WO2023109622A1 - p75细胞外结构域在类风湿性关节炎治疗和/或预防中的应用 - Google Patents

p75细胞外结构域在类风湿性关节炎治疗和/或预防中的应用 Download PDF

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WO2023109622A1
WO2023109622A1 PCT/CN2022/137395 CN2022137395W WO2023109622A1 WO 2023109622 A1 WO2023109622 A1 WO 2023109622A1 CN 2022137395 W CN2022137395 W CN 2022137395W WO 2023109622 A1 WO2023109622 A1 WO 2023109622A1
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p75ecd
group
model group
fusion
use according
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PCT/CN2022/137395
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English (en)
French (fr)
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周新富
周意
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苏州澳宗生物科技有限公司
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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
    • 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
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the invention belongs to the field of pharmacy, and in particular relates to a new application of p75 extracellular domain (p75ECD), in particular to the preparation of p75 extracellular domain (p75ECD) for treating and/or preventing rheumatoid arthritis (RA). application in medicine.
  • p75ECD p75 extracellular domain
  • p75ECD p75 extracellular domain
  • RA rheumatoid arthritis
  • RA Rheumatoid arthritis
  • RA is an autoimmune disease characterized by chronic erosive arthritis, the basic pathological changes are synovitis, acute synovial swelling, exudation, and granulocyte infiltration; In the chronic phase, the synovial membrane hyperplasia and hyperplasia forms pannus, which is the pathological basis for joint destruction, joint deformity, obstacles, and the irreversible stage of the disease.
  • RA is characterized by symmetric small joint erosive lesions, mainly involving wrist joints, metacarpophalangeal joints, proximal interphalangeal joints, knee joints, ankle joints, and toe joints, etc., and can cause local and systemic damage to bone tissue. effect.
  • RA is a chronic and progressive disease.
  • RA The global incidence of RA is 0.5%-1.0%, and it can occur in any age group. Among them, 80% of RA patients are between the ages of 20 and 45, and most of them are women, which is about three times the incidence of men [1 ] . The current incidence rate in our country is about 0.32%-0.36%.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • MDARDs disease modifying anti-rheumatic drugs
  • Methotrexate has been widely recognized by clinicians, and its potential adverse effects include oral ulcers, liver cirrhosis, hepatitis, and interstitial pneumonia and cytopenia [3] ; 3) Macromolecular biological therapeutic drugs, such as adalimumab, etanercept, golimumab, etc., play a role by blocking a specific inflammatory cytokine or surface molecule .
  • the use of macromolecular biopharmaceuticals is a major breakthrough in the treatment of RA, but it can cause local rashes, infections, tuberculosis, demyelination syndrome, etc. [4] recurrence [5] .
  • RA rheumatoid arthritis
  • CN 102233128 B discloses the application of p75NTR-ECD in the preparation of drugs for preventing and treating Alzheimer's disease.
  • CN 106794222 B discloses the use of p75 extracellular domain (p75ECD) in the preparation of medicines for treating and/or cerebral amyloid angiopathy (CAA).
  • CN107303389A discloses the use of peripheral brain-derived neurotrophic factor precursor protein (proBDNF) or a binding molecule with neutralizing or inhibiting activity in its signaling molecule in the preparation of drugs for relieving, inhibiting or treating pain.
  • proBDNF peripheral brain-derived neurotrophic factor precursor protein
  • CN 112472796 A discloses the use of p75 extracellular domain (p75ECD) or its functional fragments, variants, analogs or derivatives in the preparation of medicines for treating and/or preventing frontotemporal dementia.
  • CN 113456799A discloses the application of p75ECD in the preparation of medicine for regulating pain, wherein the pain includes inflammatory pain or surgical pain.
  • CN 104302326B (PCT/GB2013/050632) discloses the therapeutic use of p75NTR neurotrophic factor binding protein, and specifically teaches that p75NTR (NBP) is used to treat pain and/or pain symptoms.
  • CN 105873943A (PCT/GB2014/052833) discloses a fusion protein, p75NTR(NBP)-Fc fusion protein, for treating pain or pain symptoms.
  • US2009/0175859A1 relates to the treatment of tumor necrosis factor (TNF)-mediated diseases with TNF ⁇ antagonists and methotrexate, and specifically discloses a composition comprising methotrexate and anti-TNF antibodies, and wherein TNF-mediated Diseases include rheumatoid arthritis (RA), Crohn's disease, and acute and chronic transplant-associated immune diseases.
  • TNF tumor necrosis factor
  • NBP neurotrophic factor binding protein
  • BDNF brain-derived neurotrophic factor
  • NT3 or NT4/5 binding affinity Kd is 1 pM-100 nM
  • EP2667895B1 (PCT/IT2012/000018) relates to at least one p75NTR receptor inhibitor, alone or in combination with at least one TrkA receptor activator, or at least one TrkA receptor activator for the treatment of chronic inflammatory diseases in which p75NTR Receptor inhibitors are selected from polyclonal and monoclonal monospecific or bispecific p75NTR receptor-binding antibodies, chimeric molecules (whose structure includes soluble p75NTR receptor, p75NTR receptor-binding cyclic monomers and dimeric NGF-like compound), and wherein the TrkA receptor activator is selected from TrkA receptor binding polyclonal and monoclonal antibodies, cyclic monomeric and dimeric NGF analog compounds, recombinant NGF or recombinant mutant NGF.
  • said chronic inflammatory disease is selected from the group consisting of rheumatoid arthritis (RA), juvenile idiopathic arthritis, chronic inflammation of the bowel, ankylosing spondylitis, psoriasis, multiple sclerosis, lupus erythematosus, scleroderma disease, Sjögren's syndrome.
  • RA rheumatoid arthritis
  • juvenile idiopathic arthritis chronic inflammation of the bowel
  • ankylosing spondylitis psoriasis
  • multiple sclerosis multiple sclerosis
  • lupus erythematosus scleroderma disease
  • Sjögren's syndrome Sjögren's syndrome.
  • p75 neurotrophic factor extracellular domain p75ECD
  • RA rheumatoid arthritis
  • the present invention specifically relates to a method for preventing and/or treating rheumatoid arthritis disease (RA) with p75 neurotrophic factor receptor extracellular domain (p75NTR-ECD, also known as p75ECD), comprising administering p75ECD to patients in need, or involving Use of p75ECD in preparing a medicament for preventing and/or treating rheumatoid arthritis disease (RA).
  • RA rheumatoid arthritis disease
  • p75NTR-ECD also known as p75ECD
  • the present invention also relates to p75ECD for preventing and/or treating rheumatoid arthritis (RA), or a medicine for preventing and/or treating rheumatoid arthritis (RA), the medicine comprising p75ECD.
  • RA rheumatoid arthritis
  • RA rheumatoid arthritis
  • the present invention unexpectedly finds that p75ECD has very good curative effect in treating and/or preventing rheumatoid arthritis, especially in alleviating joint swelling, relieving inflammation pain and reducing joint synovial hyperplasia, synovial inflammation, cartilage damage, bone damage, etc. It has unexpected effects, which may be blocked by blocking proBDNF/p75NTR signaling pathway and inhibiting Janus Kinase 1 (Janus Kinases 1, JAK1)/transducer and activator of transcription 1 (stat1) phosphorylation and then blocking Tumor necrosis factor-a (TNF-a), interleukin-1 ⁇ (IL-1 ⁇ ), interleukin-6 (IL-6) and other inflammatory factors play a role.
  • TNF-a Tumor necrosis factor-a
  • IL-1 ⁇ interleukin-1 ⁇
  • IL-6 interleukin-6
  • treating is intended to mean that administration of p75ECD alleviates or ameliorates at least some of the signs or symptoms of RA.
  • prevention is intended to mean that the administration of p75ECD occurs prior to the diagnosis of RA and alleviates, ameliorates or delays the onset of at least some of the signs or symptoms of RA; or that the administration of p75ECD prevents Signs or symptoms of RA worsen. Accordingly, the term “prevention” is intended to include prophylactic treatment.
  • the administered p75ECD can be from any target species (eg, human, mouse, rat, rabbit, cat, dog, goat, cow, horse, non-human primate, etc.). However, in one embodiment, the p75ECD is human p75ECD. In one embodiment, the p75ECD has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2, such as the nucleotide sequence shown in SEQ ID NO: 1, or has the nucleotide sequence shown in SEQ ID NO: 2 amino acid sequence.
  • the p75ECD may be a functional fragment, variant, analog or derivative of this sequence.
  • variants, analogs or derivatives of p75ECD differ from p75ECD (or functional fragments thereof), but retain the same biological activity, particularly the ability to bind to neurotrophic factors Molecular makeup of p75ECD similarity.
  • Variants, analogs or derivatives of p75ECD (or functional fragments thereof) may have significant overall structural similarity to p75ECD (or functional fragments thereof), or may only be responsible for the One or more regions of biological activity share structural similarities.
  • a variant, analog or derivative is provided by, or is the result of, a modification of one or more amino acids of the relevant amino acid sequence.
  • the functional fragments, variants, analogs or derivatives comprising the sequence shown in SEQ ID NO: 2 are obtained by adding one or more amino acids to the amino acid sequence, deleting one or more amino acids from the amino acid sequence and/or from The amino acid sequence is provided by substitution of one or more amino acids, or is the result of the modifications described above. Amino acid inversions and other mutational changes resulting in changes in the amino acid sequence are also contemplated. Such analogs or derivatives may be obtained by introducing nucleotide changes in the polynucleotide molecule such that the desired amino acid change is obtained upon expression of the mutagenized polynucleotide molecule or by otherwise synthesizing amino acids incorporating the desired amino acid change. sequence to prepare.
  • Amino acid substitutions may include conservative or non-conservative amino acid substitutions.
  • a conservative amino acid substitution means replacing an amino acid residue with another amino acid that has similar properties and does not significantly alter the biological activity of the peptide or polypeptide.
  • Exemplary conservative amino acid substitutions are provided in Table 1 below. Specific conserved amino acids envisioned are: G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; Alkyl amino acids, which may have small changes in sequence, but do not cause any significant reduction or change in biological activity.
  • These changes may include conservative amino acid substitutions such as Gly, Ala, Val, Ile, Leu, Met; Asp, Glu, Asn, Gln; Ser, Thr; Lys, Arg, His; Phe, Tyr, Trp, His; - alkyl amino acids; and non-conservative amino acid substitutions.
  • conservative amino acid substitutions such as Gly, Ala, Val, Ile, Leu, Met; Asp, Glu, Asn, Gln; Ser, Thr; Lys, Arg, His; Phe, Tyr, Trp, His; - alkyl amino acids; and non-conservative amino acid substitutions.
  • the analogs or derivatives may include one or more amino acids not encoded by the genetic code such as carboxyglutamic acid and hydroxyproline, or may include D-amino acids in place of L- amino acid.
  • the analogue or derivative may be a mimetic such as a peptidomimetic of p75ECD (or a functional fragment thereof).
  • analogs or derivatives of p75ECD (or functional fragments thereof) do not necessarily have amino acid sequence identity and/or similarity, and in fact the analogs or derivatives may not be proteins at all.
  • peptides having amino acid sequences that are slightly altered from those shown in SEQ ID NO: 2 and SEQ ID NO: 4 may still fall within the scope of the present invention, as long as the peptide is capable of binding p75 and preventing signaling through p75 Transduction is enough.
  • natural variants (or isoforms) of p75ECD or p75ECD peptides derived from non-human species are considered to fall within the scope of the disclosed p75ECD or p75ECD-Fc fusion peptides.
  • the p75ECD comprises a p75ECD fusion, wherein the p75ECD is fused to a fusion partner.
  • the fusion partner can be any suitable fusion partner known to those skilled in the art, such as the Fc region of immunoglobulin, human serum albumin (HSA) and bovine serum albumin (BSA), etc., as long as the p75ECD remains At least some of its biological activity is sufficient. Fusion products can be produced by expressing the fusion product from a suitable construct encoding the fusion product, or by otherwise chemically linking the p75ECD to the fusion partner. The fusion partner should increase the in vivo stability and serum half-life of the p75ECD.
  • p75ECD is fused to the Fc domain of an immunoglobulin to form a p75ECD-Fc fusion.
  • the Fc domain may be any suitable Fc domain from any suitable immunoglobulin. However, in a preferred embodiment, said Fc domain is from human IgG.
  • the Fc region is capable of dimerizing to produce a dimeric form of p75ECD.
  • p75ECD may be oligomers such as dimers, trimers, tetramers, pentamers and the like.
  • the p75ECD may be a dimer.
  • the p75ECD-Fc fusion may be a p75ECD-Fc dimer.
  • the p75ECD-Fc fusion comprises a human p75ECD molecule linked to the Fc portion of human IgGl.
  • fusions may have substantially different nucleotide and amino acid sequences depending, for example, on the linkers used.
  • SEQ ID NO: 3 is the nucleotide sequence of a human p75ECD-human IgG1 Fc domain fusion (human p75ECD-Fc). Based on degeneracy, other nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:4 or variants thereof are also easily conceivable, and fall within the protection scope of the present invention.
  • the amino acid sequence of the human p75ECD-Fc fusion is provided in SEQ ID NO:4.
  • SEQ ID NO: 4 comprises the p75ECD peptide at amino acids 1-169 of SEQ ID NO: 4, followed by the linker "DKTHTCPPCP" at residues 170 to 179, followed by Fc domain at residues 180 to 396.
  • the p75ECD-Fc fusion may be a functional fragment, variant, analog or derivative of the sequence shown in SEQ ID NO:4. Functional fragments, variants, analogs or derivatives are described elsewhere herein.
  • p75ECD or a p75ECD fusion is administered to the subject as a nucleotide molecule.
  • nucleotide molecules may consist of, for example, expression vectors or expression cassettes.
  • Preferred expression vectors include viral vectors capable of effecting transduction of the desired host cell type, such as retroviral vectors, adenoviral vectors, and vectors derived from adeno-associated virus (AAV), such as those well known to those skilled in the art .
  • retroviral vectors such as retroviral vectors, adenoviral vectors, and vectors derived from adeno-associated virus (AAV), such as those well known to those skilled in the art .
  • AAV adeno-associated virus
  • Viral vectors comprising nucleotide sequences encoding p75ECD or p75ECD fusions of the present disclosure can be used to provide an in vivo source of p75ECD or p75ECD fusions at desired sites for the treatment or prevention of RA.
  • the p75ECD or p75ECD fusion is administered via a viral vector, which is known to provide a vehicle for delivering nucleotides into cells in a manner suitable for gene therapy.
  • a viral vector which is known to provide a vehicle for delivering nucleotides into cells in a manner suitable for gene therapy.
  • AAV adeno-associated viral
  • lentiviral vectors lentiviral vectors
  • herpes simplex virus vectors can be used as viral vectors as they all deliver nucleotides to cells for gene therapy purposes . Delivery of p75ECD or p75ECD fusions by viral vectors can mediate long-term expression of the agent in joints, and due to the chronic nature of RA, continued secretion of p75ECD or p75ECD fusions is desirable.
  • the use of AAV vectors is advantageous because the vectors can be administered as a single injection (e.g., intra-articular injection), can provide long-term secretion of the agent, and are also considered very safe , no pathological events have been reported in animals or humans.
  • AAV vectors are known tools for gene therapy delivery.
  • the viral vector may be an AAV vector.
  • the AAV vector can be any suitable adeno-associated virus vector known to those skilled in the art, such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and the like.
  • the AAV vector is AAV8.
  • the p75ECD-Fc was inserted into an AAV8 vector (supplied by Virovek) containing the AAV8 capsid sequence of the AAV2 phospholipase domain to enhance the potency of AAV8.
  • the sequence of the AAV8 capsid sequence with the AAV2 phospholipase domain is shown in SEQ ID NO: 5, wherein the AAV2 phospholipase domain sequence comprises nucleotides 26 to 431.
  • a “therapeutically effective amount” is any amount that induces a beneficial or therapeutic effect in a subject.
  • the dose of AAV vector for any suitable subject is between 1 x 106 to 6 x 1013 viral genomes/kg subject body weight. In one embodiment, the dose of AAV vector for any suitable subject is between 1 x 1011 and 6 x 1012 viral genomes/kg subject body weight.
  • the dose of adeno-associated viral vector administered may vary, as is well recognized by those skilled in the art.
  • a "safe" dose used in patients was 6 x 1012 viral genomes (vg)/kg body weight.
  • the dose of adenoviral vector administered to a human subject may range from 1 x 106 to 6 x 1013 vg/kg body weight.
  • the dose of adenoviral vector administered to a human subject is between 1 ⁇ 10 10 and 6 ⁇ 10 12 viral genomes/kg body weight.
  • the dose of adenoviral vector administered to a human subject is between 1 ⁇ 10 11 and 3 ⁇ 10 12 viral genomes/kg body weight.
  • the dose of adenovirus can be higher, such as 1 ⁇ 10 14 , 1 ⁇ 10 15 , 1 ⁇ 10 16 , 1 ⁇ 10 17 , 1 ⁇ 10 18 , 1 ⁇ 10 19 , 1 ⁇ 10 20 , etc. , as long as those doses are considered safe.
  • FDA Food and Drug Administration
  • a "safe" dose in mice could be 8 x 1010 viral genomes.
  • the dose of adeno-associated viral vector administered to a mouse subject may range from 1 x 105 to 1 x 1013 viral genomes.
  • the dose of adeno-associated viral vector administered to a mouse subject may range from 1 x 108 to 1 x 1012 viral genomes. In one embodiment, the dose of adeno-associated viral vector administered to a mouse subject may range from 1 x 109 to 1 x 1011 viral genomes.
  • the p75ECD or p75ECD-Fc fusion is a recombinant peptide.
  • Recombinant p75ECD or p75ECD-Fc fusion peptides can be produced using any suitable technique known to those skilled in the art, for example by in cultured bacterial systems (e.g. Escherichia coli), yeast expression systems, insect expression systems, viral Expression system or expression in eukaryotic cell expression system, or the peptide can be artificially synthesized, etc., all of which are known to those skilled in the art.
  • the dose of recombinant p75ECD or p75ECD-Fc fusion peptide administered may be any suitable amount that elicits a beneficial or therapeutic effect in a subject, and may vary with the route of administration. In one embodiment, the dosage is from about 0.01 to about 500 mg/kg body weight of the subject per day, which may be administered in single or multiple doses.
  • the dose is about 0.1 to about 250 mg/kg per day, for example, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg , 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg /kg, 25mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg, 90mg/kg, 100mg/kg, 150mg/kg, 200mg/kg , more preferably from about 0.5 to about 20 mg/kg per day.
  • the p75ECD, p75ECD fusion, p75ECD-Fc or AAV-p75ECD-Fc of this aspect of the disclosure is provided in combination with a pharmaceutically acceptable carrier, excipient and/or diluent.
  • a pharmaceutically acceptable carrier, excipients and/or diluents are well known to those skilled in the art.
  • the p75ECD, p75ECD fusion, p75ECD-Fc, or AAV-p75ECD-Fc of this aspect of the disclosure may be administered to the subject by any suitable route, such as orally, intravenously, intramuscularly, intraperitoneally, intranasally, Intracranial, intrathecal, or intraventricular routes.
  • the p75ECD or p75ECD-Fc is administered intravenously, intraperitoneally, intramuscularly, intrathecally or intracerebroventricularly.
  • a method of treating and/or preventing RA by blocking signal transduction through p75 comprising administering to a subject in need thereof a therapeutically effective amount of p75ECD or an analog thereof.
  • the p75ECD or analog thereof prevents the binding of a neurotoxic molecule selected from proBDNF and proNT3 to p75.
  • the method of this aspect uses the embodiments described above.
  • the present invention comprises the extracellular segment p75ECD of the p75NTR receptor, wherein said p75ECD consists of the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof (for example, a variant after one or more conservative amino acid substitutions).
  • p75ECD is fused to the Fc domain of immunoglobulin to form a p75ECD-Fc fusion, wherein said p75ECD-Fc is composed of the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof (for example a or variants after multiple conservative amino acid substitutions).
  • the daily dosage of p75ECD-Fc fusion protein is between 0.1 mg/kg body weight and 250 mg/kg body weight of the subject.
  • the daily dose is preferably 0.1-100 mg/kg subject body weight, for example 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 5.0 mg/kg, 10.0 mg/kg, 15mg/kg, 20mg/kg, 30mg/kg, 40mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg or 90mg/kg etc.
  • the daily dosage of the AAV vector of the p75ECD-Fc fusion protein is between 1 ⁇ 10 6 and 11 ⁇ 10 13 viral genes/kg body weight of the subject.
  • the daily dose is preferably between 1 ⁇ 10 8 and 11 ⁇ 10 13 viral genes/kg body weight of the subject.
  • the p75 extracellular domain (p75ECD) is used in combination with a second pharmacologically active compound separately, sequentially or simultaneously.
  • the second pharmacologically active compound is selected from:
  • Opioid pain relievers such as morphine, heroin, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone , meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, Propoxyphene, nelmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol ), nalbuphine or pentazocine;
  • Nonsteroidal anti-inflammatory drugs such as asprine, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen , flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofen Meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, nitrofluoro nitroflurbiprofen, olsalazine, phenylbutazone, piroxicam, sulindac, tolmetin, or zomepirac;
  • NIDs Nonsteroidal anti-inflammatory drugs
  • DMARDs Disease-modifying antirheumatic drugs
  • sulfasalazine such as sulfasalazine, D(-)-penicillamine, methotrexate, leflunomide , azathioprine, cyclosporin or hydroxychloroquine;
  • Tumor necrosis factor alpha inhibitors such as infliximab, adalimumab, etanercept, golimumab, or certolizumab ;
  • B cell inhibitors such as rituximab or belimumab
  • T-cell inhibitors such as abatacept
  • Cytokine inhibitors such as tocilizumab, sirukumab, clazakizumab, olokizumab, sarilumab, or anakinra prime (anakinra);
  • JAK1/JAK2 inhibitors such as baricitinib
  • Granulocyte-phagocyte colony-stimulating factor (GM-CSF) receptor blockers such as megim.
  • the second pharmacologically active compound is selected from etanercept.
  • the dose and frequency of administration of the preparation of the present invention can vary according to the patient's condition, and sometimes relatively short intervals and relatively high doses are required until the progression of the disease is alleviated or terminated, preferably until the patient shows partial or complete improvement of disease symptoms.
  • the drug of the present invention can be administered by intravenous, intraperitoneal, intracerebroventricular, intrathecal or intramuscular injection.
  • Figure 1 shows the total score of limb joint swelling
  • Fig. 2 shows 14 days, 24 days area under the curve (AUC) of total score of limb joint swelling degree, wherein One-way ANOVA: ***p ⁇ 0.001vs. normal control group; #p ⁇ 0.05vs. model group;# #p ⁇ 0.01vs. model group; ###p ⁇ 0.001vs. model group; T-test: $p ⁇ 0.05vs. model group; $$p ⁇ 0.01vs. model group; $$$p ⁇ 0.001vs .model group;
  • Figure 3 shows left foot pain threshold
  • Figure 4 shows the right foot pain threshold
  • Fig. 5 shows left foot pain threshold 14 days, 19 days area under the curve (AUC), wherein One-way ANOVA: ***p ⁇ 0.001vs. normal control group; #p ⁇ 0.05vs. model group; ##p ⁇ 0.01vs. model group; ###p ⁇ 0.001vs. model group;
  • Fig. 6 shows right foot pain threshold 14 days, 19 days area under the curve (AUC), wherein One-way ANOVA: ***p ⁇ 0.001vs. normal control group; #p ⁇ 0.05vs. model group; ##p ⁇ 0.01vs. model group; ###p ⁇ 0.001vs. model group;
  • Figure 7-8 is the overall view of the foot detected by uCT and the view of the ankle joint;
  • Figure 7 is the overall view of uCT detection, in which A. normal control group; B. model group; C. methotrexate-2.5mg/kg; D. etanercept-6.7mg/kg; E.anti-proBDNF- 1 ml/kg; F.p75ECD-5mg/kg; G.p75ECD-10mg/kg; H.p75ECD-15mg/kg; I.p75ECD-20mg/kg; J.p75ECD(20mg/kg)+etanercept (6.7mg/kg);
  • Figure 8 is the view of the ankle joint detected by uCT, where A. normal control group; B. model group; C. methotrexate-2.5mg/kg; D. etanercept-6.7mg/kg; E.anti-proBDNF -1ml/kg; F.p75ECD-5mg/kg; G.p75ECD-10mg/kg; H.p75ECD-15mg/kg; I.p75ECD-20mg/kg; J.p75ECD(20mg/kg)+etanercept (6.7mg/kg);
  • Figures 11-13 are pathological images of the whole palm, ankle joint and toe joint of the left hind foot;
  • Figure 11 shows the pathological images of the whole palm of the left hind foot, wherein A. normal control group; B. model group; C. methotrexate-2.5mg/kg; D. etanercept-6.7mg/kg; E. anti-proBDNF-1ml/kg; F.p75ECD-5mg/kg; G.p75ECD-10mg/kg; H.p75ECD-15mg/kg; I.p75ECD-20mg/kg; Nacept (6.7mg/kg);
  • Fig. 12 shows the pathological image of ankle joint, wherein A. normal control group; B. model group; C. methotrexate-2.5mg/kg; D. etanercept-6.7mg/kg; E.anti-proBDNF -1ml/kg; F.p75ECD-5mg/kg; G.p75ECD-10mg/kg; H.p75ECD-15mg/kg; I.p75ECD-20mg/kg; J.p75ECD(20mg/kg)+etanercept (6.7mg/kg); and
  • Figure 13 shows pathological images of toe joints, where blue arrows: articular cartilage; red arrows: joint synovial tissue; yellow arrows: articular subchondral bone tissue; black arrows: hyperplastic synovial tissue and inflammatory cells; green arrows: synovial tissue Membranous granuloma invades subchondral bone tissue; and A. normal control group; B. model group; C. methotrexate-2.5mg/kg; D.
  • Fig. 14 shows paw synovial hyperplasia score, wherein One-way ANOVA: *p ⁇ 0.05vs. model group; ***p ⁇ 0.001vs. model group; T-test: #p ⁇ 0.05vs. model group; # #p ⁇ 0.01vs. model group;
  • Figure 15 shows paw synovial inflammation score, wherein One-way ANOVA: *p ⁇ 0.05vs. model group; ***p ⁇ 0.001vs. model group; T-test: #p ⁇ 0.05vs. model group; # #p ⁇ 0.01vs. model group;
  • Figure 16 shows paw cartilage damage score, wherein One-way ANOVA: *p ⁇ 0.05vs. model group; ***p ⁇ 0.001vs. model group;
  • Figure 17 shows paw cartilage damage score, wherein One-way ANOVA: *p ⁇ 0.05vs. model group; ***p ⁇ 0.001vs. model group; T-test: #p ⁇ 0.05vs. model group; ## p ⁇ 0.01vs. model group;
  • Figure 18 shows the total injury score of paw, wherein One-way ANOVA: **p ⁇ 0.05vs. model group; ***p ⁇ 0.001vs. model group; T-test: #p ⁇ 0.05vs. model group; # #p ⁇ 0.01vs. model group; ###p ⁇ 0.001vs. model group;
  • Figure 19 shows the expression of proBDNF protein in spleen tissue, wherein T-test: #p ⁇ 0.05vs. model group; ##p ⁇ 0.01vs. model group;
  • Figure 20 shows the expression of p75NTR protein in spleen tissue, wherein T-test: #p ⁇ 0.05vs. model group; ##p ⁇ 0.01vs. model group; ###p ⁇ 0.001vs. model group;
  • Figure 21 shows the expression of pJAK1 protein in spleen tissue, wherein T-test: #p ⁇ 0.05vs. model group; ##p ⁇ 0.01vs. model group;
  • Figure 22 shows the expression of pSTAT1 protein in spleen tissue, wherein T-test: #p ⁇ 0.05vs. model group; ##p ⁇ 0.01vs. model group;
  • Figure 23 shows the expression of p75NTR mRNA in spleen tissue, wherein T-test: #p ⁇ 0.05vs. model group; ###p ⁇ 0.001vs. model group;
  • Figure 24 shows the expression of IL-1 ⁇ mRNA in spleen tissue, wherein T-test: #p ⁇ 0.05vs. model group; ###p ⁇ 0.001vs. model group;
  • Figure 25 shows the expression of IL-6mRNA in spleen tissue, wherein T-test: #p ⁇ 0.05vs. model group; ##p ⁇ 0.01vs. model group; ###p ⁇ 0.001vs. model group;
  • Figure 26 shows the expression of TNF- ⁇ mRNA in spleen tissue, wherein T-test: #p ⁇ 0.05vs. model group; ##p ⁇ 0.01vs. model group; ###p ⁇ 0.001vs. model group;
  • Figure 27 shows the content of IL-1 ⁇ in serum, wherein T-test: #p ⁇ 0.05vs. model group; ##p ⁇ 0.01vs. model group; ###p ⁇ 0.001vs. model group;
  • Figure 28 shows IL-6 content in serum, wherein T-test: #p ⁇ 0.05vs. model group; ##p ⁇ 0.01vs. model group; and
  • Figure 29 shows the content of TNF- ⁇ in serum, wherein T-test: ##p ⁇ 0.01vs. model group; ###p ⁇ 0.001vs. model group.
  • mice Female SPF grade Lewis rats, 100, were raised in a barrier system.
  • the animal's anti-environmental conditions were controlled at room temperature 20°C-26°C, humidity 40%-70%, the air in the room was changed 10-20 times per hour, and the lighting was 12/ 12-hour alternating light and dark cycle.
  • they were reared together in cages (2 to 6 animals per cage).
  • the cages were made of polysulfone plastic cages with corncob bedding. There were toys for chewing or shelter in the cages for the animals as an environment. Rich measures. Food was freely foraged, and reverse osmosis purified water was provided via water bottles for animals to drink ad libitum.
  • Model group model inducer preparation According to the number of immunized animals, determine the weight of Collagen II, dissolve it with 50mM glacial acetic acid to a final concentration of 2mg/ml; stir overnight at 4°C. On the day of immunization, mix complete Freund's adjuvant (CFA) with the prepared collagen at a ratio of 1:1, place on ice, and homogenize with a homogenizer at low speed for about 20 minutes at 3-4 gears (judgment standard: no Disperse immediately).
  • CFA complete Freund's adjuvant
  • CIA model Animals were immunized for the second time 8 days after the first immunization. After Lewis rats in the model group were anesthetized with isoflurane, the root of the tail (2-3cm away from the body, 100 ⁇ l/point) and two points on the left and right back (50 ⁇ l/point). Animals in the normal control group were injected with the same amount of normal saline.
  • Experiment grouping and administration The experiment was divided into 10 groups, group 1 was the normal group, group 2 was the model group, group 3 was the model methotrexate group, group 4 was the model etanercept group, and group 5 was the model proBDNF-positive group.
  • Cloned antibody group, group 6 to group 9 are p75ECD-Fc dosage groups, and group 10 is combined treatment group.
  • the time for the first administration of animals in each group was set on the first day after the second immunization.
  • the experimental groups and administration schemes are shown in Table 2.
  • the p75ECD-Fc protein can be prepared by referring to conventional methods in this field, especially the method disclosed in CN 102233128B;
  • the proBDNF polyclonal antibody can be prepared by referring to conventional methods in this field, especially the method disclosed in CN107303389A.
  • euthanasia was performed by isoflurane anesthesia, and the left hind limb was immersed in 10% neutral formalin (for uCT and pathological detection), and serum was collected and placed at room temperature for at least 30 minutes. Centrifuge at 5000 rpm for 5 min at 4°C, separate serum, store at -80°C, and use for detection of inflammatory factors.
  • group 1 normal group
  • group 2 model group
  • group 6-9 p75ECD-5mg/kg, 10mg/kg, 15mg/kg and 20mg/kg groups
  • group 10 combined therapy In each group
  • 6 animals were selected in each group, and the spleen tissue was taken to be quick-frozen for real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR) detection and Western blot detection.
  • Scoring began after the second immunization, once every two days, until the end of the experiment. Scoring criteria: 0 points: normal limbs; 1 point: inflammation and swelling of at least one toe joint; 2 points: inflammation and swelling of the paw; 3 points: swelling of the foot, but not involving the ankle joint; 4 points: swelling of the foot, involving the ankle joint.
  • the degree of joint swelling for each animal was calculated by adding the scores for each paw, and the total score for each animal ranged from 0 to 16 points, when the score of more than two joints was higher than 1 point or the score of more than one joint When the score is higher than 2, the model induction is considered successful.
  • PWT was measured using the method described in previous literature [7] through the von Frey fiber filament pain tester. Animals were evaluated for pain threshold once before dosing and once a week after dosing.
  • uCT analysis was performed on the part from the ankle joint to the foot to evaluate the state of joint bone damage and bone mineral density.
  • the ankle joint tissues were decalcified, embedded in paraffin, sectioned, and stained with hematoxylin-eosin according to the standard operation of histopathological techniques.
  • the synovial hyperplasia, synovial inflammation, cartilage damage and bone damage were evaluated from the ankle joint to the paw.
  • the protein expression levels of proBDNF, p75NTR, pJAK1/JAK1 and pSTAT1/STAT1 in spleen tissue were detected by Western blot.
  • the expression levels of p75NTR, TNF- ⁇ , IL-1 ⁇ and IL-6 mRNA in spleen tissue were detected by qPCR.
  • the primers were designed according to the method reported in reference [8].
  • Enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) was used to detect the contents of TNF- ⁇ , IL-1 ⁇ , IL-6 and other inflammatory factors in serum.
  • the total score of limb joints in the etanercept group was significantly different from the model group from the 5th to the 9th day; the p75ECD-5mg/kg group was significantly different from the model group from the 13th to the 19th day ;
  • the p75ECD-10mg/kg group had statistical differences compared with the model group from the 5th day to the 13th day;
  • the p75ECD-15mg/kg group had statistical differences from the 5th day to the 19th day and the 24th day compared with the model group
  • p75ECD-20mg/kg group had statistical differences compared with the model group on the 7th, 13th and 17th days;
  • the combined treatment group had statistical differences compared with the model group on the 5th to the 19th day ( table 3).
  • the area under the curve (AUC) of the joint swelling score from the 1st day to the 14th day was shown (Fig. 2, left). kg, p75ECD-20mg/kg, and the AUC area of the combined treatment group decreased significantly (p ⁇ 0.01), and the p75ECD-15mg/kg group and the combined treatment group decreased more significantly (p ⁇ 0.001); from the 1st day to the 28th day, the joint
  • the area under the curve (AUC) of the swelling score showed (Figure 2, right).
  • the AUC area of the etanercept-6.7mg/kg group, different dose groups of p75ECD and the combined treatment group were significantly reduced (p ⁇ 0.01) , in which p75ECD-15mg/kg group and combined treatment group decreased more significantly (p ⁇ 0.001); at the same time, compared with etanercept-6.7mg/kg group or p75ECD-Fc-20mg/kg monotherapy, both
  • the combined treatment group was more advantageous in improving the joint swelling score of the extremities.
  • the above results suggest that different doses of p75ECD used alone or in combination with etanercept can improve the joint swelling of CIA-induced RA model animals, and the p75ECD-15mg/kg group and the combined treatment group have better effects.
  • Figure 5 shows that, compared with the model group, the area under the curve (AUC) of the p75ECD-5mg/kg group, the p75ECD-10mg/kg group, the p75ECD-15mg/kg group and the combined treatment group on the 14th day increased significantly, and the p75ECD-10mg/kg kg group and combined treatment group increased significantly (p ⁇ 0.01); p75ECD-10mg/kg group, p75ECD-15mg/kg group and combined treatment group on the 19th day area under the curve significantly increased, p75ECD-10mg/kg group increased more Significantly (p ⁇ 0.01);
  • Figure 6 shows that compared with the model group, except for anti-proBDNF, the areas under the curves of all other groups increased significantly on day 14 and day 19. The above results suggest that different doses of p75ECD used alone or in combination with etanercept can improve inflammatory pain in CIA-induced RA model animals.
  • Figure 7 and Figure 8 are the overall view of the foot and the ankle joint detected by uCT.
  • the CT value (HU) and bone density of the model group were significantly decreased (p ⁇ 0.001); compared with the model group, the p75ECD-5mg/kg group, p75ECD-10mg/kg group and p75ECD-15mg/kg group
  • the CT value (HU) and bone density of the p75ECD-20mg/kg group were all significantly increased (p ⁇ 0.05) (p ⁇ 0.001); Figure 10).
  • Figures 11-13 are pathological images of the whole palm, ankle joint and toe joint of the left hind foot, respectively.
  • Paw synovial hyperplasia score, synovial inflammation score, cartilage damage score, bone damage score and total damage score are shown in Table 6.
  • Figure 14 shows that in the paw synovial hyperplasia score, the model group was significantly higher than the normal group (p ⁇ 0.001); after giving 4 drug treatments, compared with the model group, the methotrexate-2.5mg/kg group Significantly reduce the synovial hyperplasia score of the foot (p ⁇ 0.05), and the p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg groups all significantly reduce the synovial hyperplasia score of the foot (p ⁇ 0.01, p ⁇ 0.05, p ⁇ 0.01).
  • Figure 15 shows that in the paw synovial inflammation score, the model group was significantly higher than the normal group (p ⁇ 0.001); after giving 4 drug treatments, compared with the model group, the methotrexate-2.5mg/kg group Significantly reduced the synovitis score of the foot (p ⁇ 0.05), and the scores of the p75ECD-10mg/kg, p75ECD-15mg/kg, and p75ECD-20mg/kg groups were all significantly reduced (p ⁇ 0.01, p ⁇ 0.05, p ⁇ 0.01 ).
  • Figure 16 shows that in the paw cartilage damage score, the model group was significantly higher than the normal group (p ⁇ 0.001); after giving 4 times of drug treatment, compared with the model group, the methotrexate-2.5mg/kg group Significantly reduced paw cartilage damage score (p ⁇ 0.05), p75ECD-5mg/kg, p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg group scores were significantly reduced (p ⁇ 0.05, p ⁇ 0.05, p ⁇ 0.001, p ⁇ 0.05).
  • Figure 17 shows that in the paw bone injury score, the model group was significantly higher than the normal group (p ⁇ 0.001); after giving 4 drug treatments, compared with the model group, the methotrexate-2.5mg/kg group was significantly significantly reduced the paw bone injury score (p ⁇ 0.05, Figure 17), and the p75ECD-5mg/kg, p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg group scores were significantly reduced (p ⁇ 0.05, p ⁇ 0.05, p ⁇ 0.001, p ⁇ 0.05).
  • Figure 18 shows that in the total score of paw damage, the model group was significantly higher than the normal group (p ⁇ 0.001); at the same time, after 4 drug treatments, compared with the model group, methotrexate-2.5mg/kg The total score of the group was significantly reduced (p ⁇ 0.05, Figure 18), and the total score of the p75ECD-5mg/kg, p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg groups were all significantly reduced (p ⁇ 0.05, p ⁇ 0.01, p ⁇ 0.01, p ⁇ 0.001).
  • FIG. 19 shows that, compared with the normal group, the expression of proBDNF in the model group increased significantly (p ⁇ 0.01); compared with the model group, except the p75ECD-5mg/kg group, the proBDNF of the other groups decreased significantly (p ⁇ 0.05), Among them, p75ECD-15mg/kg group decreased more significantly (p ⁇ 0.01).
  • Figure 20 shows that, compared with the normal group, the expression of p75NTR in the model group was significantly increased (p ⁇ 0.001); compared with the model group, p75NTR in each group was significantly decreased (p ⁇ 0.05, p ⁇ 0.01).
  • Figure 21 shows that, compared with the normal group, the phosphorylated JAK1 in the model group increased significantly (p ⁇ 0.01); compared with the model group, the phosphorylated JAK1 in each group decreased significantly (p ⁇ 0.05), wherein p75ECD-15mg/ The kg group decreased more significantly (p ⁇ 0.01).
  • Figure 22 shows that compared with the normal group, the phosphorylated STAT1 of the model group increased significantly (p ⁇ 0.05); compared with the model group, each group decreased significantly (p ⁇ 0.05), and the combined treatment group decreased more significantly (p ⁇ 0.05). ⁇ 0.01).
  • Table 8 shows the expression levels of p75NTR, IL-1 ⁇ , IL-6 and TNF- ⁇ mRNA in spleen tissues.
  • the results showed that: compared with the normal control group, the expression level of p75NTR mRNA in the spleen tissue of the animals in the model group was significantly increased (p ⁇ 0.001); kg, p75ECD-15mg/kg, p75ECD-20mg/kg, and the combined treatment group decreased more significantly (p ⁇ 0.001) ( Figure 23); compared with the normal control group, the expression level of IL-1 ⁇ mRNA in the spleen tissue of the model group increased significantly ( p ⁇ 0.001), compared with the model group, except the anti-proBDNF group, the expression levels of IL-1 ⁇ mRNA in the spleen tissues of the animals in the other groups were significantly reduced, and the p75ECD dosage groups and combined treatment groups were more significantly reduced (p ⁇ 0.001) ( Figure 24); compared with the normal control group, the expression level of IL-6 mRNA in
  • Table 8 Expression levels of p75NTR, IL-1 ⁇ , IL-6 and TNF- ⁇ mRNA in spleen tissue (MEAN ⁇ SD)
  • methotrexate can significantly reduce the paw injury score of female Lewis CIA model rats at a dose of 2.5 mg/kg, showing a significant curative effect.
  • Etanercept at a dose of 6.7mg/kg can reduce the joint swelling score of the limbs of female Lewis CIA model rats (day 5-9) and improve the pain threshold (PWT);
  • PWT pain threshold
  • p75ECD at 5mg/kg, 10mg/kg, 15mg /kg, 20mg/kg 4 dose groups showed good drug efficacy in body weight, PWT, ankle joint volume and diameter, swelling score, and could significantly reduce the paw injury score of model rats in pathology, exerting a Very good therapeutic effect, among which 15mg/kg has the best curative effect, better than methotrexate 2.5mg/kg.

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Abstract

提供p75细胞外结构域(p75ECD)在治疗和/或预防类风湿性关节炎(RA)中的应用。

Description

p75细胞外结构域在类风湿性关节炎治疗和/或预防中的应用 技术领域
本发明属于制药领域，具体涉及一种p75细胞外结构域(p75ECD)的新用途，尤其涉及p75细胞外结构域(p75ECD)在制备用于治疗和/或预防类风湿性关节炎(RA)的药物中的应用。
背景技术
类风湿性关节炎(Rheumatoid arthritis，RA)是一种以慢性侵蚀性关节炎为主要特征的自身免疫性疾病，基本病理改变是滑膜炎、急性期滑膜肿胀、渗出，粒细胞浸润；慢性期滑膜增生肥厚，形成血管翳，后者是造成关节破坏、关节畸形、障碍、使疾病进入不可逆阶段的病理基础。RA的特征是对称性的小关节侵蚀性病变，主要累及腕关节、掌指关节、近端指间关节、膝关节、踝关节及足趾关节等，对骨骼组织会产生局部和全身性破坏性作用。RA是一种慢性、进展性疾病，虽然不会直接危及生命，但起病2年即可导致骨关节的破坏，表现为受累关节的肿胀、疼痛、功能下降，病变呈持续、反复发作的过程。早期未经正确及时治疗，往往导致关节的破坏和畸形，最终导致关节畸形、功能丧失，严重影响患者的生活质量，是造成我国人群丧失劳动力和致残的主要原因之一。
RA的全球发病率为0.5％-1.0％，在任何年龄段均可发病，其中80％的RA患者的发病年龄在20至45岁之间，女性居多，约为男性发病率的3倍 [1]。我国目前的发病率约为0.32％-0.36％。
目前临床上治疗RA的药物包括：1)非甾体类抗炎药(non-steroidal anti-inflammatory drugs,NSAIDs)，如阿司匹林、吲哚美辛等，NSAIDs可缓解RA患者关节肿痛症状，但不能控制RA的活动和发展，并且长期服用后会有明显的不良反应，包括中枢系统 症状、消化系统症状、心血管损伤和肝肾功能不全等 [2]；2)改善疾病的抗风湿药(disease modifying anti-rheumatic drugs,MDARDs)，如甲氨蝶呤、来氟米特等，甲氨蝶呤得到了临床医生的广泛认可，潜在的不利影响包括口腔溃疡、肝硬化、肝炎、间质性肺炎和细胞减少症 [3]；3)大分子生物类治疗药物，如阿达木单抗、依那西普、戈利木单抗等，通过阻断某种特定炎症细胞因子或表面分子而发挥作用。大分子生物药的使用是RA治疗中的一个重大突破，但会造成局部皮疹、感染、结核、失髓鞘综合征等 [4]，比如，阿达木单抗可能导致严重的感染，从而导致疾病复发 [5]。除药物治疗外，临床上还经常采用滑膜切除术治疗RA，但存在创伤大、患者依从性差等局限。综合来看，在过去的十几年里，虽然RA的治疗已取得了很大进步，但无论是药物治疗还是手术治疗仍然存在着诸多局限性。因此，开发治疗和/或预防RA的药物具有重要的社会和经济意义。
RA的发病机制复杂，涉及大量不同的细胞类型和信号通路。研究发现，RA的发生发展中，炎症网络是关键因素，其中炎症反应、T细胞和B细胞、自身抗体、细胞因子以及其他炎症介质是炎症网络重要的组成成分。发明人前期研究发现，脑源性神经营养因子前体(proBDNF)是一种炎症介质，在调节疼痛方面起着重要的作用 [6]，可作为炎症介质调节疼痛，但proBDNF/p75NTR信号通路在类风湿性关节炎(RA)中的作用尚不清楚。
CN 102233128 B公开了p75NTR-ECD在制备防治阿尔茨海默病的药物中的应用。CN 106794222 B公开了p75细胞外结构域(p75ECD)在制备用于治疗和/或脑淀粉样血管病(CAA)的药物中的用途。CN107303389A公开了外周脑源性神经营养因子前体蛋白(proBDNF)或其信号传递分子中具有中和或抑制活性的结合分子在制备用于缓解、抑制或治疗疼痛的药物中的应用。CN 112472796 A公开了p75细胞外结构域(p75ECD)或其功能性片段、变体、类似物或衍生物在制备用于治疗和/或预防额颞叶痴呆的药物中的用途。CN 113456799A公开了一种p75ECD在制备用于调节疼痛的药物中的应用，其中所述疼痛包括炎性疼痛或手术疼痛。CN 104302326B(PCT/GB2013/050632)公开了p75NTR神经营养因子结合蛋白的治疗性用途，具体教导了p75NTR(NBP)用于治疗疼痛和/或疼痛症状。CN 105873943A(PCT/GB2014/052833)公开了一种融合蛋白，即p75NTR(NBP)-Fc融合蛋白，用于治疗疼痛或疼痛症状。
US2009/0175859A1涉及TNFα拮抗剂和甲氨蝶呤治疗肿瘤坏死因子(TNF)介导的疾病，并具体公开了一种组合物，其包括甲氨蝶呤和抗-TNF抗体，以及其中TNF介导的疾病包括类风湿性关节炎(RA)、克雷恩氏病(Crohn’s disease)以及急性和慢性与移植相关的免疫性疾病。
US2018/0170995A1(PCT/EP2016/056049)公开了一种p75NTR神经营养因子结合蛋白(NBP)-Fc融合蛋白，用于治疗疼痛和/或疼痛症状，以及用于治疗包括湿疹、银屑病、皮炎、神经变性、亨廷顿病、老年痴呆症、自闭症、癌症、乳腺癌、类风湿性关节炎(RA)、骨关节炎、膀胱炎和子宫内膜异位等在内的34种疾病，其中p75NTR(NBP)与神经生长因子(NGF)、脑源性神经营养因子(BDNF)、NT3或NT4/5之一结合(结合亲和性Kd为1pM-100nM)。
EP2667895B1(PCT/IT2012/000018)涉及至少一种p75NTR受体抑制剂，单独或与至少一种TrkA受体活化剂组合，或至少一种TrkA受体活化剂用于治疗慢性炎性疾病，其中p75NTR受体抑制剂选自多克隆和单克隆单特异性或双特异性p75NTR受体结合抗体，嵌合分子(其结构包括可溶性p75NTR受体，p75NTR受体结合环状单体和二聚体NGF类似物化合物)，以及其中TrkA受体活化剂选自TrkA受体结合多克隆和单克隆抗体，环状单体和二聚体NGF类似物化合物，重组NGF或重组突变的NGF。根据该发明，所述慢性炎性疾病选自类风湿性关节炎(RA)，青少年特发性关节炎，肠慢性炎症，强直 性脊柱炎，银屑病，多发性硬化，红斑狼疮，硬皮病，舍格伦综合征。
然而，p75神经营养因子细胞外结构域(p75ECD)在类风湿性关节炎(RA)预防和/或治疗中的作用在全世界范围内未见报道，发明人首次发现p75ECD可作为预防和治疗类RA的药物。
发明内容
本发明具体涉及p75神经营养因子受体细胞外结构域(p75NTR-ECD，又称p75ECD)预防和/或治疗类风湿性关节炎疾病(RA)的方法，包括给需要的患者施用p75ECD，或者涉及p75ECD在制备用于预防和/或治疗类风湿性关节炎疾病(RA)的药物中的用途。
本发明还涉及p75ECD用于预防和/或治疗类风湿性关节炎疾病(RA)，或者用于预防和/或治疗类风湿性关节炎疾病(RA)的药物，该药物包含p75ECD。
本发明出人意料地发现p75ECD治疗和/或预防类风湿性关节炎具有非常好的疗效，特别是在缓解关节肿胀、缓解炎症疼痛以及降低关节滑膜增生、滑膜炎症、软骨损伤、骨损伤等方面具有意想不到的效果，可能通过阻断proBDNF/p75NTR信号通路以及抑制Janus激酶1(Janus Kinases 1,JAK1)/转导与转录激活因子1(signal transducerand activator of transcription 1,stat1)磷酸化继而阻断肿瘤坏死因子-a(TNF-a)，白介素-1β(IL-1β)，白介素-6(IL-6)等炎性因子而发挥作用。
在本文中使用时，术语“治疗”意欲是指p75ECD的给药减轻或改善了RA的至少某些迹象或症状。
在本文中使用时，术语“预防”意欲是指p75ECD的给药发生在RA的诊断之前，并减轻、改善了RA的至少某些迹象或症状或者延 迟其发作；或者是指p75ECD的给药阻止了RA的迹象或症状的恶化。因此，术语“预防”意欲包括预防性治疗。
给药的p75ECD可以来自于任何目标物种(例如人类、小鼠、大鼠、兔、猫、狗、山羊、奶牛、马、非人类灵长动物等)。然而，在一个实施方式中，所述p75ECD是人类p75ECD。在一个实施方式中，所述p75ECD具有编码SEQ ID NO:2所示氨基酸序列的核苷酸序列，例如SEQ ID NO:1所示的核苷酸序列，或具有SEQ ID NO:2所示的氨基酸序列。
Figure PCTCN2022137395-appb-000001
Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys
Lys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn
Gln Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val
Val Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu
Gln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg
Cys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala
Cys Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp
LysGln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp
Glu Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp
Thr Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu Ile Pro Gly Arg Trp Ile Thr(SEQ ID NO:2)
在替代实施方式中，所述p75ECD可以是该序列的功能性片段、变体、类似物或衍生物。例如，p75ECD(或其功能性片段)的变体、类似物或衍生物由不同于p75ECD(或其功能性片段)，但在生物活性、特别是与促神经营养因子结合的能力上保留了与p75ECD的相似性的分子构成。p75ECD(或其功能性片段)的变体、类似物或衍生物可以与p75ECD(或其功能性片段)具有显著的总体结构相似性，或者仅仅与p75ECD(或其功能性片段)的负责所述生物活性的一个或多个区域具有结构相似性。通常，变体、类似物或衍生物由相关氨基酸序列的一个或多个氨基酸的修饰提供，或者是所述修饰的结果。例如，包含SEQ ID NO:2所示序列的功能性片段、变体、类似物或衍生物通过向该氨基酸序列添加一个或多个氨基酸、从该氨基酸序列缺失一个或多个氨基酸和/或从该氨基酸序列置换一个或多个氨基酸来提供，或者是上述修饰的结果。也涵盖了氨基酸的反转和引起氨基酸序列改变的其他突变变化。这种类似物或衍生物可以通过在多核苷酸分子中引入核苷酸变化使得在诱变的多核苷酸分子表达后获得所需氨基酸变化或通过以其他方式合成掺有所需氨基酸变化的氨基酸序列来制备。氨基酸的置换可以包括保守或非保守氨基酸置换。保守氨 基酸置换意味着将氨基酸残基用具有相似特性并且不显著改变所述肽或多肽的生物活性的另一个氨基酸替换。示例性的保守氨基酸置换被提供在下面的表1中。设想的具体保守氨基酸是：G，A，V，I，L，M；D，E；N，Q；S，T；K，R，H；F，Y，W，H；以及P，Nα-烷基氨基酸，其可能在序列上具有少量变化，但是不引起生物活性的任何显著降低或变化。这些变化可以包括保守氨基酸置换例如Gly，Ala，Val，Ile，Leu，Met；Asp，Glu，Asn，Gln；Ser，Thr；Lys，Arg，His；Phe，Tyr，Trp，His；和Pro，Nα-烷基氨基酸；以及非保守氨基酸置换。
表1：示例性的保守氨基酸置换
Figure PCTCN2022137395-appb-000002
*指示优选的保守置换
当通过合成制备类似物或衍生物时，所述类似物或衍生物可能包 括不由遗传密码编码的一个或多个氨基酸例如羧基谷氨酸和羟脯氨酸，或者可能包括D-氨基酸代替L-氨基酸。因此，所述类似物或衍生物可以是p75ECD(或其功能性片段)的模拟物例如肽模拟物。然而，p75ECD(或其功能性片段)的类似物或衍生物不必定具有氨基酸序列同一性和/或相似性，并且事实上，类似物或衍生物可能完全不是蛋白质。
应该理解，与SEQ ID NO:2和SEQ ID NO:4所示的氨基酸序列具有少量改变的氨基酸序列的肽(例如通过一个或多个，例如1个，2个，3个，4个，5个，6个，7个，8个，9个，10个等保守氨基酸置换产生的变体)，仍可能落于本发明的范围之内，只要所述肽能够结合p75并阻止通过p75的信号转导即可。具体来说，p75ECD的天然变体(或同工型)或源自于非人类物种的p75ECD肽被认为落入所公开的p75ECD或p75ECD-Fc融合肽的范围之内。
在一个实施方式中，p75ECD包含p75ECD融合体，其中p75ECD被融合到融合配偶体。所述融合配偶体可以是本领域技术人员已知的任何适合的融合配偶体，例如免疫球蛋白的Fc区、人血清白蛋白(HSA)和牛血清白蛋白(BSA)等，只要所述p75ECD保留至少一些它的生物活性即可。融合产物可以通过从编码所述融合产物的适合的构建物表达所述产物，或通过以其他方式将p75ECD化学连接到融合配偶体来生产。所述融合配偶体应该提高所述p75ECD在体内的稳定性和血清半衰期。在一个实施方式中，将p75ECD融合到免疫球蛋白的Fc结构域，以形成p75ECD-Fc融合体。所述Fc结构域可以是来自于任何适合的免疫球蛋白的任何适合的Fc结构域。然而，在优选实施方式中，所述Fc结构域来自于人类IgG。
在一个实施方式中，所述Fc区能够二聚化以产生p75ECD的二聚体形式。然而，其他融合配偶体可能能够形成其他类型的寡聚体，例如三聚体、四聚体、五聚体等。因此，p75ECD可能是寡聚体例如 二聚体、三聚体、四聚体、五聚体等。在一个实施方式中，所述p75ECD可以是二聚体。在一个实施方式中，所述p75ECD-Fc融合体可以是p75ECD-Fc二聚体。
在一个实施方式中，所述p75ECD-Fc融合体包含连接到人类IgG1的Fc部分的人类p75ECD分子。本领域技术人员将会认识到，取决于例如所使用的连接物，融合体可能具有大量不同的核苷酸和氨基酸序列。然而，在一个实施方式中，SEQ ID NO:3为人类p75ECD-人类IgG1Fc结构域融合体(人类p75ECD-Fc)的核苷酸序列。基于简并性，其他编码SEQ ID NO:4所示的氨基酸序列或其变体的核苷酸序列也是容易想到的，并落入本发明保护范围。
Figure PCTCN2022137395-appb-000003
Figure PCTCN2022137395-appb-000004
类似地，在一个实施方式中，人类p75ECD-Fc融合体的氨基酸序列被提供在SEQ ID NO:4中。在一个实施方式中，SEQ ID NO:4包含在SEQ ID NO:4的第1-169位氨基酸处的p75ECD肽，随后是在第170至179位残基处的连接物“DKTHTCPPCP”，随后是在第180至396位残基处的Fc结构域。然而，应该认识到，p75ECD-Fc融合体可以是由SEQ ID NO:4所示序列的功能性片段、变体、类似物或衍生物。本文中别处描述了功能性片段、变体、类似物或衍生物。
Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys
Lys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn 
Gln Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val
Val Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu 
Gln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg 
Cys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala
Cys Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp
Lys Gln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp
Glu Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp 
Thr Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys
Glu Glu Ile Pro Gly Arg Trp Ile Thr Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys(SEQ ID NO:4)
在一个实施方式中，将p75ECD或p75ECD融合体作为核苷酸分子给药到所述对象。这种核苷酸分子可以由例如表达载体或表达盒构成。优选的表达载体包括能够实现所需宿主细胞类型的转导的病毒载体，例如反转录病毒载体、腺病毒载体和源自于腺相关病毒(AAV)的载体，例如本领域技术人员公知的那些。包含编码本公开的p75ECD或p75ECD融合体的核苷酸序列的病毒载体可用于在所需位点处提供p75ECD或p75ECD融合体的体内来源，用于RA的治疗或预防。
在优选实施方式中，将p75ECD或p75ECD融合体通过病毒载体 给药，已知所述病毒载体提供以适合于基因疗法的方式将核苷酸递送到细胞内的工具。正如本领域技术人员将会理解的，腺相关病毒(AAV)载体、慢病毒载体或单纯性疱疹病毒载体可以用作病毒载体，因为它们都能将核苷酸递送到细胞用于基因治疗的目的。通过病毒载体递送p75ECD或p75ECD融合体可以介导药剂在关节中的长期表达，并且由于RA的慢性本质，p75ECD或p75ECD融合体的持续分泌是合乎需要的。在一个实施方式中，AAV载体的使用是有利的，这是因为所述载体可以作为单次注射(例如关节内注射)给药，可以提供药剂的长期分泌，并且此外也被认为是非常安全的，在动物或人类中尚未报道过病理事件。AAV载体已知是用于基因疗法递送的工具。因此，在一个实施方式中，病毒载体可以是AAV载体。所述AAV载体可以是本领域技术人员已知的任何适合的腺相关病毒载体，例如AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12等。在一个实施方式中，所述AAV载体是AAV8。
将p75ECD-Fc插入到AAV8载体(由Virovek提供)中，所述载体含有AAV2磷脂酶结构域的AAV8衣壳序列，以提高AAV8的效能。具有AAV2磷脂酶结构域的AAV8衣壳序列的序列如SEQ ID NO:5所示，其中所述AAV2磷脂酶结构域序列包含第26至431位核苷酸。
Figure PCTCN2022137395-appb-000005
Figure PCTCN2022137395-appb-000006
“治疗有效量”是在对象中引发有益或治疗效果的任何量。在一个实施方式中，用于任何适合的对象的AAV载体的剂量在1×10 6至6×10 13个病毒基因组/kg对象体重之间。在一个实施方式中，用于任何适合的对象的AAV载体的剂量在1×10 11至6×10 12个病毒基因组/kg对象体重之间。
正如本领域技术人员充分认识到的，给药的腺相关病毒载体的剂量可以变化。例如，对于人类对象来说，在美国国立卫生研究院(U.S.National Institutes of Health)的机构的临床试验中，患者中使用的“安全”剂量为6×10 12个病毒基因组(vg)/kg体重。然而，对于本文中描述的方法来说，给药到人类对象的腺病毒载体的剂量可以在1×10 6至6×10 13vg/kg体重的范围内。在一个实施方式中，给药到人类对象的腺病毒载体的剂量在1×10 10至6×10 12个病毒基因组/kg体重之间。在一个实施方式中，给药到人类对象的腺病毒载体的剂量在1×10 11至3×10 12个病毒基因组/kg体重之间。在一个实施方式中，腺病毒的剂量可以更高，例如1×10 14、1×10 15、1×10 16、1×10 17、1×10 18、1×10 19、1×10 20等，只要这些剂量被认为是安全的即可。
可以使用用于在物种间转换药物等价剂量的美国食品和药物监督管理局(US Food and Drug Administration)(FDA)标准，在身体表面积的基础上将人类对象中使用的AAV载体的剂量转换成适合用于其他动物的剂量[以mg/kg为单位的人类等价剂量＝以mg/kg为单位的动物剂量×(以kg为单位的动物体重/以kg为单位的人类体重)0.33](食品和药物监督管理局，2003)。因此，在小鼠中的“安全”剂量可以是8×10 10个病毒基因组。然而，本领域技术人员将会认识到，给药到小鼠对象的腺相关病毒载体的剂量可以在1×10 5至1×10 13个病毒基因组的范围内。在一个实施方式中，给药到小鼠对象的腺相关病毒载体的剂量可以在1×10 8至1×10 12个病毒基因组的范围内。在一个实施方式中，给药到小鼠对象的腺相关病毒载体的剂量可以在1×10 9至1×10 11个病毒基因组的范围内。
在本发明的这一方面的一个实施方式中，p75ECD或p75ECD-Fc融合体是重组肽。重组p75ECD或p75ECD-Fc融合肽可以使用本领域技术人员已知的任何适合的技术来生产，例如通过在培养的细菌系统(例如大肠杆菌(Escherichia coli))、酵母表达系统、昆虫表达系统、病毒表达系统或在真核细胞表达系统中表达，或者所述肽可以人工合成等，所有这些都为本领域技术人员所知。
在一个实施方式中，给药的重组p75ECD或p75ECD-Fc融合肽的剂量可以是在对象中引发有益或治疗效果的任何适合的量，并且可能随着给药途径而变。在一个实施方式中，所述剂量为每天约0.01至约500mg/kg对象体重，其可以在单剂或多剂中给药。优选地，所述剂量为每天约0.1至约250mg/kg，例如，0.5mg/kg，1mg/kg，2mg/kg，3mg/kg，4mg/kg，5mg/kg，6mg/kg，7mg/kg，8mg/kg，9mg/kg，10mg/kg，11mg/kg，12mg/kg，13mg/kg，14mg/kg，15mg/kg，16mg/kg，17mg/kg，18mg/kg，19mg/kg，20mg/kg，25mg/kg，30mg/kg，35mg/kg，40mg/kg，50mg/kg，60mg/kg，70mg/kg，80mg/kg，90mg/kg，100mg/kg，150mg/kg，200mg/kg，更优选为每天约0.5至约20mg/kg。
在一个实施方式中，本公开的这一方面的p75ECD、p75ECD融合体、p75ECD-Fc或AAV-p75ECD-Fc与可药用载体、赋形剂和/或稀释剂相组合提供。适合的可药用载体、赋形剂和/或稀释剂对于本领域技术人员来说是公知的。
本公开的这一方面的p75ECD、p75ECD融合体、p75ECD-Fc或AAV-p75ECD-Fc可以通过任何适合的途径给药到所述对象，例如口服、静脉内、肌内、腹膜内、鼻内、颅内、鞘内或脑室内途径。然而，优选地，所述p75ECD或p75ECD-Fc被静脉内、腹膜内、肌内、鞘内或脑室内给药。
另一方面，提供了一种通过阻断经过p75的信号转导来治疗和/或预防RA的方法，所述方法包括向需要的对象给药治疗有效量的p75ECD或其类似物。在一个实施方式中，所述p75ECD或其类似物阻止选自proBDNF和proNT3的神经毒性分子与p75的结合。这一方面的方法使用上面描述的实施方式。
根据本发明，它包括p75NTR受体的胞外段p75ECD，其中所述p75ECD由SEQ ID NO:2所述的氨基酸序列或其变体(例如一个或多个保守氨基酸置换后的变体)组成。
根据本发明，它包括p75ECD被融合到免疫球蛋白的Fc结构域，以形成p75ECD-Fc融合体，其中所述p75ECD-Fc由SEQ ID NO:4所示的氨基酸序列或其变体(例如一个或多个保守氨基酸置换后的变体)组成。
根据本发明，p75ECD-Fc融合蛋白的日给药剂量在0.1mg/kg对象体重至250mg/kg对象体重之间。其中对治疗而言，日剂量优选为0.1-100mg/kg对象体重，例如0.1mg/kg，0.5mg/kg，1.0mg/kg，2.0mg/kg，3.0mg/kg,5.0mg/kg，10.0mg/kg，15mg/kg，20mg/kg，30mg/kg,40mg/kg,50mg/kg,60mg/kg,70mg/kg,80mg/kg或90mg/kg等。
根据本发明，p75ECD-Fc融合蛋白的AAV载体的日给药剂量在1×10 6至11×10 13个病毒基因/kg对象体重之间。其中对治疗而言，日剂量优选为1×10 8至11×10 13个病毒基因/kg对象体重之间。
根据本发明，p75细胞外结构域(p75ECD)与第二药理活性化合物组合时分别、依次或同时使用。优选地，第二药理活性化合物选自：
阿片类镇痛药，例如吗啡(morphine)、海洛因(heroin)、氢吗啡酮(hydromorphone)、羟吗啡酮(oxymorphone)、左啡诺(levorphanol)、左洛啡烷(levallorphan)、美沙酮(methadone)、哌替啶(meperidine)、芬 太尼(fentanyl)、可卡因(cocaine)、可待因(codeine)、双氢可待因(dihydrocodeine)、羟考酮(oxycodone)、氢可酮(hydrocodone)、右丙氧芬(propoxyphene)、纳美芬(nelmefene)、烯丙吗啡(nalorphine)、纳洛酮(naloxone)、纳曲酮(naltrexone)、丁丙诺啡(buprenorphine)、布托啡诺(butorphanol)、纳布啡(nalbuphine)或喷他佐辛(pentazocine)；
非甾体抗炎药(NASID)，例如阿司匹林(asprine)、双氯芬酸(diclofenac)、二氟尼柳(diflusinal)、依托度酸(etodolac)、芬布芬(fenbufen)、非诺洛芬(fenoprofen)、氟苯柳(flufenisal)、氟比洛芬(flurbiprofen)、布洛芬(ibuprofen)、吲哚美辛(indomethacin)、酮几布洛芬(ketoprofen)、酮咯酸(ketorolac)、甲氯芬那酸(meclofenamic acid)、甲芬那酸(mefenamic acid)、美洛昔康(meloxicam)、萘丁美酮(nabumetone)、萘普生(naproxen)、尼美舒利(nimesulide)、硝基氟比洛芬(nitroflurbiprofen)、奥沙拉秦(olsalazine)、保泰松(phenylbutazone)、吡罗昔康(piroxicam)、舒林酸(sulindac)、托美丁(tolmetin)或佐美酸(zomepirac)；
改善疾病的抗类风湿类药物(DMARDs)，例如柳氮磺胺吡啶(sulfasalazine)、D-青霉胺(D(-)-penicillamine)、甲氨蝶呤(methotrexate)、来氟米特(leflunomide)、硫唑嘌呤(azathioprine)、环孢素(cycloporin)或羟氯喹(hydroxychloroquine)；
肿瘤坏死因子α抑制剂，例如英夫利昔单抗(infliximab)、阿达木单抗(adalimumab)、依那西普(etanercept)、戈利木单抗(golimumab)或赛托珠单抗(certolizumab)；
B细胞抑制剂，例如利妥昔单抗(rituximab)或贝利木单抗(belimumab)；
T细胞抑制剂，例如阿巴西普(abatacept)；
细胞因子抑制剂例如托珠单抗(tocilizumab)、西鲁库姆(sirukumab)、克拉扎珠单抗(clazakizumab)、奥西单抗(olokizumab)、沙利鲁单抗(sarilumab)或阿那白滞素(anakinra)；
JAK1/JAK2抑制剂，例如巴瑞克替尼(baricitinib)；
粒细胞-吞噬细胞集落刺激因子(GM-CSF)受体阻断剂，例如马 维李单抗(mavrilimumab)。
根据本发明，第二药理活性化合物选自依那西普。
本发明制剂给药的剂量和频率可以根据患者病情而变化，有时需要以相对短的间隔、相对高剂量，直至减轻或终止疾病的进展，优选直至患者表现出疾病症状的部分或完全改善。本发明的药物可以通过静脉内、腹膜内、脑室内、鞘内或肌内注射来给药。
附图简述
为了更清楚地描述本发明的技术方案，下面将结合附图作简要介绍。显而易见，这些附图仅是本申请记载的一些具体实施方式。本发明包括但不限于这些附图。
图1示出四肢关节肿胀程度总评分；
图2示出四肢关节肿胀程度总评分14天、24天曲线下面积(AUC)，其中One-way ANOVA:***p<0.001vs.正常对照组；#p<0.05vs.模型组；##p<0.01vs.模型组；###p<0.001vs.模型组；T-test:$p<0.05vs.模型组；$$p<0.01vs.模型组；$$$p<0.001vs.模型组；
图3示出左足疼痛阈值；
图4示出右足疼痛阈值；
图5示出左足疼痛阈值14天、19天曲线下面积(AUC)，其中One-way ANOVA:***p<0.001vs.正常对照组；#p<0.05vs.模型组；##p<0.01vs.模型组；###p<0.001vs.模型组；
图6示出右足疼痛阈值14天、19天曲线下面积(AUC)，其中One-way ANOVA:***p<0.001vs.正常对照组；#p<0.05vs.模型组；##p<0.01vs.模型组；###p<0.001vs.模型组；
图7-8为uCT检测足整体视图及踝关节视图；其中
图7为uCT检测整体视图，其中A.正常对照组；B.模型组；C.甲氨蝶呤-2.5mg/kg；D.依那西普-6.7mg/kg；E.anti-proBDNF-1 ml/kg；F.p75ECD-5mg/kg；G.p75ECD-10mg/kg；H.p75ECD-15mg/kg；I.p75ECD-20mg/kg；J.p75ECD(20mg/kg)+依那西普(6.7mg/kg)；
图8为uCT检测踝关节视图，其中A.正常对照组；B.模型组；C.甲氨蝶呤-2.5mg/kg；D.依那西普-6.7mg/kg；E.anti-proBDNF-1ml/kg；F.p75ECD-5mg/kg；G.p75ECD-10mg/kg；H.p75ECD-15mg/kg；I.p75ECD-20mg/kg；J.p75ECD(20mg/kg)+依那西普(6.7mg/kg)；
图9为左后足CT值(HU)，其中One-way ANOVA:*p<0.05vs.模型组；***p<0.001vs.模型组；T-test:p＝0.06vs.模型组；###p<0.001vs.模型组；
图10为左后足骨密度，其中One-way ANOVA:*p<0.05vs.模型组；***p<0.001vs.模型组；T-test:p＝0.06vs.模型组；###p<0.001vs.模型组；
图11-13分别为左后足全掌、踝关节及趾关节病理图像；其中
图11示出左后足全掌病理图像，其中A.正常对照组；B.模型组；C.甲氨蝶呤-2.5mg/kg；D.依那西普-6.7mg/kg；E.anti-proBDNF-1ml/kg；F.p75ECD-5mg/kg；G.p75ECD-10mg/kg；H.p75ECD-15mg/kg；I.p75ECD-20mg/kg；J.p75ECD(20mg/kg)+依那西普(6.7mg/kg)；
图12示出踝关节病理图像，其中A.正常对照组；B.模型组；C.甲氨蝶呤-2.5mg/kg；D.依那西普-6.7mg/kg；E.anti-proBDNF-1ml/kg；F.p75ECD-5mg/kg；G.p75ECD-10mg/kg；H.p75ECD-15mg/kg；I.p75ECD-20mg/kg；J.p75ECD(20mg/kg)+依那西普(6.7mg/kg)；以及
图13示出趾关节病理图像，其中蓝色箭头：关节软骨；红色箭头：关节滑膜组织；黄色箭头：关节软骨下骨组织；黑色箭头：增生之滑膜组织及炎症细胞；绿色箭头：滑膜肉芽肿侵入软骨下骨组织；以及其中A.正常对照组；B.模型组；C.甲氨蝶呤-2.5mg/kg；D.依那西普-6.7mg/kg；E.anti-proBDNF-1ml/kg；F.p75ECD-5mg/kg；G.p75ECD-10mg/kg；H.p75ECD-15mg/kg；I.p75ECD-20mg/kg；J.p75ECD(20mg/kg)+依那西普(6.7mg/kg)；
图14示出足爪滑膜增生评分，其中One-way ANOVA:*p<0.05vs.模型组；***p<0.001vs.模型组；T-test:#p<0.05vs.模型组；##p<0.01vs.模型组；
图15示出足爪滑膜炎症评分，其中One-way ANOVA:*p<0.05vs.模型组；***p<0.001vs.模型组；T-test:#p<0.05vs.模型组；##p<0.01vs.模型组；
图16示出足爪软骨损伤评分，其中One-way ANOVA:*p<0.05vs.模型组；***p<0.001vs.模型组；
图17示出足爪软骨损伤评分，其中One-way ANOVA:*p<0.05vs.模型组；***p<0.001vs.模型组；T-test:#p<0.05vs.模型组；##p<0.01vs.模型组；
图18示出足爪总损伤评分，其中One-way ANOVA:**p<0.05vs.模型组；***p<0.001vs.模型组；T-test:#p<0.05vs.模型组；##p<0.01vs.模型组；###p<0.001vs.模型组；
图19示出脾脏组织中proBDNF蛋白表达情况，其中T-test:#p<0.05vs.模型组；##p<0.01vs.模型组；
图20示出脾脏组织中p75NTR蛋白表达情况，其中T-test:#p<0.05vs.模型组；##p<0.01vs.模型组；###p＜0.001vs.模型组；
图21示出脾脏组织中pJAK1蛋白表达情况，其中T-test:#p<0.05vs.模型组；##p<0.01vs.模型组；
图22示出脾脏组织中pSTAT1蛋白表达情况，其中T-test:#p<0.05vs.模型组；##p<0.01vs.模型组；
图23示出脾脏组织中p75NTR mRNA表达情况，其中其中T-test：#p＜0.05vs.模型组；###p<0.001vs.模型组；
图24示出脾脏组织中IL-1βmRNA表达情况，其中其中T-test：#p＜0.05vs.模型组；###p<0.001vs.模型组；
图25示出脾脏组织中IL-6mRNA表达情况，其中其中T-test：#p＜0.05vs.模型组；##p<0.01vs.模型组；###p<0.001vs.模型组；
图26示出脾脏组织中TNF-αmRNA表达情况，其中其中T-test：#p＜0.05vs.模型组；##p<0.01vs.模型组；###p<0.001vs.模型组；
图27示出血清中IL-1β含量，其中T-test：#p＜0.05vs.模型组；##p<0.01vs.模型组；###p<0.001vs.模型组；
图28示出血清中IL-6含量，其中T-test：#p＜0.05vs.模型组；##p<0.01vs.模型组；以及
图29示出血清中TNF-α含量，其中T-test：##p<0.01vs.模型组；###p<0.001vs.模型组。
具体实施方式
为了进一步理解本发明，下面将结合实施例对本发明的优选方案进行描述。这些描述只是举例说明本发明的技术方案的特征和优点，而非限制本发明的保护范围。
实验目的
评价静脉注射p75ECD-Fc融合蛋白对胶原(CIA)诱导的类风湿性关节炎模型Lewis雌性大鼠的治疗作用。
实验动物、造模及分组
雌性SPF级Lewis大鼠，100只，饲养于屏障体系中，动物防环境条件控制为室温20℃～26℃，湿度40％～70％，房间内每小时空气更换10～20次，照明12/12小时明暗循环交替。入组实验后进行合笼饲养(每笼2～6只)，笼具为聚砜材质的塑料笼具，带有玉米芯垫料，笼具中有供啃咬或遮蔽的玩具给动物作为环境丰富措施。自由觅食，通过水瓶提供反渗透纯化水，供动物自由饮用。
模型组模型诱导剂配制：根据所需免疫动物数量，确定称取Collagen II的质量，用50mM冰醋酸溶解，至终浓度为2mg/ml；4℃搅拌过夜。免疫当天，按照1:1吸取完全弗氏佐剂(CFA)与配置好的胶原混合，放置冰上，用匀浆机3-4档低速匀浆20分钟左右(判断标准：滴入清水中不立即分散)。
CIA模型建立：动物自第一次免疫后间隔8天后进行二次免疫造模。模型组Lewis大鼠用异氟烷麻醉后，尾根(距身体2-3cm,100μl/点)和后背左右两点(50μl/点)。正常对照组动物注射等量生理盐水。
实验分组及给药：实验分为10组，组1为正常组、组2为模型组、组3为模型甲氨蝶呤组、组4为模型依那西普组、组5为模型proBDNF多克隆抗体组、组6至组9为p75ECD-Fc各剂量组、组10为联合治疗组。每组动物初次给药时间定于第二次免疫后第一天，实验分组及给药方案详见表2。
表2：实验分组和给药方案
Figure PCTCN2022137395-appb-000007
备注1：p75ECD-Fc蛋白可以参考本领域常规方法制备得到，特别参考CN 102233128B公开的方法制备；
备注2：proBDNF多克隆抗体可以参考本领域常规方法制备得到，特别参考CN107303389A公开的方法制备。
实验终点(最后1次给药后第三天)，采用异氟烷麻醉安乐死，取左后肢浸于10％中性福尔马林中(用于uCT和病理检测)，采集 血清，常温放置至少30min，4℃，5000rpm离心5min，分离血清，-80℃保存，用于炎症因子检测。另在第1组(正常组)、第2组(模型组)、第6-9组(p75ECD-5mg/kg,10mg/kg,15mg/kg和20mg/kg组)以及第10组(联合治疗组)中每组选6只动物，取脾脏组织速冻用于检测实时荧光定量PCR(Quantitative Real-time PCR,qPCR)检测和Westernblot检测。
检测指标：
1、关节肿胀评分
在第二次免疫后开始评分，每两天1次，直至实验终点。评分标准：0分：四肢正常；1分：至少一个趾关节发炎、肿胀；2分：爪子发炎、肿胀；3分：足爪肿胀，但不累及踝关节；4分：足爪肿胀，累及到踝关节。每只动物的关节肿胀程度通过每个爪子的得分相加而成，每只动物的总分从0分到16分，当多于两个关节的得分高于1分或多于一个关节的得分高于2分时，视为模型诱导成功。
2、爪缩阈(Paw Withdrawal Threshold,PWT)
PWT测量采用先前文献介绍的方法 [7]通过von Frey纤维丝测痛仪进行。给药前一次、给药后每周一次，评价动物的疼痛阈值。
3、超声断层成像(Ultrasonic Computed Tomography,uCT)
实验终点，对踝关节至足爪部分进行uCT分析，评估关节骨损伤状态和骨密度。
4、组织病理观察
uCT检测后，按组织病理学技术标准操作，对踝关节组织进行脱钙、石蜡包埋、切片、以苏木精-伊红染色。评价踝关节至足爪滑膜增生、滑膜炎症、软骨损伤、骨损伤程度。
5、Westernblot检测
采用Westernblot检测脾脏组织中proBDNF、p75NTR、pJAK1/JAK1和pSTAT1/STAT1蛋白表达水平。
6、qPCR检测
采用qPCR检测脾脏组织p75NTR、TNF-α、IL-1β和IL-6mRNA表达水平。引物参考文献报道[8]的方法设计。
7、ELISA检测
采用酶联免疫吸附测定(Enzyme linked immunosorbent assay,ELISA)检测血清中TNF-α、IL-1β、IL-6等炎症因子含量。
实验结果
1、关节肿胀评分
实验第3天各造模组动物开始进入发病初期阶段，第17天到达发病顶峰并持续到实验终点。模型组关节肿胀程度总评分从实验第5天开始与正常组相比有显著差异。依那西普组四肢关节总评分在第5天至第9天与模型组相比有统计学差异；p75ECD-5mg/kg组在第13天至第19天与模型组相比有统计学差异；p75ECD-10mg/kg组在第5天至第13天与模型组相比有统计学差异；p75ECD-15mg/kg组在第5天至第19天以及第24天与模型组相比有统计学差异；p75ECD-20mg/kg组在第7、第13和第17天与模型组相比有统计学差异；联合治疗组在第5天至第19天与模型组相比有统计学差异(表3)。第1天到第14天关节肿胀评分曲线下面积(AUC)显示(图2，左)，与模型组比较，依那西普-6.7mg/kg组、p75ECD-10mg/kg、p75ECD-15mg/kg、p75ECD-20mg/kg以及联合治疗组的AUC面积显著降低(p＜0.01)，其中p75ECD-15mg/kg组与联合治疗组下降更明显(p＜0.001)；第1天到第28天关节肿胀评分曲线下面积(AUC)显示(图2，右)，与模型组比较，依那西普-6.7mg/kg组、p75ECD不同剂量组以及联合治疗组的AUC面积显著降低(p＜0.01)，其中p75ECD-15mg/kg组与联合治疗组下降更明显(p＜0.001)；同时，与依那西普-6.7mg/kg 组或p75ECD-Fc-20mg/kg单药治疗相比，二者联合治疗组在改善四肢关节肿胀评分方面更有优势。上述结果提示不同剂量的p75ECD单独使用或与依那西普连用可以改善CIA诱导的RA模型动物的关节肿胀，其中p75ECD-15mg/kg组与联合治疗组效果更好。
Figure PCTCN2022137395-appb-000008
2、爪缩阈(PWT)
测量动物PWT结果请见表4，图3和图4显示：第5天模型组动物左右足PWT均下降，除anti-proBDNF组外，其余各给药组均提高了左足PWT，并且p75ECD-10mg/kg组和p75ECD-15mg/kg组在第1天至第5天保持平稳。动物右足PWT检测中，除anti-proBDNF组外，其余各组PWT均比模型组提高。图5显示，与模型组比较，p75ECD-5mg/kg组、p75ECD-10mg/kg组、p75ECD-15mg/kg组和联合治疗组第14天的曲线下面积(AUC)显著上升，p75ECD-10mg/kg组和联合治疗组上升更明显(p＜0.01)；p75ECD-10mg/kg组、p75ECD-15mg/kg组和联合治疗组第19天的曲线下面积显著上升，p75ECD-10mg/kg组上升更明显(p＜0.01)；图6显示，与模型组比较，除anti-proBDNF外，其余各组14天和19天的曲线下面积均显著上升。上述结果提示，不同剂量的p75ECD单独使用或与依那西普联用可以改善CIA诱导的RA模型动物的炎症疼痛。
Figure PCTCN2022137395-appb-000009
3、uCT检测
图7和图8为uCT检测足整体视图及踝关节视图。与正常组相比，模型组CT值(HU)、骨密度均显著下降(p＜0.001)；与模型组比较，p75ECD-5mg/kg组、p75ECD-10mg/kg组和p75ECD-15mg/kg组的CT值(HU)、骨密度均显著升高(p＜0.001)；p75ECD-20mg/kg组的CT值(HU)、骨密度均显著升高(p＜0.05)(表5，图9-图10)。
表5：uCT检测(MEAN±SD)
Figure PCTCN2022137395-appb-000010
注：One-way ANOVA:*p<0.05vs.模型组；***p<0.001vs.模型组；T-test:p＝0.06vs.模型组； ###p<0.001vs.模型组
4、组织病理检测
图11-13分别为左后足全掌、踝关节及趾关节病理图像。
足爪滑膜增生评分、滑膜炎症评分、软骨损伤评分、骨损伤评分以及总损伤评分见表6。
表6：足爪损伤评分(MEAN±SD)
Figure PCTCN2022137395-appb-000011
Figure PCTCN2022137395-appb-000012
注：One-way ANOVA:*p<0.05vs.模型组；**p<0.01vs.模型组；***p<0.001vs.模型组；T-test:#p<0.05vs.模型组；##p<0.01vs.模型组。
图14显示在足爪滑膜增生评分中，模型组较正常组显著性升高(p<0.001)；给予4次药物治疗后，与模型组相比，甲氨蝶呤-2.5mg/kg组显著性降低足爪滑膜增生评分(p<0.05)，p75ECD-10mg/kg、p75ECD-15mg/kg、p75ECD-20mg/kg组均显著性降低足爪滑膜增生评分(p<0.01，p<0.05，p<0.01)。
图15显示在足爪滑膜炎症评分中，模型组较正常组显著性升高(p<0.001)；给予4次药物治疗后，与模型组相比，甲氨蝶呤-2.5mg/kg组显著性降低足爪滑膜炎评分(p<0.05)，p75ECD-10mg/kg、p75ECD-15mg/kg、p75ECD-20mg/kg组评分均显著性降低(p<0.01，p<0.05，p<0.01)。
图16显示在足爪软骨损伤评分中，模型组较正常组显著性升高(p<0.001)；给予4次的药物治疗后，与模型组相比，甲氨蝶呤-2.5mg/kg组显著性降低足爪软骨损伤评分(p<0.05)，p75ECD-5mg/kg、p75ECD-10mg/kg、p75ECD-15mg/kg、p75ECD-20mg/kg组评分均显 著性降低(p<0.05，p<0.05，p<0.001，p<0.05)。
图17显示在足爪骨损伤评分中，模型组较正常组显著性升高(p<0.001)；给予4次药物治疗后，与模型组相比，甲氨蝶呤-2.5mg/kg组显著性降低足爪骨损伤评分(p<0.05，图17)，p75ECD-5mg/kg、p75ECD-10mg/kg、p75ECD-15mg/kg、p75ECD-20mg/kg组评分均显著性降低(p<0.05，p<0.05，p<0.001，p<0.05)。
图18显示在足爪损伤总评分中，模型组较正常组显著性升高(p<0.001)；同时，给予4次药物治疗后，与模型组相比，甲氨蝶呤-2.5mg/kg组总评分显著性降低(p<0.05，图18)，p75ECD-5mg/kg、p75ECD-10mg/kg、p75ECD-15mg/kg、p75ECD-20mg/kg组总评分均显著性降低(p<0.05，p<0.01，p<0.01，p<0.001)。
5、脾脏组织中proBDNF、p75NTR、pJAK1、pSTAT1检测(westernblot)
脾脏组织中proBDNF、p75NTR、pJAK1、pSTAT1蛋白表达情况请见表7。图19显示，与正常组比较，模型组proBDNF表达显著性上升(p<0.01)；与模型组比较，除p75ECD-5mg/kg组外，其余各组proBDNF均显著性下降(p<0.05)，其中p75ECD-15mg/kg组下降更明显(p＜0.01)。图20显示，与正常组比较，模型组p75NTR表达显著性上升(p<0.001)；与模型组比较，各组p75NTR均显著性下降(p<0.05,p＜0.01)。图21显示，与正常组比较，模型组磷酸化的JAK1显著性上升(p<0.01)；与模型组比较，各组磷酸化的JAK1均显著性下降(p<0.05)，其中p75ECD-15mg/kg组下降更明显(p＜0.01)。图22显示，与正常组比较，模型组磷酸化的STAT1显著性上升(p<0.05)；与模型组比较，各组均显著性下降(p<0.05)，其中联合治疗组下降更明显(p＜0.01)。
表7：脾脏组织中proBDNF、p75NTR、pJAK1、pSTAT1/蛋白表达情况(MEAN±SEM)
Figure PCTCN2022137395-appb-000013
注：T-test:#p＜0.05vs.模型组；##p<0.01vs.模型组；###p<0.001vs.模型组
6、脾脏组织中p75NTR、IL-1β、IL-6和TNF-αmRNA表达水平(qPCR)
表8示出脾脏组织中p75NTR、IL-1β、IL-6和TNF-αmRNA表达水平。结果显示：与正常对照组比较，模型组动物脾脏组织中p75NTR mRNA表达水平显著上升(p＜0.001)，与模型组比较，各组动物脾脏组织中p75NTR mRNA表达水平显著降低，其中p75ECD-10mg/kg、p75ECD-15mg/kg、p75ECD-20mg/kg以及联合治疗组下降更明显(p＜0.001)(图23)；与正常对照组比较，模型组动物脾脏组织中IL-1βmRNA表达水平显著上升(p＜0.001)，与模型组比较，除anti-proBDNF组外，其余各组动物脾脏组织中IL-1βmRNA表达水平均显著降低，其中p75ECD各剂量组和联合治疗组降低更明显(p＜0.001)(图24)；与正常对照组比较，模型组动物脾脏组织中IL-6 mRNA表达水平显著上升(p＜0.001)，与模型组比较，各组动物脾脏组织中IL-6 mRNA表达水平均显著降低，其中甲氨蝶呤-2.5mg/kg组、依那西普-6.7mg/kg组、p75ECD-15mg/kg组以及联合治疗组降低更明显(p＜0.001)(图25)；与正常对照组比较，模型组动物脾脏组织中TNF-αmRNA表达水平显著上升(p＜0.001)，与模型组比较，各组动物 脾脏组织中IL-6 mRNA表达水平均显著降低，其中甲氨蝶呤-2.5mg/kg组、依那西普-6.7mg/kg组、p75ECD-10mg/kg组、p75ECD-15mg/kg组以及联合治疗组降低更明显(p＜0.001)(图26)。
表8：脾脏组织中p75NTR、IL-1β、IL-6和TNF-αmRNA表达水平(MEAN±SD)
Figure PCTCN2022137395-appb-000014
注：T-test:#p＜0.05vs.模型组；##p＜0.01vs.模型组；###p＜0.001vs.模型组
7、血清中IL-1β、IL-6和TNF-α含量(ELISA)
血清中IL-1β、IL-6和TNF-α水平见表9。结果显示，与正常对照组比较，模型组IL-1β、IL-6和TNF-α显著升高，与模型组比较，除anti-proBDNF组外，其余各组动物血清中IL-1β含量均显著下降，其中p75ECD-15mg/kg组下降更明显(p＜0.001)(图27)；与模型组比较，甲氨蝶呤-2.5mg/kg、依那西普-6.7mg/kg和anti-proBDNF组动物血清中IL-6含量无显著差异，而p75ECD各剂量组以及联合治疗组动物血清中IL-6含量显著下降(p＜0.05,)，其中p75ECD-15mg组下降更明显(p＜0.01)(图28)；与模型组比较，除anti-proBDNF组外，其他各组动物血清中TNF-α含量均显著下降(p＜0.01,p＜0.001)(图29)。
表9：血清中炎症因子含量(pg/ml)(MEAN±SD)
Figure PCTCN2022137395-appb-000015
注：T-test:#p＜0.05vs.模型组；##p＜0.01vs.模型组；###p＜0.001vs.模型组
上述结果显示，通过建立雌性Lewis大鼠CIA模型，甲氨蝶呤在2.5mg/kg的剂量下可以显著降低雌性Lewis CIA模型大鼠的足爪损伤评分，呈现显著性的疗效作用。依那西普在6.7mg/kg的剂量下可以降低雌性Lewis CIA模型大鼠四肢关节肿胀评分(第5-9天)以及提高疼痛阈值(PWT)；p75ECD在5mg/kg、10mg/kg、15mg/kg、20mg/kg4个剂量组在体重、PWT、踝关节容积及直径、肿胀评分中均表现较好的药效并且在病理中均可以显著降低造模大鼠的足爪损伤评分，发挥出很好的治疗效果，其中以15mg/kg疗效最佳，药效较甲氨蝶呤2.5mg/kg更好。
以上具体实施方式的说明只是用于帮助理解本发明的核心思想。应当指出，对于本领域的普通技术人员而言，在不脱离本发明原理的前提下，还可以对本发明的技术方案进行若干改进和修饰，但这些改进和修饰也落入本发明权利要求请求保护的范围内。
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Claims (14)

  1. p75细胞外结构域(p75ECD)或其功能性片段、变体、类似物或衍生物在制备用于治疗和/或预防类风湿性关节炎(RA)的药物中的用途。
  2. 权利要求1所述的用途，其中所述p75ECD由SEQ ID NO:2所示的氨基酸序列或其变体组成。
  3. 权利要求1所述的用途，其中所述p75ECD被融合到免疫球蛋白的Fc结构域，以形成p75ECD-Fc融合体，其中所述p75ECD-Fc由SEQ ID NO:4所示的氨基酸序列或其变体组成。
  4. 权利要求3所述的用途，其中所述p75ECD-Fc融合体以p75ECD-Fc融合体腺相关病毒(AAV)载体的形式给药。
  5. 权利要求3所述的用途，其中所述p75ECD-Fc融合体是重组肽。
  6. 权利要求1所述的用途，其中所述p75ECD通过静脉内、腹膜内、脑室内、鞘内或肌内注射来给药。
  7. 权利要求4所述的用途，其中所述p75ECD-Fc融合体AAV载体以1×10 6至11×10 13个病毒基因/kg对象体重之间的剂量给药。
  8. 权利要求4所述的用途，其中所述p75ECD-Fc融合体AAV载体以1×10 8至11×10 13个病毒基因/kg对象体重之间的剂量给药。
  9. 权利要求5所述的用途，其中所述p75ECD-Fc融合体重组肽以0.1mg/kg对象体重至250mg/kg对象体重之间的剂量给药。
  10. 权利要求1所述的用途，其中所述p75ECD与第二药理活性化合物组合时分别、依次或同时使用。
  11. 权利要求10所述的用途，其中所述第二药理化合物选自：阿片类镇痛药；非甾体抗炎药；类固醇激素；改善疾病的抗类风湿类药物(DMARDs)；肿瘤坏死因子α抑制剂；B细胞抑制剂；T细胞抑制剂；细胞因子抑制剂；细胞因子抑制剂；JAK1/JAK2抑制剂；粒细胞-吞噬细胞集落刺激因子(GM-CSF)受体阻断剂。
  12. 权利要求11所述的用途，其中所述阿片类镇痛药选自吗啡、海洛因、氢吗啡酮、羟吗啡酮、左啡诺、左洛啡烷、美沙酮、哌替啶、芬太尼、可卡因、可待因、双氢可待因、羟考酮、氢可酮、右丙氧芬、纳美芬、烯丙吗啡、纳洛酮、纳曲酮、丁丙诺啡、布托啡诺、纳布啡或喷他佐辛；
    所述非甾体抗炎药选自阿司匹林、双氯芬酸、二氟尼柳、依托度酸、芬布芬、非诺洛芬、氟苯柳、氟比洛芬、布洛芬、吲哚美辛、酮洛芬、酮咯酸、甲氯芬那酸、甲芬那酸、美洛昔康、萘丁美酮、萘普生、尼美舒利、硝基氟比洛芬、奥沙拉秦、保泰松、吡罗昔康、舒林酸、托美丁或佐美酸；
    所述改善疾病的抗类风湿类药物(DMARDs)选自柳氮磺胺吡啶、D-青霉胺、甲氨蝶呤、来氟米特、硫唑嘌呤、环孢素或羟氯喹；
    所述肿瘤坏死因子α抑制剂选自英夫利昔单抗、阿达木单抗、依那西普、戈利木单抗或赛托珠单抗；
    所述B细胞抑制剂选自利妥昔单抗或贝利木单抗；
    所述T细胞抑制剂选自阿巴西普；
    所述细胞因子抑制剂选自托珠单抗、西鲁库姆、克拉扎珠单抗、奥西单抗、沙利鲁单抗或阿那白滞素；
    所述JAK1/JAK2抑制剂选自巴瑞克替尼；
    所述粒细胞-吞噬细胞集落刺激因子(GM-CSF)受体阻断剂选自 马维李单抗。
  13. 权利要求12所述的用途，其中所述肿瘤坏死因子α抑制剂选自依那西普。
  14. 权利要求1所述的用途，其中所述药物用于治疗和/或预防RA的对象是人类。
PCT/CN2022/137395 2021-12-15 2022-12-08 p75细胞外结构域在类风湿性关节炎治疗和/或预防中的应用 WO2023109622A1 (zh)

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