hybridization experiment was performed according to the instruction manual of the RNAscope in situ hybridization kit.
  • the Wnt5a probes were all labeled with Cy3 dye.
  • the DRG sections continue to undergo immunohistochemical staining in the dark.
  • Tissue sections were blocked with blocking solution at room temperature for 1 to 2 hours, and chicken anti-NF200 (1:1000) was added and incubated at 4°C for 18 to 20 hours. Afterwards, wash the slices 4 times with PBS, 10 minutes each time.
  • Add corresponding Alexa 488 fluorescent dye-coupled secondary antibody (1:500, Jackson ImmunoResearch) was placed at room temperature and incubated in the dark for 2 hours. Wash the slices 4 times with PBS, 10 minutes each time.
  • Mount the slides with mounting medium containing antifade agent dry at room temperature in the dark, and observe under a fluorescence microscope or store at -20°C. The experimental results obtained are shown in Figure 2.
  • Immunohistochemical staining uses fluorescence microscopy to obtain tissue images of different groups under the same shooting conditions. Image J software and NIS Element AR software were used for quantitative analysis of fluorescence signal intensity. Select 4 to 6 areas without fluorescence signals in each picture, and the average value of the obtained signal values is used as the background intensity of the background signal.
  • fluorescence signal of a cell is ⁇ 2X (background intensity+standard deviation), it is regarded as a positive cell; when a clear outline of the nucleus can be seen in a positive cell, it is used for quantitative analysis of cell area. 779 Wnt5a+ neurons from at least 3 mice were counted, and the distribution proportion of Wnt5a+ neurons in different area intervals was calculated.
  • Wnt5a+ neurons and NF200+ type A DRG neurons have basically the same distribution characteristics.
  • Immunohistochemical staining was used to quantify the Wnt5a fluorescence signal intensity in DCR sections of STZ-treated and non-STZ-treated mice. Among them, each group counted 9 DRG slices from 3 mice, each containing 137 to 166 Wnt5a + neurons, for quantitative statistics of Wnt5a fluorescence signal intensity.
  • mice After the mice were deeply anesthetized with isoflurane, their heads were quickly decapitated and the blood was discarded.
  • the mouse L3-L6 DRG tissue was isolated and used for total RNA extraction.
  • the specific operation method of RNA extraction was carried out according to the instructions of the RNA extraction kit, and then the RNA was eluted with 40 ⁇ l RNase-free H 2 O. After determining the RNA concentration, 300ng of total RNA was taken for cDNA synthesis.
  • the cDNA synthesis operation was performed according to the instructions of the HiScript II Q RT SuperMix kit.
  • the synthesized cDNA was used for real-time fluorescence quantitative PCR (qRT-PCR) to detect the expression changes of the corresponding genes.
  • the reaction mixture is: 10 ⁇ l 2X Premix Ex Taq TM II, 1 ⁇ l 10 ⁇ M Primer F, 1 ⁇ l 10 ⁇ M Primer R, 7 ⁇ l ddH 2 O, 1 ⁇ l cDNA, total volume 20 ⁇ l.
  • the reaction conditions are: 95°C for 2min; 95°C for 10s, 60°C for 50s, read the plate, 40cycles; 65°C ⁇ 95°C gradient temperature, 0.5°C/cycle, read the melt curve.
  • Gapdh was used as an internal reference gene, and the relative expression changes of the target genes were calculated using the 2 - ⁇ Ct method. Gapdh was used as the internal reference gene in the qRT-PCR experiment, and ACTIN was used as the loading control in the western blot experiment.
  • the SDS-PAGE stacking gel concentration is 5%, and the separation gel concentration is 10% or 12%.
  • mice After the mice were deeply anesthetized, a midline incision was made from the head to the occipital tuberosity, and the muscles from the occipital bone to the atlas were bluntly separated to expose the white dura mater.
  • the cerebrospinal fluid was extracted with a glass electrode from the cerebellomedullary cistern. 10 to 20 ⁇ l of cerebrospinal fluid was collected from each mouse. Centrifuge at 1000 g for 5 min at 4°C to remove cells. Take the supernatant and quickly store it at -80°C. cerebrospinal fluid samples with obvious blood contamination were discarded. Take 5 ⁇ l of cerebrospinal fluid and mix with 5 ⁇ l of PBS, then add 1/3 volume of 4X sample processing solution.
  • the upper bar graph represents the quantitative statistics of Wnt5a secretion levels.
  • ACTIN is detected as a negative control
  • CSF marker protein TTR Transthyretin
  • the lower part shows the results of western blot detection of Wnt5a, ACTIN, and TTR.
  • mice After the mice were deeply anesthetized with isoflurane, their heads were quickly decapitated and the blood was discarded. After surface disinfection with 75% alcohol, all DRG tissues of the mice were quickly separated on ice and placed in ice-cold PBS. After discarding the PBS, add 1 ml of enzyme solution (Collagenase A 20 mg/100 ml, Dispase II 300 mg/100 ml dissolved in PBS) for digestion at 37°C for 1 hour; centrifuge at 500g for 5 minutes to collect the digested tissue. Add 2 ml of DMEM culture medium (containing 10% FBS, 1X Pen/Strep), and mechanically pipet the tissue until there are no obvious clumps.
  • enzyme solution Collagenase A 20 mg/100 ml, Dispase II 300 mg/100 ml dissolved in PBS
  • the entire cell suspension was transferred, spread on the upper layer of 15% BSA solution, and centrifuged at 500 g for 10 min. Gently withdraw the upper solution, leaving only the cell pellet at the bottom of the centrifuge tube.
  • the cell suspension is dropped on a culture dish or cell slide pre-coated with poly-D-Lys and laminin, and placed in a 37°C CO 2 incubator for 30 minutes to allow DRG neurons to adhere to the cell slide at a higher cell density. Chip. Subsequently, an appropriate volume of Neurobasal medium was added, placed in a 37°C CO 2 incubator for overnight culture, and then used for corresponding experiments.
  • DRG neurons were cultured at 37°C for 24 hours, the medium was discarded and rinsed three times with preheated Neurobasal basal medium at 37°C.
  • Add 2 ml Neurobasal medium (containing 0.5% B27, 1% penicillin and streptomycin) to each dish, and culture it in a 37°C CO 2 incubator for 12 hours. Collect the culture medium and centrifuge at 1000g for 5 minutes at 4°C to remove cells and cell debris. Collect the supernatant, add 10% trichloroacetic acid (TCA), and shake gently at 4°C for 10 minutes to precipitate proteins in the culture medium.
  • TCA trichloroacetic acid
  • Figure 9 is a representative image of three independent repeated experiments. Detection of ACTIN was used as negative control and loading control.
  • Wnt5a protein in the DRG cell culture medium The increased intensity is due to the secretion of Wnt5a from STZ-treated DRG neurons into the culture medium.
  • the increased intensity of Wnt5a protein in the STZ-treated DRG cell lysates is due to its inability to be secreted into the culture medium and therefore accumulates in the culture medium. in cells.
  • mice were placed in the behavioral testing room to adapt for 1 hour every day.
  • the behavioral testing room should be kept quiet, with sufficient lighting, a temperature of 22 ⁇ 2°C, and a humidity of 40-60%. All behavioral experiments were tested and analyzed using a single-blind method.
  • mice On the day of the experiment, the mice were placed into a transparent observation grid with a length of 10cm, a width of 10cm, and a height of 13cm. The observation grid was placed on a wire mesh with a height of 30cm.
  • von Frey fiber 0.02-2g
  • a mouse is considered responsive if it shows behaviors such as lifting, shaking, and licking its feet within 3 seconds after being stimulated.
  • Dixon’s up-down method first use 0.16g von Frey wire to stimulate. When the mouse responds, select the adjacent low-intensity von Frey wire to stimulate. If the mouse does not respond, select the high-intensity von Frey wire to stimulate.
  • the foot withdrawal threshold was measured before STZ injection and 4 weeks after injection. After confirming the mechanical allodynia of mice, gradient doses of Box5 or Anti-Wnt5a IgG were injected intrathecally (i.t.) to evaluate the effect of antagonizing Wnt5a on STZ-induced mechanical allodynia behavior. Inject Saline or Goat IgG as a negative control. The results are shown in Figure 11.
  • Trpv1-ChR2/EYFP mice were injected with Vehicle or STZ, DRG tissues were taken for primary culture of DRG neurons.
  • Whole-cell patch-clamp recording technique was used to detect the effect of STZ treatment on the excitability of Trpv1+DRG neurons.
  • Box5 was perfused to detect the effect of antagonizing Wnt5a on the excitability of Trpv1+DRG neurons.
  • Whole-cell patch clamp recordings Use MultiClamp 700B amplifier and Digidata 1550B digitizer to perform whole-cell patch clamp recordings of primary cultured DRG neurons and HEK293T cells.
  • the glass electrode was drawn using P-97 microelectrode drawing instrument. When used for whole-cell recording, the electrode tip resistance value is controlled at 4–6 ⁇ .
  • inward currents of different strengths 600 ms were sequentially injected into the cells through the recording electrodes to record the action potential firing of DRG neurons. The results are shown in Figures 13 and 14.
  • inward currents of different strengths 600ms were sequentially injected into the cells through the recording electrodes to record the action potential firing of DRG neurons.
  • the results are shown in Figure 15.
  • the required amount of inward current to activate DRG neurons is shown in Figure 15.
  • the minimum current is Rheobase.
  • Wnt5a flox/flox mice aged 4-5 weeks were used as Wnt5a CKO mice after intrathecal injection of AAV9-CAG-Cre-mCherry (10 11 VG) virus; injection of AAV9-CAG-mCherry (10 11 VG) Viral Wnt5a flox/flox mice served as WT controls.
  • Von The Frey test was used to determine the foot withdrawal threshold of mice; after that, STZ 180 mg/kg body weight was injected intraperitoneally to construct a type I diabetes model. 7 days after STZ injection, blood was collected from the tail vein, and the random blood glucose level was measured with an ONETOUCH@UltraEasy blood glucose meter.
  • FIG. 16 is a timeline showing the experimental protocol for Wnt5a CKO mouse preparation, STZ modeling, behavioral testing, and tissue sample collection.
  • WT and Wnt5a CKO mice were injected intraperitoneally with STZ (180 mg/kg body weight) to induce type I diabetes model; before STZ injection and 1 to 6 weeks after injection, the Von frey test was performed to measure the mechanical allodynia of the mice and to evaluate Wnt5a Effect of knockout on STZ-induced allodynia behavior.
  • Real-time place escape/avoidance used to assess the emotional component of neuropathic pain in mice.
  • the Real-time PEA test box consists of two connected boxes with a length of 30cm, a width of 28cm, and a height of 50cm, distinguished by black and white horizontal and vertical stripes. Put the mouse into the test box, and use ANY-maze software to record the mouse movement trajectory.
  • the Real-time PEA experiment is divided into three consecutive stages, namely Pre-stimulation, Stimulation, and Post-stimulation.
  • Pre-stimulation phase without any stimulation, the mouse moves freely between the two boxes for 10 minutes; Stimulation phase: according to the preference shown by the mouse in the Pre-stimulation phase, when the mouse enters the preferred side Use 0.4g von Frey wire to stimulate the soles of its hind paws. When the mouse enters the non-preferred side, no stimulation is applied, and the mouse is allowed to move between the two boxes for 10 minutes; Post-stimulation stage: under the condition of no stimulation The mice moved freely between the two boxes for 10 min. Analyze the three stages of Pre, Stimulation and Post, and the total time for mice to enter the Stimulation side. Calculate the time difference between the mouse entering the Stimulation side before and after stimulation, that is, Pre-Post, as the mouse shows aversive escape after being stimulated. Aversion time. The results are shown in Figure 18.
  • the DRG and spinal cord lumbar enlargement tissue of WT and Wnt5a CKO mice were collected, total RNA was extracted, and cDNA was synthesized. PCR experiments were then performed to detect the expression levels of Wnt5a and Cre in DRG and spinal cord tissue.
  • the PCR reaction mixture is: 10 ⁇ l 2X Premix Taq TM II, 1 ⁇ l 10 ⁇ M Primer F, 1 ⁇ l 10 ⁇ M Primer R, 7 ⁇ l ddH 2 O, 1 ⁇ l cDNA, total volume 20 ⁇ l.
  • the reaction conditions were: 94°C for 3 minutes; 94°C for 10 seconds, 58°C for 15 seconds, 72°C for 30 seconds, 35 cycles; 72°C for 5 minutes.
  • C-E Intrathecally injected AAV9 virus mainly infects DRG neurons in DRG tissue.
  • Virus-infected mCherry/Cre signal and neuronal marker Nissl Wnt5a CKO mouse DRG tissue sections were used for immunohistochemical staining to evaluate the expression of AAV9-Cre/mCherry in DRG.
  • the tissue sections were blocked with blocking solution at room temperature for 1 to 2 hours, and an antibody was added: goat anti-IBA1 (macrophage marker protein IBA1) (1:1000) or rabbit anti-GS (satellite glial cell marker protein GS) (1:10000). ) and incubate at 4°C for 18 to 20 hours. Afterwards, wash the slices 4 times with PBS, 10 minutes each time. Add corresponding Alexa 488 fluorescent dye-conjugated secondary antibody (1:500, Jackson ImmunoResearch) or fluorescently labeled dye NeuroTrace 640/660-Nissl (1:800), and incubate at room temperature in the dark for 2 hours. Wash the slices 4 times with PBS, 10 minutes each time. Mount the slides with mounting medium containing antifade agent, dry at room temperature in the dark, and observe under a fluorescence microscope or store at -20°C.
  • AAV9-Cre/mCherry co-localizes with DRG neurons, but does not co-localize with the macrophage marker protein IBA1 and satellite glial cell marker protein GS, indicating that the AAV9 virus injected intrathecally DRG neurons are mainly infected in DRG tissue.
  • Tissue sections were blocked with blocking solution at room temperature for 1 to 2 hours, and rabbit anti-PGP9.5 (1:2000) was added and incubated at 4°C for 18 to 20 hours. Afterwards, wash the slices 4 times with PBS, 10 minutes each time. Add corresponding Alexa Cy3 fluorescent dye-coupled secondary antibody (1:500, Jackson ImmunoResearch) was placed at room temperature and incubated in the dark for 2 hours. Wash the slices 4 times with PBS, 10 minutes each time. Mount the slides with mounting medium containing antifade agent, dry at room temperature in the dark, and observe under a fluorescence microscope or store at -20°C.
  • tissue sections in the Meissner's corpuscles area were selected for quantitative statistics of intra-epidermal nerve terminal branches (IENF) in the epidermis.
  • IENF intra-epidermal nerve terminal branches
  • Z-axis superposition (20 ⁇ m) of each tissue section the composite image was taken for quantitative analysis of PGP9.5+ nerve terminals.
  • the complete PGP9.5+ nerve fiber branches extending from the dermis to the epidermis were included in the quantitative statistics of IENF.
  • Example 2 Wnt5a activates DRG nociceptive sensory neurons and enhances spinal dorsal horn excitability. Synaptic transmission, causing neuropathic pain phenotype
  • WT Naive mice were intrathecally injected with gradient doses of Wnt5a, and the Von frey test was performed to evaluate the mechanical allodynia caused by exogenous Wnt5a. The results are shown in Figure 26. Intrathecal injection of Saline was used as a negative control.
  • WT Naive mice were taken and intrathecally injected with 10ng Wnt5a, and Real-time PEA test was performed to evaluate the pain aversion caused by exogenous Wnt5a. The results are shown in Figure 27. Intrathecal injection of Saline was used as a negative control.
  • AdvillinCre:GCaMP6 flox/- DRG were primary cultured and used in calcium imaging experiments to detect the neuronal calcium response induced by Wnt5a by monitoring the fluorescence changes of GCamp6 protein.
  • primary cultured Under normal extracellular fluid perfusion, DRG neurons were administered 0.1-10ng/ml Wnt5a, 1 ⁇ M Capsaicin, etc. using the ALA-VM8 automatic drug delivery system for 15-30 seconds, and were washed with ECS for 5 minutes between two administrations.
  • Cell images were taken at an excitation wavelength of 470nm, and the changes in intracellular fluorescence intensity were continuously recorded by a high-speed scanning camera Flash4.0LT at a speed of 0.5fps.
  • Whole-cell patch clamp recordings Use MultiClamp 700B amplifier and Digidata 1550B digitizer to perform whole-cell patch clamp recordings of primary cultured DRG neurons.
  • 1ng/ml Wnt5a was administered by the ALA-VM8 automatic drug delivery system for 5 seconds, and the action potential firing of DRG small neurons (diameter ⁇ 25 ⁇ m) induced by Wnt5a was recorded.
  • Perfuse 1 ⁇ m Cap as a positive control. The results are shown in Figure 31.
  • Whole-cell patch clamp recordings Use MultiClamp 700B amplifier and Digidata 1550B digitizer to perform whole-cell patch clamp recordings of primary cultured DRG neurons.
  • 1ng/ml Wnt5a was administered for 5 s by the ALA-VM8 automatic drug delivery system, and the inward current of DRG small neurons (diameter ⁇ 25 ⁇ m) induced by Wnt5a was recorded.
  • Perfusion with 0.1 ⁇ m Cap served as a positive control. The results are shown in Figure 32.
  • Wnt5a can induce DRG small neurons to generate inward current.
  • mice (age 4 to 8 weeks) were used for spinal cord slice recording.
  • Urethane 1.5-2.0g/kg body weight
  • the mice were quickly decapitated and the spinal cord of the mouse lumbar segment (L4-L5) was quickly separated on ice and immediately placed in an oxygenated, in pre-cooled sectioning liquid.
  • the spinal cord slices were placed in oxygenated (95% O2, 5% CO2) slicing fluid and incubated at room temperature for more than 2 hours, and then patch-clamp recording was performed.
  • the P-97 microelectrode drawing instrument is used to draw the glass electrode, and the resistance value of the electrode tip is controlled at 5 ⁇ 10 M ⁇ .
  • the cell membrane potential was clamped at -70mV in voltage clamp mode, and the spontaneous excitatory postsynaptic current (sEPSC) of interneurons or SOM+ interneurons in the spinal cord dorsal horn lamina II layer was recorded.
  • the signal acquisition frequency is 10kHz and the filtering frequency is 2kHz. The results are shown in Figure 33 and Figure 34.
  • Figure 33 left: Representative picture of electrode recording of SOM+ neurons in the dorsal horn of the spinal cord. Red arrows indicate electrode-clamped SOM+ neurons. Right: Representative images of sEPSC recordings. a, b show partial enlarged images of sEPSCs recorded before and after Wnt5a (10ng/mL) administration.
  • Wnt5a can enhance the excitatory synaptic transmission of SOM+ interneurons in the spinal dorsal horn.
  • type I diabetes models were induced by intraperitoneal injection of STZ (180 mg/kg body weight); 4-5 weeks after STZ injection, DRG tissues were harvested for primary culture of neurons and whole-cell patch clamp electrophysiological recordings were performed. .
  • current clamp mode inward currents of different strengths (600 ms) were sequentially injected into the cells through the recording electrodes, and the action potential firing of DRG neurons was recorded.
  • Perfuse 10 ⁇ g/ml Box5 to evaluate the effect of Box5 on the excitability of DRG neurons. The perfused vehicle served as a negative control.
  • This example verified that GPR177-mediated Wnt5a secretion in DRG is involved in type II diabetic neuralgia, and confirmed that diabetic neuralgia can be significantly improved by antagonizing Wnt5a (by administering Wnt5a neutralizing antibodies or Wnt5a inhibitors).
  • WT and db/db mice were intrathecally injected with 5 ⁇ g Box5 (E) or 4 ⁇ g anti-Wnt5a antibody (F).
  • E 5 ⁇ g Box5
  • F 4 ⁇ g anti-Wnt5a antibody
  • the von Frey test was performed before injection and 0.5h to 1d after injection to evaluate the effect of antagonistic Wnt5a on type II diabetes model mice. Effects of mechanical touch on allodynia behavior. The results are shown in Figure 38 and Figure 39.
  • Example 4 Wnt5a selectively activates TRPV1 channels and can induce TRPV1 single-channel electrical activity in HEK293T cells.
  • HEK293T cells were cultured in DMEM medium (containing 10% FBS, 1X Pen/Strep) in a 37°C CO 2 incubator. After the cells grew to a density of more than 90%, they were digested with 0.25% Trypsin at 37°C for 2 minutes and then passaged. 24 hours after passage, when the cell density reaches 50% to 60%, Lipofectamine 2000 is used for plasmid processing. DNA transfection.
  • a 60mm culture dish prepare 2 to 3 ⁇ g of plasmid DNA and 125 ⁇ l of opti-MEM culture medium mixture for each dish, and prepare 4 to 6 ⁇ l of Lipofectamine 2000 and 125 ⁇ l of opti-MEM culture medium mixture. Mix separately and mix at a ratio of 1:1. , let it stand at room temperature for 5 to 10 minutes. Add the mixture dropwise to the petri dish, shake gently to mix, and incubate in the incubator for 24 to 48 hours before using for corresponding experiments.
  • HEK293T cells were transfected with plasmid DNA, they were cultured in a 37°C CO 2 incubator for 24-36 hours, and incubated with calcium dye Fluo-2AM (2 ⁇ M, containing 0.02% F127) for 30 min at 37°C, followed by calcium imaging experiments.
  • the cell images were continuously and alternately excited by a high-speed continuous monochromatic light source at the excitation wavelength of 340/380nm, and the changes in intracellular fluorescence intensity were continuously recorded by a high-speed scanning camera Flash4.0LT at a speed of 0.5fps, at 340nm/380nm.
  • the ratio of the lower fluorescence signals represents the intensity of the calcium signal.
  • the top is a picture of HEK293T cells transfected with DsRed-N1 empty plasmid; the bottom is a picture of HEK293T cells transfected with Trpv1-DsRed-N1 plasmid.
  • Wnt5a selectively activates TRPV1 channels when transfected with different types of TRP channels.
  • HEK293T cells were transfected with plasmid Trpv1-EGFP-N1, they were cultured in a 37°C CO 2 incubator for 24 to 36 hours, and then whole-cell patch clamp recording experiments were performed. In voltage clamp mode, the cell membrane potential was clamped at -70mV, and the inward current generated by HE293T cells activated by 10ng/ml Wnt5a was recorded. 100nM Cap was perfused as a positive control. Wnt5a and Cap were administered for 10 seconds respectively, with an interval of 5 minutes between two administrations. The results are shown in Figure 43.
  • the left side is a representative picture of Wnt5a-evoked inward current.
  • the right side shows quantitative statistics of the amplitude of Wnt5a-evoked inward current.
  • Wnt5a activates TRPV1 to induce inward current.
  • Trpv1-EGFP Trpv2-EGFP
  • Trpv3-EGFP Trpa1-EGFP
  • Trpa1-EGFP whole-cell patch clamp recording experiments were performed.
  • voltage clamp mode the cell membrane potential was clamped at -70mV, and the inward current generated by HE293T cells activated by 10ng/ml Wnt5a was recorded.
  • the left side is a representative picture of Wnt5a-induced single-channel current under different voltage clamps.
  • the right side is the I/V curve of single channel current induced by Wnt5a.
  • the potential binding sites between Wnt5a and TRPV1 were determined, and a variety of antagonistic peptides and control peptides were designed. It was confirmed that the antagonistic peptides can inhibit the iCa2+ activity induced by Wnt5a. In vivo experiments further verified that the antagonistic peptides can improve diabetes. Allodynia phenotype in mice.
  • the HHpred server https://toolkit.tuebingen.mpg.de/#/ was used to model the homology structure of Wnt5a protein using the WNT signaling complex structure (PDB ID: 6AHY) as a template.
  • PDB ID: 6AHY WNT signaling complex structure
  • the Wnt5a and mTRPV1 structural models were further refined through the Rosetta molecular modeling component relax application; the molecular model with the lowest energy score was selected for further molecular structure modeling; a total of 20,000 molecular models were generated for protein docking experiments.
  • 1000 docking models with the lowest total energy score (Rosetta energy term name: score) were screened; from these 1000 models, 10 models with the lowest interface score (Rosetta energy term name) were further screened.
  • residue_energy application uses the residue_energy application to calculate the total binding energy of potential binding sites between Wnt5a and mTRPV1 channels.
  • Amino acid residues with a total binding energy greater than -0.6R.E.U (Rosetta energy unit) are represented in dark red.
  • antagonist peptides were designed to block the interaction between TRPV1 and Wnt5a. All possible binding sites in the sequence of the antagonist peptide AA601-25 were mutated to alanine residues as a control peptide.
  • Control(Ctl)peptide is a mutated form of polypeptide AA601-25, in which the possible binding site is replaced with alanine.
  • sequence information of AA601-25 is: EDGKNNSLPVESPPHKCRGSACRPG (SEQ ID NO.3).
  • sequence information of AA453-72 is: AYYRPVEGLPPYKLNNTVGD (SEQ ID NO.1).
  • sequence information of AA644-56 is: TCLELFKFTIGMG (SEQ ID NO.6).
  • sequence information of humanized AA601-25 is: EDGKNDSLPSESTSHRWRGPACRPP (SEQ ID NO.5).
  • WT mice were intrathecally injected with 1ng Wnt5a and different antagonist peptides; before intrathecal injection and 0.5h-3d after injection, Von frey test was performed to measure the mechanical allodynia of mice and evaluate different forms and doses of antagonism. Effect of peptides on Wnt5a-induced allodynia behavior. Inject the vehicle as a negative control. The results are shown in Figure 49 and Figure 50.
  • the antagonist peptide AA601-25 inhibits Wnt5a-induced iCa2+ activity, but does not affect Cap-induced iCa2+ activity.
  • AdvillinCre:GCaMP6 flox/- DRG was taken for primary culture and used for calcium imaging experiments.
  • Wnt5a or Cap For the first time, mix it with the antagonist peptide AA601-25. After perfusion for 15 seconds, use normal extracellular fluid to rinse for 5 minutes; perfuse Wnt5a or Cap alone for 15 seconds.
  • the left side is the real-time change curve of iCa2+ activity induced by Wnt5a (1ng/ml) or Cap (1 ⁇ M); the right side is ⁇ Fmax/F0 in iCa2+ activity induced by Wnt5a (1ng/ml) or Cap (1 ⁇ M).
  • the antagonist peptide AA601-25 inhibits the iCa2+ activity induced by Wnt5a, but does not affect the iCa2+ activity induced by Cap.
  • the antagonist peptide AA601-25 can improve the allodynia phenotype of diabetic mice.
  • the type I diabetes model was induced by intraperitoneal injection of STZ (180 mg/kg body weight); before STZ injection and 4 weeks after injection, the Von Frey test was performed to measure the mechanical allodynia of the mice.
  • Gradient doses of the antagonist peptide AA601-25 were injected intrathecally.
  • the Von frey test was performed to measure the mechanical allodynia of mice and evaluate the effect of AA601-25 on STZ-induced allodynia.
  • Intrathecal injection of vehicle served as a negative control. The results are shown in Figure 52.
  • Trpv1 can block the allodynia phenotype induced by Wnt5a.
  • Trpv1 Knocking out Trpv1 blocks the Wnt5a-induced allodynia phenotype.
  • WT and Trpv1 -/- mice were intrathecally injected with 1ng Wnt5a; before Wnt5a injection and 0.5h-2d after injection, Von frey test was performed to measure the mechanical allodynia of mice and evaluate the effect of Trpv1 knockout on Wnt5a induction. Effects of allodynia behavior. The results are shown in Figure 53.
  • Trpv1 blocks the allodynia phenotype induced by Wnt5a.
  • Trpv1 Knocking out Trpv1 can improve the STZ-induced neuropathic pain phenotype.
  • Trpv1 -/- mice were injected intraperitoneally with STZ (180 mg/kg body weight) to induce type I diabetes model; before STZ injection and 1-6 weeks after injection, the Von frey test was performed to measure the mechanical allodynia of the mice. To evaluate the effect of Trpv1 knockout on STZ-induced allodynia behavior. The results are shown in Figure 54.
  • Trpv1 can improve the neuropathic pain phenotype induced by STZ.
  • GPR177-Wnt5a-TRPV1 signaling axis can be used as a potential target for clinical treatment of DNP
  • GPR177 In human DRG, GPR177 is expressed in NF200+ DRG neurons and co-expressed with WNT5A mRNA.
  • the expression of GPR177 and WNT5A mRNA in human DRG tissue sections was detected using in situ hybridization experiments, and the Dapb probe was used as a negative control.
  • the human DRG was fixed in 4% PFA for 2 to 4 days and dehydrated in 30% sucrose solution for more than 2 days until the tissue block precipitated.
  • the fully dehydrated tissue blocks were rinsed 2 to 3 times in PBS, trimmed off excess tissue, quickly frozen and embedded in OCT, and sectioned in a freezing microtome. DRG tissue was cut into 10 ⁇ m thickness for patch staining.
  • the in situ hybridization experiment was performed according to the instruction manual of the RNAscope in situ hybridization kit.
  • the DRG sections continue to undergo immunohistochemical staining in the dark.
  • Tissue sections were blocked with blocking solution at room temperature for 1 to 2 hours, and chicken anti-NF200 (1:1000) was added and incubated at 4°C for 18 to 20 hours. Afterwards, wash the slices 4 times with PBS, 10 minutes each time. Add corresponding Alexa 488 fluorescent dye-coupled secondary antibody (1:500, Jackson ImmunoResearch) was placed at room temperature and incubated in the dark for 2 hours. Wash the slices 4 times with PBS, 10 minutes each time. Mount the slides with mounting medium containing antifade agent, dry at room temperature in the dark, and observe under a fluorescence microscope or store at -20°C. The results are shown in Figure 55.
  • GPR177 is expressed in NF200+ DRG neurons in human DRG tissue sections and is co-expressed with WNT5A mRNA.
  • NIS Element AR software was used for quantitative analysis of fluorescence signal intensity. Select 4 to 6 areas with no fluorescence signal in each picture, and the average value of the obtained signal values is used as the background intensity of the background signal.
  • the fluorescence signal of a cell is ⁇ 2X (background intensity+standard deviation), it is regarded as a positive cell; when a clear outline of the nucleus can be seen in a positive cell, it is used for quantitative analysis of cell area.
  • the cell areas of GPR177 + neurons and all DRG neurons were counted, and their distribution ratios in different area intervals were calculated. The results are shown in Figure 56.
  • GPR177 + neurons are mainly distributed in medium to large diameter human DRG neurons.
  • NIS Element AR software was used for quantitative analysis of fluorescence signal intensity. Select 4 to 6 areas with no fluorescence signal in each picture, and the average value of the obtained signal values is used as the background intensity of the background signal. When the fluorescence signal of a cell is ⁇ 2X (background intensity+standard deviation), it is regarded as a positive cell; when a clear outline of the nucleus can be seen in a positive cell, it is used for quantitative analysis of cell area. Statistics were obtained from at least 4 DRG slices, and the proportion of WNT5A + , NF200 + neurons among GPR177 + neurons was calculated. The results are shown in Figure 57.
  • No DNP represents diabetic patients without neuropathic pain symptoms (NRS score ⁇ 2).
  • DNP represents diabetic patients with neuropathic pain (NRS score ⁇ 4).
  • Detection ACTIN was used as a negative control, and Ponceau staining was used as a loading control.
  • HEK293T cells were transfected with human Trpv1-EGFP plasmid, they were cultured in a 37°C CO 2 incubator for 24 to 36 hours, and then whole-cell patch clamp recording experiments were performed. In voltage clamp mode, the cell membrane potential was clamped at -70mV, and the inward current generated by HE293T cells activated by 10ng/ml Wnt5a was recorded. 100nM Cap was perfused as a positive control. The results are shown in Figure 60.

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Abstract

L'invention concerne un régulateur Wnt5a et un utilisateur de celui-ci. En étudiant la pathogenèse de DNP, une nouvelle cible analgésique, Wnt5a, est découverte, fournissant une nouvelle stratégie d'intervention pour le traitement clinique de la douleur neuropathique ; un régulateur basé sur la nouvelle cible analgésique, Wnt5a, est développé, et peut soulager efficacement la douleur neuropathique.
PCT/CN2023/080207 2022-03-15 2023-03-08 Régulateur wnt5a et son utilisation WO2023174116A1 (fr)

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