CN116688094A - Use of p75 extracellular domain in the treatment and/or prevention of rheumatoid arthritis - Google Patents

Abstract

The invention belongs to the field of pharmacy, and particularly relates to application of a p75 extracellular domain (p 75 ECD) in treatment and/or prevention of Rheumatoid Arthritis (RA).

Description

Use of p75 extracellular domain in the treatment and/or prevention of rheumatoid arthritis

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a novel application of a p75 extracellular domain (p 75 ECD), in particular to an application of the p75 extracellular domain (p 75 ECD) in preparing a medicament for treating and/or preventing Rheumatoid Arthritis (RA).

Background

Rheumatoid arthritis (Rheumatoid arthritis, RA) is an autoimmune disease characterized by chronic erosive arthritis, the basic pathological changes being synovitis, acute synovial swelling, exudation, granulocyte infiltration; chronic synovial hyperplasia is hypertrophic, and pannus is formed, which is the pathological basis for causing joint destruction, joint deformity, disorder and disease to enter into irreversible stage. RA is characterized by symmetrical facet joint erosive lesions, mainly involving the wrist, metacarpophalangeal, proximal interphalangeal, knee, ankle, and toe joints, and produces local and systemic destructive effects on skeletal tissue. RA is a chronic, progressive disease that, although not directly life threatening, can lead to destruction of the bone joint in 2 years, manifested as swelling, pain, and decline of function of the affected joint, with lesions in a sustained, recurrent process. Early and incorrect treatment in time often leads to joint destruction and deformity, and finally to joint deformity and loss of function, which seriously affects the life quality of patients.

The global incidence of RA is 0.5% -1.0%, and it is onset at any age, with 80% of RA patients ranging in age from 20 to 45 years, women being most numerous, about 3 times the incidence of men ^[1] . The current incidence rate of China is about 0.32% -0.36%.

Current drugs for the clinical treatment of RA include: 1) Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and indomethacin can relieve joint pain symptoms of RA patients, but cannot control RA activity and development, and have obvious adverse effects after long-term administration, including central system symptoms, digestive system symptoms, cardiovascular injury, liver and kidney dysfunction, etc ^[2] The method comprises the steps of carrying out a first treatment on the surface of the 2) Antirheumatic drugs (MDARDs) for ameliorating diseases, such as methotrexate, leflunomide, etc., which are widely accepted by clinicians, potential adverse effects include canker sore, liver cirrhosis, hepatitis, interstitial pneumonia and cytopenia ^[3] The method comprises the steps of carrying out a first treatment on the surface of the 3) Macromolecular biological therapeutic agents, e.g. adalimumabMonoclonal antibodies, etanercept, golimumab, and the like, act by blocking certain specific inflammatory cytokines or surface molecules. The use of macromolecular biological drugs is a major breakthrough in RA treatment, but can cause local rash, infection, tuberculosis, demyelinating syndrome and the like ^[4] For example, adalimumab may cause severe infections, leading to disease recurrence ^[5] . In addition to drug therapy, synovial resection is often used clinically to treat RA, but has the limitations of large trauma, poor patient compliance, and the like. Taken together, although RA has advanced significantly over the last decade, there are still many limitations to both drug and surgical therapies. Therefore, the development of drugs for the treatment and/or prevention of RA is of great social and economic significance.

The pathogenesis of RA is complex involving a large number of different cell types and signaling pathways. It was found that inflammatory networks are key factors in the development of RA, with inflammatory responses, T and B cells, autoantibodies, cytokines and other inflammatory mediators being important components of the inflammatory network. Previous studies by the inventors have found that brain-derived neurotrophic factor precursor (proBDNF) is an inflammatory mediator and plays an important role in the regulation of pain ^[6] Can be used as inflammatory mediators to regulate pain, but the role of the proBDNF/p75NTR signaling pathway in Rheumatoid Arthritis (RA) is not yet clear.

CN 102233128B discloses the use of p75NTR-ECD in the preparation of a medicament for the prevention and treatment of alzheimer's disease. CN 106794222B discloses the use of the p75 extracellular domain (p 75 ECD) for the preparation of a medicament for the treatment and/or Cerebral Amyloid Angiopathy (CAA). CN107303389a discloses the use of a binding molecule with neutralizing or inhibitory activity in a peripheral brain-derived neurotrophic factor precursor protein (probnf) or signaling molecule thereof for the preparation of a medicament for alleviating, inhibiting or treating pain. CN 112472796A discloses the use of the p75 extracellular domain (p 75 ECD) or a functional fragment, variant, analogue or derivative thereof for the manufacture of a medicament for the treatment and/or prevention of frontotemporal dementia. CN113456799a discloses the use of a p75ECD in the manufacture of a medicament for modulating pain, wherein the pain comprises inflammatory pain or surgical pain. CN 104302326B

(PCT/GB 2013/050632) discloses therapeutic uses of p75NTR neurotrophin binding proteins, specifically teaching the use of p75NTR (NBP) for the treatment of pain and/or pain symptoms. CN 105873943a (PCT/GB 2014/052833) discloses a fusion protein, i.e. a p75NTR (NBP) -Fc fusion protein, for use in the treatment of pain or pain symptoms.

US2009/0175859A1 relates to tnfα antagonists and methotrexate for the treatment of Tumor Necrosis Factor (TNF) mediated diseases 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 immune diseases associated with transplantation.

US2018/0170995A1 (PCT/EP 2016/056049) discloses a p75NTR neurotrophic factor binding protein (NBP) -Fc fusion protein for use in the treatment of pain and/or pain symptoms, and for use in the treatment of 34 diseases including eczema, psoriasis, dermatitis, neurodegeneration, huntington's disease, senile dementia, autism, cancer, breast cancer, rheumatoid Arthritis (RA), osteoarthritis, cystitis, endometriosis and the like, wherein p75NTR (NBP) binds to one of Nerve Growth Factor (NGF), brain Derived Neurotrophic Factor (BDNF), NT3 or NT4/5 (binding affinity Kd is 1pM-100 nM).

EP2667895B1 (PCT/IT 2012/000018) 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 use in the treatment of chronic inflammatory diseases, wherein the p75NTR receptor inhibitor is selected from polyclonal and monoclonal monospecific or bispecific p75NTR receptor binding antibodies, chimeric molecules (the structure of which comprises soluble p75NTR receptors, p75NTR receptor binding cyclic monomer and dimer NGF analog compounds), and wherein the TrkA receptor activator is selected from TrkA receptor binding polyclonal and monoclonal antibodies, cyclic monomer and dimer NGF analog compounds, recombinant NGF or recombinant mutated NGF. According to the invention, the chronic inflammatory disease is selected from the group consisting of Rheumatoid Arthritis (RA), juvenile idiopathic arthritis, chronic inflammation of the intestines, ankylosing spondylitis, psoriasis, multiple sclerosis, lupus erythematosus, scleroderma, sjogren's syndrome.

However, the role of the p75 neurotrophic factor extracellular domain (p 75 ECD) in the prevention and/or treatment of Rheumatoid Arthritis (RA) has not been reported worldwide, and the inventors have found for the first time that p75ECD can be used as a medicament for the prevention and treatment of RA-like diseases.

Disclosure of Invention

The present invention relates in particular to a method of preventing and/or treating rheumatoid arthritis disease (RA) by the p75 neurotrophic factor receptor extracellular domain (p 75NTR-ECD, also known as p75 ECD), comprising administering p75ECD to a patient in need thereof, or to the use of p75ECD in the manufacture of a medicament for preventing and/or treating rheumatoid arthritis disease (RA).

The invention also relates to a p75ECD for use in the prevention and/or treatment of rheumatoid arthritis disease (RA), or a medicament for the prevention and/or treatment of rheumatoid arthritis disease (RA), the medicament comprising a p75ECD.

The present invention surprisingly found that p75ECD has a very good therapeutic effect in the treatment and/or prevention of rheumatoid arthritis, in particular unexpected effects in the relief of joint swelling, in the relief of inflammatory pain and in the reduction of joint synovial hyperplasia, synovial inflammation, cartilage damage, bone damage etc., possibly by blocking the pro BDNF/p75NTR signaling pathway and inhibiting Janus kinase 1 (Janus Kinases 1, JAK 1)/transduction and transcriptional activator 1 (signal transducerand activator of transcription 1, stat 1) phosphorylation followed by blocking tumor necrosis factor-a

(TNF-a), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and other inflammatory factors.

As used herein, the term "treatment" is intended to mean that administration of the p75ECD reduces or ameliorates at least some signs or symptoms of RA.

As used herein, the term "preventing" is intended to mean that administration of the p75ECD occurs prior to diagnosis of RA and alleviates, ameliorates, or delays onset of at least some signs or symptoms of RA; or that administration of the p75ECD prevents signs or exacerbations of RA. Thus, the term "preventing" is intended to include prophylactic treatment.

The administered p75ECD can be from any species of interest (e.g., human, mouse, rat, rabbit, cat, dog, goat, cow, horse, non-human primate, etc.). However, in one embodiment, the p75ECD is a human p75ECD. In one embodiment, the p75ECD has a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO. 2, such as the nucleotide sequence set forth in SEQ ID NO. 1, or has the amino acid sequence set forth in SEQ ID NO. 2.

aaggaggcat gccccacagg cctgtacaca cacagcggtg agtgctgcaaagcctgcaac

ctgggcgagg gtgtggccca gccttgtgga gccaaccaga ccgtgtgtga gccctgcctggacagcgtga cgttctccga cgtggtgagc gcgaccgagc cgtgcaagccgtgcaccgag

tgcgtggggc tccagagcat gtcggcgccg tgcgtggagg ccgacgacgccgtgtgccgc

tgcgcctacg gctactacca ggatgagacg actgggcgct gcgaggcgtgccgcgtgtgc

gaggcgggct cgggcctcgt gttctcctgc caggacaagc agaacaccgtgtgcgaggag

tgccccgacg gcacgtattc cgacgaggcc aaccacgtgg acccgtgcctgccctgcacc

gtgtgcgagg acaccgagcg ccagctccgc gagtgcacac gctgggccgacgccgagtgc

gaggagatcc ctggccgttg gattaca(SEQ ID NO:1)

Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys CysLys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly AlaAsn

Gln Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp ValVal Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly LeuGln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys ArgCys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys GluAla

Cys Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys GlnAsp

LysGln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser AspGlu Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys GluAsp

Thr Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala GluCys Glu Glu Ile Pro Gly Arg Trp Ile Thr(SEQ ID NO:2)

In alternative embodiments, the p75ECD may be a functional fragment, variant, analog or derivative of the sequence. For example, a variant, analog or derivative of the p75ECD (or a functional fragment thereof) is composed of a molecule that differs from the p75ECD (or a functional fragment thereof) but retains similarity to the p75ECD in biological activity, particularly in its ability to bind to a neurotrophic factor. Variants, analogs, or derivatives of the p75ECD (or functional fragment thereof) may have significant overall structural similarity to the p75ECD (or functional fragment thereof), or structural similarity only to one or more regions of the p75ECD (or functional fragment thereof) responsible for the biological activity. Typically, a variant, analogue or derivative is provided by, or is the result of, a modification of one or more amino acids of the relevant amino acid sequence. For example, functional fragments, variants, analogues or derivatives comprising the sequence shown in SEQ ID NO. 2 are provided 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 replacing one or more amino acids from the amino acid sequence, or as a result of such modifications. Inversions of amino acids and other mutational changes that cause changes in amino acid sequence are also contemplated. Such analogs or derivatives may be prepared by introducing nucleotide changes into a polynucleotide molecule such that a desired amino acid change is obtained upon expression of the mutagenized polynucleotide molecule or by otherwise synthesizing an amino acid sequence incorporating the desired amino acid change. Substitutions of amino acids may include conservative or non-conservative amino acid substitutions. Conservative amino acid substitutions mean the replacement of 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 envisaged are: g, A, V, I, L, M; d, E; n, Q; s, T; k, R, H; f, Y, W, H; and P, nα -alkyl amino acids, which may have small changes in sequence, but do not cause any significant decrease 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; and Pro, nα -alkyl amino acids; non-conservative amino acid substitutions.

Table 1: exemplary conservative amino acid substitutions

* Indicating preferred conservative substitutions

When the analogue or derivative is prepared synthetically, the analogue or derivative may include one or more amino acids not encoded by the genetic code, such as carboxyglutamic acid and hydroxyproline, or may include a D-amino acid instead of an L-amino acid. Thus, the analogue or derivative may be a mimetic of the p75ECD (or a functional fragment thereof), such as a peptide mimetic. However, an analogue or derivative of the p75ECD (or a functional fragment thereof) does not necessarily have amino acid sequence identity and/or similarity, and in fact, the analogue or derivative may not be a protein at all.

It will be appreciated that peptides having amino acid sequences that are slightly altered from the amino acid sequences shown in SEQ ID No. 2 and SEQ ID No. 4 (e.g., variants produced by one or more conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.), are still possible within the scope of the invention, provided that the peptide is capable of binding p75 and preventing signal transduction through p 75. In particular, 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.

In one embodiment, the p75ECD comprises a p75ECD fusion, wherein the p75ECD is fused to a fusion partner. The fusion partner may be any suitable fusion partner known to the skilled person, such as an Fc region of an immunoglobulin, human Serum Albumin (HSA), bovine Serum Albumin (BSA), etc., as long as the p75ECD retains at least some of its biological activity. Fusion products can be produced by expressing the product from a suitable construct encoding the fusion product, or by otherwise chemically linking the p75ECD to a fusion partner. The fusion partner should increase the stability and serum half-life of the p75ECD in vivo. In one embodiment, the 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, the Fc domain is derived from a human IgG.

In one embodiment, the Fc region is capable of dimerizing to produce a dimeric form of the p75 ECD. However, other fusion partners may be capable of forming other types of oligomers, such as trimers, tetramers, pentamers, and the like. Thus, the p75ECD may be an oligomer such as a dimer, trimer, tetramer, pentamer, or the like. In one embodiment, the p75ECD may be a dimer. In one embodiment, the p75ECD-Fc fusion may be a p75ECD-Fc dimer.

In one embodiment, the p75ECD-Fc fusion comprises a human p75ECD molecule linked to the Fc portion of human IgG 1. One skilled in the art will recognize that fusions may have a large number of different nucleotide and amino acid sequences depending on, for example, the linker used. However, in one embodiment, SEQ ID NO. 3 is the nucleotide sequence of a human p75 ECD-human IgG1 Fc domain fusion (human p75 ECD-Fc). Based on the degeneracy, other nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO. 4 or variants thereof are also conceivable and fall within the scope of the invention.

aaggaggcat gccccacagg cctgtacaca cacagcggtg agtgctgcaaagcctgcaac

ctgggcgagg gtgtggccca gccttgtgga gccaaccaga ccgtgtgtga gccctgcctggacagcgtga cgttctccga cgtggtgagc gcgaccgagc cgtgcaagccgtgcaccgag

tgcgtggggc tccagagcat gtcggcgccg tgcgtggagg ccgacgacgccgtgtgccgc

tgcgcctacg gctactacca ggatgagacg actgggcgct gcgaggcgtgccgcgtgtgc

gaggcgggct cgggcctcgt gttctcctgc caggacaagc agaacaccgtgtgcgaggag

tgccccgacg gcacgtattc cgacgaggcc aaccacgtgg acccgtgcctgccctgcacc

gtgtgcgagg acaccgagcg ccagctccgc gagtgcacac gctgggccgacgccgagtgc

gaggagatcc ctggccgttg gattacagac aaaactcaca catgcccacc gtgcccagcacctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc

atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccctgaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccgcgggaggagc agtacaacag cacgtaccgg gtggtcagcg tcctcaccgtcctgcaccag

gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagcccccatcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggtgtacaccctg

cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcctggtcaaaggc

ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccggagaacaactac

aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc

gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggctctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa(SEQ IDNO:3)

Similarly, in one embodiment, the amino acid sequence of a human p75ECD-Fc fusion is provided in SEQ ID NO. 4. In one embodiment, SEQ ID NO. 4 comprises the p75ECD peptide at amino acids 1-169 of SEQ ID NO. 4, followed by a linker "DKTTCPPCP" at residues 170 to 179, followed by an Fc domain at residues 180 to 396. However, it will be appreciated that the p75ECD-Fc fusion may be a functional fragment, variant, analogue or derivative of the sequence shown by SEQ ID NO. 4. Functional fragments, variants, analogs, or derivatives are described elsewhere herein.

Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu CysCys

Lys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala AsnGln Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp ValVal Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly LeuGln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys ArgCys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu AlaCys Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln AspLys Gln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser AspGlu Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu AspThr Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu CysGlu 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 PhePro 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 PheAsn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys ProArg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu ThrVal Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys LysVal

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 ArgAsp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys GlyPhe 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 SerPhe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln GlnGly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn HisTyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys(SEQ ID NO:4)

In one embodiment, the p75ECD or p75ECD fusion is administered to the subject as a nucleotide molecule. Such nucleotide molecules may be constituted by, for example, expression vectors or expression cassettes. Preferred expression vectors include viral vectors, such as retroviral vectors, adenoviral vectors, and vectors derived from adeno-associated virus (AAV), such as those known to those skilled in the art, which are capable of achieving transduction of the desired host cell type. Viral vectors comprising a nucleotide sequence encoding the p75ECD or p75ECD fusion of the disclosure can be used to provide an in vivo source of the p75ECD or p75ECD fusion at a desired site for treatment or prevention of RA.

In a preferred embodiment, the p75ECD or p75ECD fusion is administered by a viral vector, which is known to provide a means of delivering nucleotides into cells in a manner suitable for gene therapy. As will be appreciated by those skilled in the art, adeno-associated virus (AAV), lentiviral or herpes simplex virus vectors may be used as viral vectors, as they are both capable of delivering nucleotides to cells for gene therapy purposes. Delivery of the p75ECD or p75ECD fusion by viral vectors can mediate long term expression of the agent in the joint, and sustained secretion of the p75ECD or p75ECD fusion is desirable due to the chronic nature of RA. In one embodiment, 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, in addition, are considered very safe, and no pathological events have been reported in animals or humans. AAV vectors are known as tools for gene therapy delivery. Thus, in one embodiment, the viral vector may be an AAV vector. The AAV vector may be any suitable adeno-associated viral vector known to those skilled in the art, such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and the like. In one embodiment, the AAV vector is AAV8.

The p75ECD-Fc was inserted into an AAV8 vector (supplied by Virovek) containing AAV8 capsid sequences of the AAV2 phospholipase domain to increase the efficacy of AAV 8. The sequence of AAV8 capsid sequence having AAV2 phospholipase domain is shown in SEQ ID NO. 5, wherein said AAV2 phospholipase domain sequence comprises nucleotide 26 to 431.

atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac

aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac

cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg

ggaaagaaga gaccggtaga gccatcaccc cagcgttctc cagactcctc tacgggcatc

ggcaagaaag gccaacagcc cgccagaaaa agactcaatt ttggtcagac tggcgactca

gagtcagttc cagaccctca acctctcgga gaacctccag cagcgccctc tggtgtgggacctaatacaa tggctgcagg cggtggcgca ccaatggcag acaataacga aggcgccgacggagtgggta gttcctcggg aaattggcat tgcgattcca catggctggg cgacagagtc

atcaccacca gcacccgaac ctgggccctg cccacctaca acaaccacct ctacaagcaaatctccaacg ggacatcggg aggagccacc aacgacaaca cctacttcgg ctacagcacc

ccctgggggt attttgactt taacagattc cactgccact tttcaccacg tgactggcag

cgactcatca acaacaactg gggattccgg cccaagagac tcagcttcaa gctcttcaac

atccaggtca aggaggtcac gcagaatgaa ggcaccaaga ccatcgccaa taacctcacc

agcaccatcc aggtgtttac ggactcggag taccagctgc cgtacgttct cggctctgcc

caccagggct gcctgcctcc gttcccggcg gacgtgttca tgattcccca gtacggctac

ctaacactca acaacggtag tcaggccgtg ggacgctcct ccttctactg cctggaatac

tttccttcgc agatgctgag aaccggcaac aacttccagt ttacttacac cttcgaggac

gtgcctttcc acagcagcta cgcccacagc cagagcttgg accggctgat gaatcctctg

attgaccagt acctgtacta cttgtctcgg actcaaacaa caggaggcac ggcaaatacg

cagactctgg gcttcagcca aggtgggcct aatacaatgg ccaatcaggc aaagaactggctgccaggac cctgttaccg ccaacaacgc gtctcaacga caaccgggca aaacaacaat

agcaactttg cctggactgc tgggaccaaa taccatctga atggaagaaa ttcattggct

aatcctggca tcgctatggc aacacacaaa gacgacgagg agcgtttttt tcccagtaac

gggatcctga tttttggcaa acaaaatgct gccagagaca atgcggatta cagcgatgtc

atgctcacca gcgaggaaga aatcaaaacc actaaccctg tggctacaga ggaatacggtatcgtggcag ataacttgca gcagcaaaac acggctcctc aaattggaac tgtcaacagc

cagggggcct tacccggtat ggtctggcag aaccgggacg tgtacctgca gggtcccatctgggccaaga ttcctcacac ggacggcaac ttccacccgt ctccgctgat gggcggcttt

ggcctgaaac atcctccgcc tcagatcctg atcaagaaca cgcctgtacc tgcggatcct

ccgaccacct tcaaccagtc aaagctgaac tctttcatca cgcaatacag caccggacag

gtcagcgtgg aaattgaatg ggagctgcag aaggaaaaca gcaagcgctg gaaccccgag

atccagtaca cctccaacta ctacaaatct acaagtgtgg actttgctgt taatacagaa

ggcgtgtact ctgaaccccg ccccattggc acccgttacc tcacccgtaa tctgtaa(SEQ ID NO:5)

A "therapeutically effective amount" is any amount that elicits a beneficial or therapeutic effect in a subject. In one embodiment, the dose of AAV vector for any suitable subject is 1X 10 ⁶ Up to 6X 10 ¹³ Between viral genomes/kg body weight of the subject. In one embodiment, the dose of AAV vector for any suitable subject is 1X 10 ¹¹ Up to 6X 10 ¹² Between viral genomes/kg body weight of the subject.

As is well recognized by those skilled in the art, the dosage of the adeno-associated viral vector administered may vary. For example, in clinical trials in institutions of the national institutes of health (U.S. national Institutes of Health) for human subjects, a "safe" dose of 6X 10 is used in patients ¹² Individual diseaseVirulence genome (vg)/kg body weight. However, for the methods described herein, the dose of adenovirus vector administered to a human subject may be 1X 10 ⁶ Up to 6X 10 ¹³ In the range of vg/kg body weight. In one embodiment, the adenovirus vector is administered to a human subject at a dose of 1X 10 ¹⁰ Up to 6X 10 ¹² Between viral genomes/kg body weight. In one embodiment, the adenovirus vector is administered to a human subject at a dose of 1X 10 ¹¹ Up to 3X 10 ¹² Between viral genomes/kg body weight. In one embodiment, the dose of adenovirus may be higher, for example 1X 10 ¹⁴ 、1×10 ¹⁵ 、1×10 ¹⁶ 、1×10 ¹⁷ 、1×10 ¹⁸ 、1×10 ¹⁹ 、1×10 ²⁰ Etc., as long as the dosages are deemed safe.

The dose of AAV vector used in a human subject can be converted to a dose suitable for other animals on the basis of body surface area [ human equivalent dose in mg/kg = animal dose in mg/kg x (animal body weight in kg/human body weight in kg) 0.33 using the us food and drug administration (US Food and Drug Administration) (FDA) standard for converting drug equivalent doses between species](food and drug administration, 2003). Thus, the "safe" dose in mice may be 8×10 ¹⁰ And the viral genome. However, one skilled in the art will recognize that the dose of adeno-associated viral vector administered to a mouse subject may be 1X 10 ⁵ Up to 1X 10 ¹³ Within the individual viral genomes. In one embodiment, the dose of adeno-associated viral vector administered to the mouse subject may be 1X 10 ⁸ Up to 1X 10 ¹² Within the individual viral genomes. In one embodiment, the dose of adeno-associated viral vector administered to the mouse subject may be 1X 10 ⁹ Up to 1X 10 ¹¹ Within the individual viral genomes.

In one embodiment of this aspect of the invention, the p75ECD or p75ECD-Fc fusion is a recombinant peptide. The recombinant p75ECD or p75ECD-Fc fusion peptide may be produced using any suitable technique known to those skilled in the art, for example by expression in a cultured bacterial system, such as E.coli (Escherichia coli), a yeast expression system, an insect expression system, a viral expression system or in a eukaryotic cell expression system, or the peptide may be synthesized synthetically, etc., all of which are known to those skilled in the art.

In one embodiment, the dose of recombinant p75ECD or p75ECD-Fc fusion peptide administered can 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 dose is about 0.01 to about 500mg/kg of subject body weight per day, which may be administered in a single dose or in multiple doses. Preferably, the dose is about 0.1 to about 250mg/kg per day, for example, 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, more preferably about 0.5 to about 20mg/kg per day.

In one embodiment, the p75ECD, p75ECD fusion, p75ECD-Fc, or AAV-p75ECD-Fc of this aspect of the disclosure are provided in combination with a pharmaceutically acceptable carrier, excipient, and/or diluent. Suitable pharmaceutically acceptable carriers, 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 oral, intravenous, intramuscular, intraperitoneal, intranasal, intracranial, intrathecal or intraventricular route. Preferably, however, the p75ECD or p75ECD-Fc is administered intravenously, intraperitoneally, intramuscularly, intrathecally or intraventricularly.

In another aspect, a method of treating and/or preventing RA by blocking signal transduction through p75 is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of a p75ECD or analog thereof. In one embodiment, the p75ECD or analog thereof prevents binding of a neurotoxic molecule selected from the group consisting of probnf and probt 3 to p 75. The method of this aspect uses the embodiments described above.

According to the invention it comprises the extracellular portion p75ECD of a p75NTR receptor, wherein said p75ECD consists of the amino acid sequence set forth in SEQ ID NO. 2 or a variant thereof (e.g.one or more conservative amino acid substitution variants).

According to the invention it comprises a p75ECD fused to an Fc domain of an immunoglobulin to form a p75ECD-Fc fusion, wherein said p75ECD-Fc consists of the amino acid sequence shown in SEQ ID NO. 4 or a variant thereof (e.g.one or more conservative amino acid substitution variants).

According to the invention, the p75ECD-Fc fusion protein is administered in a daily dose of between 0.1mg/kg body weight of the subject and 250mg/kg body weight of the subject. Wherein for treatment the daily dose is preferably 0.1-100mg/kg of the subject's body weight, e.g. 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 or 90mg/kg etc.

According to the present invention, the AAV vector of the p75ECD-Fc fusion protein is administered at a daily dose of 1X 10 ⁶ To 11X 10 ¹³ Between individual viral genes/kg body weight of the subject. Wherein for treatment the daily dose is preferably 1X 10 ⁸ To 11X 10 ¹³ Between individual viral genes/kg body weight of the subject.

According to the invention, the p75 extracellular domain (p 75 ECD) is used separately, sequentially or simultaneously in combination with a second pharmacologically active compound. Preferably, the second pharmacologically active compound is selected from:

opioid analgesics such as morphine (morphine), heroin (herman), hydromorphone (hydromorphone), oxymorphone (oxymorphone), levorphanol (levorphanol), levorphanol (levalorphan), methadone (methadone), meperidine (meperidine), fentanyl (fentanyl), ***e (codeine), codeine (codeine), dihydrocodeine (dihydrocodeine), oxycodone (oxycodone), hydrocodone (hydromorphone), dextrorphan (lipoxyphenne), nalmefene (nalmefene), allylmorphine (nalorphine), naloxone (naloxone), naltrexone (buprenorphine), buprenorphine (butorphanol), buprofen (nalmefene), buprenorphine (nalmefene) or pentazocine (pentazocine);

Non-steroidal anti-inflammatory drugs (nasds), such as aspirin (aspriline), diclofenac (triclofenac), diflunisal (diflunisal), etodolac (etodolac), fenbufen (fenbufen), fenoprofen (fenoprofen), flubensal (flubenisal), flurbiprofen (flubiprofen), ibuprofen (ibuprofen), indomethacin (indomethacin), ketoprofen (ketoprofen), ketorolac (torolac), meclofenamic acid (meclofenamic acid), mefenamic acid (mefenamic acid), meloxicam (meloxicam), nabumetone (naproxen), nimesulide (nimesulide), niflumiprofen (niflumiprofen), salazine (tazosin), piroxicam (mezocine), or zazocine (mevalonate);

anti-rheumatoid drugs (DMARDs) for ameliorating diseases, such as sulfasalazine (sulfasalazine), D-penicillamine (D (-) -penicillamine), methotrexate (methotrextrate), leflunomide (leflunomide), azathioprine (azathioprine), cyclosporin (cyclosporin), or hydroxychloroquine (hydroxychlorperaquinone);

tumor necrosis factor alpha inhibitors such as infliximab, adalimumab, etanercept, golimumab, or certolizumab;

B-cell inhibitors such as rituximab (rituximab) or belimumab (belimumab);

t cell inhibitors such as abatacept;

cytokine inhibitors such as tolizumab, cetirizine Lu Kum (sirukumab), cladzazumab (clazakizumab), oxhizumab (olokizumab), sha Lilu mab (sarilumab) or anakinra;

JAK1/JAK2 inhibitors, such as barricitinib;

granulocyte-phagocyte colony stimulating factor (GM-CSF) receptor blockers, such as Ma Weili monoclonal antibodies (mavrilimumab).

According to the application, the second pharmacologically active compound is selected from etanercept.

The dosage and frequency of administration of the formulations of the application may vary depending on the patient's condition, and it is sometimes desirable to administer relatively high doses at relatively short intervals until the progression of the disease is reduced or terminated, preferably until the patient exhibits a partial or complete improvement in the symptoms of the disease. The medicament of the present application may be administered by intravenous, intraperitoneal, intraventricular, intrathecal or intramuscular injection.

Drawings

In order to more clearly describe the technical solution of the present application, a brief description will be given below with reference to the accompanying drawings. It is apparent that these drawings are merely some of the specific embodiments of the present application. The present application includes, but is not limited to, these figures.

FIG. 1 shows total score of extent of swelling of joints of limbs;

figure 2 shows total score of degree of joint swelling in extremities for 14 days, area under 24 days curve (AUC), where One-way ANOVA: p <0.001vs. normal control group; model group, #p <0.05 vs; model group # p <0.01 vs; model group # # p <0.001 vs; t-test $ p <0.05vs. model set; model group of $ p <0.01 vs; model group, $p <0.001 vs;

FIG. 3 shows a left foot pain threshold;

FIG. 4 shows a right foot pain threshold;

fig. 5 shows the area under the curve (AUC) for 14 days, 19 days of left foot pain threshold, where One-way ANOVA p <0.001vs. normal control; model group, #p <0.05 vs; model group # p <0.01 vs; model group # # p <0.001 vs;

fig. 6 shows the right foot pain threshold for 14 days, area under the 19 day curve (AUC), where One-way ANOVA p <0.001vs. normal control; model group, #p <0.05 vs; model group # p <0.01 vs; model group # # p <0.001 vs;

FIGS. 7-8 are overall views of the uCT examination foot and ankle views; wherein the method comprises the steps of

FIG. 7 is an overall view of the uCT assay, wherein A. Normal control group; B. a model group; C. methotrexate-2.5 mg/kg; D. etanercept-6.7 mg/kg; E.anti-proBDNF-1ml/kg; F.p75ECD-5mg/kg; G.p75ECD-10mg/kg; H.p75ECD-15mg/kg; I.p75ECD-20mg/kg; p75ECD (20 mg/kg) +etanercept (6.7 mg/kg);

FIG. 8 is a view of the ankle joint for uCT examination, wherein A. Normal control group; B. a model group; C. methotrexate-2.5 mg/kg; D. etanercept-6.7 mg/kg; E.anti-proBDNF-1ml/kg; F.p75ECD-5mg/kg; G.p75ECD-10mg/kg; H.p75ECD-15mg/kg; I.p75ECD-20mg/kg; p75ECD (20 mg/kg) +etanercept (6.7 mg/kg);

FIG. 9 is a left hind foot CT value (HU) with One-way ANOVA p <0.05vs. model group; * P <0.001vs. model group; t-test, p=0.06 vs; model group # # p <0.001 vs;

FIG. 10 is the left hind paw bone density, wherein One-way ANOVA: <0.05vs. model group; * P <0.001vs. model group; t-test, p=0.06 vs; model group # # p <0.001 vs;

FIGS. 11-13 are images of the left hind foot full-sole, ankle joint, and toe joint pathology, respectively; wherein the method comprises the steps of

Fig. 11 shows a left hind foot full-palm pathology image, wherein a. normal control group; B. a model group; C. methotrexate-2.5 mg/kg; D. etanercept-6.7 mg/kg; E.anti-proBDNF-1ml/kg; F.p75ECD-5mg/kg; G.p75ECD-10mg/kg; H.p75ECD-15mg/kg; I.p75ECD-20mg/kg; p75ECD (20 mg/kg) +etanercept (6.7 mg/kg);

fig. 12 shows an ankle pathology image, wherein a. normal control group; B. a model group; C. methotrexate-2.5 mg/kg; D. etanercept-6.7 mg/kg; E.anti-proBDNF-1ml/kg; F.p75ECD-5mg/kg; G.p75ECD-10mg/kg; H.p75ECD-15mg/kg; I.p75ECD-20mg/kg; p75ECD (20 mg/kg) +etanercept (6.7 mg/kg); and

Fig. 13 shows a toe joint pathology image, wherein a. normal control group; B. a model group; C. methotrexate-2.5 mg/kg; D. etanercept-6.7 mg/kg; E.anti-proBDNF-1ml/kg; F.p75ECD-5mg/kg; G.p75ECD-10mg/kg; H.p75ECD-15mg/kg; I.p75ECD-20mg/kg; p75ECD (20 mg/kg) +etanercept (6.7 mg/kg);

FIG. 14 shows paw synovial hyperplasia scores, wherein One-way ANOVA:. P <0.05vs. model group; * P <0.001vs. model group; t-test: #p <0.05vs. model group; model group # p <0.01 vs;

FIG. 15 shows paw synovial inflammation scores, wherein One-way ANOVA:. P <0.05vs. model group; * P <0.001vs. model group; t-test: #p <0.05vs. model group; model group # p <0.01 vs;

FIG. 16 shows paw cartilage damage scores, where One-way ANOVA:. P <0.05vs. model group; * P <0.001vs. model group;

FIG. 17 shows paw cartilage damage scores, where One-way ANOVA:. P <0.05vs. model group; * P <0.001vs. model group; t-test: #p <0.05vs. model group; model group # p <0.01 vs;

fig. 18 shows paw total injury score, wherein One-way ANOVA: <0.05 vs; * P <0.001vs. model group; t-test: #p <0.05vs. model group; model group # p <0.01 vs; model group # # p <0.001 vs;

FIG. 19 shows the expression of proBDNF protein in spleen tissue, where T-test: #p <0.05vs. model group; model group # p <0.01 vs;

FIG. 20 shows p75NTR protein expression in spleen tissue, where T-test: #p <0.05vs. model group; model group # p <0.01 vs; model group # # p <0.001 vs;

FIG. 21 shows pJAK1 protein expression in spleen tissue, wherein T-test: #p <0.05vs. model group; model group # p <0.01 vs;

FIG. 22 shows pSTAT1 protein expression in spleen tissue, where T-test: #p <0.05vs. model group; model group # p <0.01 vs;

FIG. 23 shows p75NTR mRNA expression in spleen tissue, wherein T-test: model group with #p less than 0.05 vs; model group # # p <0.001 vs;

FIG. 24 shows IL-1β mRNA expression in spleen tissue, wherein T-test: model group with #p less than 0.05 vs; model group # # p <0.001 vs;

FIG. 25 shows IL-6mRNA expression in spleen tissue, wherein T-test: model group with #p less than 0.05 vs; model group # p <0.01 vs; model group # # p <0.001 vs;

FIG. 26 shows TNF- α mRNA expression in spleen tissue, wherein T-test: model group with #p less than 0.05 vs; model group # p <0.01 vs; model group # # p <0.001 vs;

FIG. 27 shows IL-1β content in serum, wherein T-test: model group with #p less than 0.05 vs; model group # p <0.01 vs; model group # # p <0.001 vs;

FIG. 28 shows IL-6 content in serum, wherein T-test: model group with #p less than 0.05 vs; model group # p <0.01 vs; and

FIG. 29 shows TNF- α content in serum, wherein T-test: model group # p <0.01 vs; # # p <0.001vs.

Detailed Description

For a further understanding of the present invention, preferred embodiments of the present invention will be described below with reference to examples. The description is only illustrative of the features and advantages of the technical solution of the invention and does not limit the scope of protection of the invention.

Purpose of experiment

The therapeutic effect of intravenous injection of p75ECD-Fc fusion protein on Collagen (CIA) -induced rheumatoid arthritis model Lewis female rats was evaluated.

Experimental animals, modeling and grouping

Female SPF-class Lewis rats, 100 animals are raised in a barrier system, the environment-friendly conditions of the animals are controlled to be between 20 and 26 ℃ at room temperature and 40 to 70 percent of humidity, the animals are replaced 10 to 20 times in each hour of air in the room, and the lighting is performed for 12/12 hours in a light-dark circulation mode. After the group experiment, cage closing feeding (2-6 cages) is carried out, wherein the cages are made of polysulfone plastic cages with corncob padding, and toys for biting or shielding are arranged in the cages to provide rich measures for the environment for animals. Free foraging, providing reverse osmosis purified water through a water bottle, and providing free drinking for animals.

Model group model inducer preparation: determining and weighing the mass of Collagen II according to the number of the required immunized animals, dissolving with 50mM glacial acetic acid, and keeping the final concentration to 2mg/ml; stir overnight at 4 ℃. On the day of immunization, complete Freund's Adjuvant (CFA) was aspirated at 1:1, mixed with prepared collagen, placed on ice, and homogenized at 3-4 speed for about 20 minutes using a homogenizer (criterion: not immediately dispersed by instillation into fresh water).

And (3) establishing a CIA model: animals were subjected to secondary immunization modelling 8 days after the first immunization. Model group Lewis rats were anesthetized with isoflurane, and were placed at both the tail root (2-3 cm from the body, 100. Mu.l/point) and the back side (50. Mu.l/point). Normal control animals were injected with an equivalent amount of physiological saline.

Experimental grouping and dosing: the experiment was divided into 10 groups, group 1 being the normal group, group 2 being the model group, group 3 being the model methotrexate group, group 4 being the model etanercept group, group 5 being the model proBDNF polyclonal antibody group, groups 6 to 9 being the p75ECD-Fc dose groups, group 10 being the combination treatment group. The initial dosing time for each group of animals was fixed on the first day after the second immunization, and the experimental groupings and dosing schedule are detailed in table 2.

Table 2: experimental grouping and dosing regimen

Remarks 1: the p75ECD-Fc protein can be prepared by methods conventional in the art, particularly by methods disclosed in CN 102233128B;

Remarks 2: the proBDNF polyclonal antibody can be prepared by referring to the conventional methods in the art, in particular, the method disclosed in CN 107303389A.

At the end of the experiment (the third day after the last 1 administration), the left hind limb is euthanized by isoflurane anesthesia, immersed in 10% neutral formalin (for uCT and pathology detection), serum is collected, placed at normal temperature for at least 30min, centrifuged at 5000rpm for 5min, and the serum is separated and stored at-80 ℃ for inflammatory factor detection. In addition, 6 animals were selected from each of group 1 (normal group), group 2 (model group), 6-9 (p 75ECD-5mg/kg,10mg/kg,15mg/kg and 20mg/kg group) and 10 (combination treatment group), and spleen tissue was quick frozen for detection of real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR) detection and Westernblot detection.

Detecting the index:

1. joint swelling score

Scoring was started after the second immunization, 1 every two days, until the end of the experiment. Scoring criteria: 0 point: the limbs are normal; 1, the method comprises the following steps: inflammation and swelling of at least one of the toe joints; 2, the method comprises the following steps: paw inflammation, swelling; 3, the method comprises the following steps: paw swelling, but not ankle joint involvement; 4, the following steps: paw swelling involves the ankle joint. The degree of joint swelling of each animal was added by the score of each paw, with the total score for each animal ranging from 0 to 16 points, and model induction was considered successful when the score for more than two joints was greater than 1 point or the score for more than one joint was greater than 2 points.

2. Paw withdrawal threshold (Paw Withdrawal Threshold PWT)

PWT measurement is performed by the method described in the previous literature ^[7] By von Frey fiber yarn pain measuring instrument. Animals were evaluated for pain threshold once a week before and after dosing.

3. Ultrasonic tomography (Ultrasonic Computed Tomography, uCT)

At the end of the experiment, the ankle to paw portion was subjected to uCT analysis to evaluate the bone injury status and bone density of the joint.

4. Tissue pathology observation

After the uCT detection, the ankle joint tissues are decalcified, embedded in paraffin, sectioned and stained with hematoxylin-eosin according to the standard histopathological technique. The extent of ankle to paw synovial hyperplasia, synovial inflammation, cartilage injury, and bone injury was evaluated.

5. Westernblot detection

The expression levels of proBDNF, p75NTR, pJAK1/JAK1 and pSTAT1/STAT1 proteins in spleen tissues were detected by Westernblot.

6. qPCR detection

Spleen tissue p75NTR, TNF- α, IL-1β and IL-6mRNA expression levels were detected using qPCR. Primer reference report [8] method design.

7. ELISA detection

The enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) is adopted to detect the content of inflammatory factors such as TNF-alpha, IL-1 beta, IL-6 and the like in serum.

Experimental results

1. Joint swelling score

Each model animal started to enter the initial stage of onset on day 3 of the experiment, reached the peak of onset on day 17 and continued to the end of the experiment. The total score for the degree of joint swelling in the model group was significantly different from that in the normal group from the day 5 of the experiment. The total joint scores of the etanercept group were statistically different from the model group on days 5 to 9; the p75ECD-5mg/kg group was statistically different from the model group on days 13 to 19; the p75ECD-10mg/kg group was statistically different from the model group on days 5 to 13; the p75ECD-15mg/kg group was statistically different from the model group on days 5 to 19 and 24; the p75ECD-20mg/kg group was statistically different from the model group on days 7, 13 and 17; the combination treatment groups were statistically different from the model group on days 5 to 19 (table 3). Area under the joint swelling score curve (AUC) from day 1 to day 14 is shown (FIG. 2, left), with significant decrease in AUC area (p < 0.01) for etanercept-6.7 mg/kg, p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg, and combination treatment groups, as compared to model groups, where the decrease in p75ECD-15mg/kg was more pronounced (p < 0.001) with the combination treatment group; area under the joint swelling score curve (AUC) from day 1 to day 28 is shown (fig. 2, right), with significant decrease in AUC area for etanercept-6.7 mg/kg, different doses of p75ECD, and combination treatment group (p < 0.01), with a more significant decrease in p75ECD-15mg/kg versus combination treatment group (p < 0.001) compared to the model group; meanwhile, compared with etanercept-6.7 mg/kg group or p75ECD-Fc-20mg/kg single drug treatment, the two combined treatment groups have more advantages in improving the limb joint swelling score. The above results suggest that different doses of p75ECD alone or in combination with etanercept can improve joint swelling in CIA-induced RA model animals, with the p75ECD-15mg/kg group being more effective than the combination treatment group.

2. Paw Withdrawal Threshold (PWT)

Results of measuring animal PWT are shown in table 4, fig. 3 and 4 show: on day 5, the animals of the model group had both left and right feet PWT lowered, the left foot PWT was raised by each of the other administration groups except the anti-proBDNF group, and the p75ECD-10mg/kg group and the p75ECD-15mg/kg group remained stable on days 1 to 5. In the detection of the PWT of the right foot of the animal, the PWT of each group except the anti-proBDNF group is improved compared with the model group. FIG. 5 shows that the area under the curve (AUC) on day 14 was significantly increased in the p75ECD-5mg/kg, p75ECD-10mg/kg, p75ECD-15mg/kg and combination treatment groups, and the p75ECD-10mg/kg and combination treatment groups were more significantly increased (p < 0.01) as compared to the model group; the area under the curve of the 19 th day of the p75ECD-10mg/kg group, the p75ECD-15mg/kg group and the combined treatment group is obviously increased, and the increase of the p75ECD-10mg/kg group is more obvious (p is less than 0.01); FIG. 6 shows that the area under the curve was significantly increased for each of the remaining groups, except for anti-proBDNF, for 14 days and 19 days, compared to the model group. The above results suggest that different doses of p75ECD alone or in combination with etanercept can improve CIA-induced inflammatory pain in RA model animals.

3. uCT detection

Fig. 7 and 8 are views of the overall view of the foot and ankle joint of the uCT examination. Compared with the normal group, the CT value (HU) and the bone density of the model group are obviously reduced (p < 0.001); compared with the model group, the CT values (HU) and the bone densities of the p75ECD-5mg/kg group, the p75ECD-10mg/kg group and the p75ECD-15mg/kg group are all obviously increased (p < 0.001); both the CT value (HU) and bone density were significantly increased (p < 0.05) in the p75ECD-20mg/kg group (Table 5, FIGS. 9-10).

Table 5: uCT detection (MEAN+ -SD)

Note that: one-way ANOVA p<0.05vs. model group; * P<Model group 0.001 vs; t-test, p=0.06 vs; ^### p<model group 0.001vs

4. Histopathological examination

Fig. 11-13 are images of the pathology of the left hind foot full sole, ankle joint and toe joint, respectively.

Table 6 shows paw synovial hyperplasia score, synovial inflammation score, cartilage injury score, bone injury score, and total injury score.

Table 6: paw injury score (MEAN+ -SD)

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Note that: one-way ANOVA 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.

Figure 14 shows that the model group was significantly elevated (p < 0.001) compared to the normal group in paw synovial hyperplasia score; after 4 drug treatments, methotrexate-2.5 mg/kg significantly reduced paw synovial hyperplasia scores (p < 0.05), p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg significantly reduced paw synovial hyperplasia scores (p <0.01, p <0.05, p < 0.01) compared to the model group.

Figure 15 shows that the model group was significantly elevated (p < 0.001) compared to the normal group in paw synovial inflammation scores; after 4 drug treatments, methotrexate-2.5 mg/kg significantly reduced paw synovitis score (p < 0.05), p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg scores were all significantly reduced (p <0.01, p <0.05, p < 0.01) compared to the model group.

Figure 16 shows that the model group was significantly elevated (p < 0.001) compared to the normal group in paw cartilage damage scores; following 4 drug treatments, methotrexate-2.5 mg/kg significantly reduced paw cartilage damage scores (p < 0.05), p75ECD-5mg/kg, p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg scores were all significantly reduced (p <0.05, p <0.001, p < 0.05) compared to the model group.

Figure 17 shows that the model group was significantly elevated (p < 0.001) compared to the normal group in paw bone injury score; following 4 drug treatments, methotrexate-2.5 mg/kg group significantly reduced paw bone injury scores (p <0.05, figure 17), p75ECD-5mg/kg, p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg group scores were all significantly reduced (p <0.05, p <0.001, p < 0.05) compared to the model group.

Figure 18 shows that model groups were significantly elevated (p < 0.001) over normal groups in paw injury total scores; meanwhile, after 4 drug treatments, the total score of the methotrexate-2.5 mg/kg group was significantly reduced (p <0.05, FIG. 18), p75ECD-5mg/kg, p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg group was significantly reduced (p <0.05, p <0.01, p < 0.001) compared to the model group.

5. Detection of proBDNF, p75NTR, pJAK1, pSTAT1 in spleen tissue (western blot)

Expression of proBDNF, p75NTR, pJAK1, pSTAT1 proteins in spleen tissue is shown in Table 7. FIG. 19 shows that the model group showed a significant increase in proBDNF expression (p < 0.01) compared to the normal group; each of the other groups showed a significant decrease in proBDNF (p < 0.05) except for the p75ECD-5mg/kg group, with a more pronounced decrease in p75ECD-15mg/kg group (p < 0.01) compared to the model group. Figure 20 shows that model group p75NTR expression was significantly elevated (p < 0.001) compared to normal group; each group had a significant decrease in p75NTR (p <0.05, p < 0.01) compared to the model group. Figure 21 shows that model group phosphorylated JAK1 is significantly elevated compared to normal group (p < 0.01); each group had a significant decrease in phosphorylated JAK1 (p < 0.05), with a more pronounced decrease in p75ECD-15mg/kg group (p < 0.01) compared to the model group. Fig. 22 shows that model group phosphorylated STAT1 is significantly increased (p < 0.05) compared to normal group; each group showed a significant decrease (p < 0.05) compared to the model group, with the combination treatment group showing a more pronounced decrease (p < 0.01).

Table 7: expression of proBDNF, p75NTR, pJAK1, pSTAT 1/protein in spleen tissue (MEAN.+ -. SEM)

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Note that: t-test, #p <0.05 vs; model group # p <0.01 vs; model group # # p <0.001vs

6. P75NTR, IL-1 beta, IL-6 and TNF-alpha mRNA expression levels in spleen tissue (qPCR)

Table 8 shows the expression levels of p75NTR, IL-1. Beta., IL-6 and TNF-. Alpha.mRNA in spleen tissue. The results show that: the expression level of p75NTR mRNA was significantly increased in spleen tissue of animals in the model group (p < 0.001) compared to the normal control group, and significantly decreased in spleen tissue of animals in each group compared to the model group, wherein the decrease in p75ECD-10mg/kg, p75ECD-15mg/kg, p75ECD-20mg/kg, and the combination treatment group was more pronounced (p < 0.001) (FIG. 23); the expression level of IL-1β mRNA in spleen tissues of animals in the model group was significantly increased (p < 0.001) compared to the normal control group, and the expression level of IL-1β mRNA in spleen tissues of animals in the other groups except the anti-proBDNF group was significantly decreased compared to the model group, wherein the decrease in each dose group and the combination treatment group of p75ECD was more significant (p < 0.001) (FIG. 24); IL-6mRNA expression levels were significantly increased in spleen tissues of animals in the model group (p < 0.001) compared to the normal control group, and were significantly decreased in spleen tissues of animals in each group compared to the model group, wherein the decrease in methotrexate-2.5 mg/kg group, etanercept-6.7 mg/kg group, p75ECD-15mg/kg group, and the combination treatment group was more pronounced (p < 0.001) (FIG. 25); the levels of TNF-. Alpha.mRNA expression in spleen tissue were significantly increased in the animals of the model group (p < 0.001) compared to the normal control group, and the levels of IL-6mRNA expression were significantly decreased in the spleen tissue of each group compared to the model group, in which methotrexate-2.5 mg/kg group, etanercept-6.7 mg/kg group, p75ECD-10mg/kg group, p75ECD-15mg/kg group, and the combination treatment group were significantly decreased (p < 0.001) (FIG. 26).

Table 8: p75NTR, IL-1 beta, IL-6 and TNF-alpha mRNA expression levels (MEAN+ -SD) in spleen tissue

Note that: t-test, #p < 0.05 vs; model group # p < 0.01 vs; model group with # # p < 0.001vs

7. IL-1 beta, IL-6 and TNF-alpha content in serum (ELISA)

IL-1 beta, IL-6 and TNF-alpha levels in serum are shown in Table 9. The results showed that the model group showed a significant increase in IL-1β, IL-6 and TNF- α compared to the normal control group, and that the serum IL-1β content was significantly decreased in each of the animals except the anti-proBDNF group compared to the model group, wherein the decrease in p75ECD-15mg/kg group was more pronounced (p < 0.001) (FIG. 27); compared to the model group, there was no significant difference in IL-6 content in serum of animals from the methotrexate-2.5 mg/kg, etanercept-6.7 mg/kg and anti-proBDNF groups, whereas IL-6 content was significantly reduced in serum of animals from the p75ECD groups and the combination treatment group (p < 0.05), wherein the reduction in p75ECD-15mg group was more pronounced (p < 0.01) (FIG. 28); compared with the model group, the serum TNF-alpha content of animals of each group was significantly reduced (p < 0.01, p < 0.001) except for the anti-proBDNF group (FIG. 29).

Table 9: inflammatory factor content (pg/ml) in serum (mean.+ -. SD)

Note that: t-test, #p < 0.05 vs; model group # p < 0.01 vs; model group with # # p < 0.001vs

The results show that by establishing a CIA model of a female Lewis rat, methotrexate can obviously reduce the paw injury score of the CIA model of the female Lewis rat at the dosage of 2.5mg/kg, and the CIA model of the female Lewis rat has obvious curative effect. Etanercept at a dose of 6.7mg/kg may decrease the limb joint swelling score (days 5-9) and increase the pain threshold (PWT) in female Lewis CIA model rats; the p75ECD has better drug effect in the weight, PWT, ankle joint volume and diameter and swelling scores in 4 dosage groups of 5mg/kg, 10mg/kg, 15mg/kg and 20mg/kg, can obviously reduce the paw injury score of a model rat in pathology, and exerts a good treatment effect, wherein the 15mg/kg curative effect is optimal, and the drug effect is better than that of methotrexate 2.5 mg/kg.

The foregoing description of the specific embodiments is provided as an aid in understanding the core concepts of the invention. It should be noted that it is possible for a person skilled in the art to make several improvements and modifications to the solution according to the invention without departing from the principle of the invention, but these improvements and modifications are also within the scope of the invention as claimed.

Reference is made to:

[1]Chuang M,Ketharnathan S.Rheumatoid Arthritis:The stride from research to clinical practice[J].Int J Mol Sci,2016,17(6):900-910.

[2] zhang Lingling, wei Wei pharmaceutical research for the treatment of autoimmune diseases [ J ]. Chinese pharmacological bulletins, 2019,35 (2): 149-156.

[3]Cansu DU,Teke HU,Bodakci E,et al.How should we manage low-dose methotrexate-induced pancytopenia in patients with rheumatoid arthritis？[J].Clin.Rheumatol,2018,37(12):3419-3425.

[4]Yang M,Feng X,Ding J,et al.Nanotherapeutics relieve rheumatoid arthritis[J].J Control Release,2017,252:108-124.

[5] Wu Weiyong and Pang Xuefeng clinical treatment of rheumatoid arthritis, pharmaceutical community, 2020,9 (17): 43.

[6]Luo,C.,Zhong,X.L.,Zhou,F.H.,Li,J.Y.,Zhou,P.,Xu,J.M.,Song,B.,Li,C.Q.,Zhou,X.F.,and Dai,R.P.(2016)Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator.Scientific reports,2016,6,27171.

[7]Chaplan,S.R.,et al.Quantitative assessment of tactile allodynia in the rat paw.J Neurosci Methods,1994,53(1):55-63.

[8]Chun-Rui Yang,Hong-Jun Ding,Miao Yu,et al.proBDNF/p75NTR promotes rheumatoid arthritis and inflammatory response by activating proinflammatory cytokines.The FASEB J,2022,DOI:10.1096/fj/202101558R.

Claims (14)

1. Use of the p75 extracellular domain (p 75 ECD) or a functional fragment, variant, analogue or derivative thereof for the manufacture of a medicament for the treatment and/or prophylaxis of Rheumatoid Arthritis (RA).
2. 2. The use according to claim 1, wherein the p75ECD consists of the amino acid sequence set forth in SEQ ID No. 2 or a variant thereof.
3. 3. The use of claim 1, wherein the p75ECD is fused to an Fc domain of an immunoglobulin to form a p75ECD-Fc fusion, wherein the p75ECD-Fc consists of the amino acid sequence set forth in SEQ ID No. 4 or a variant thereof.
4. 4. The use of claim 3, wherein the p75ECD-Fc fusion is administered in the form of a p75ECD-Fc fusion adeno-associated virus (AAV) vector.
5. 5. The use of claim 3, wherein the p75ECD-Fc fusion is a recombinant peptide.
6. 6. The use of claim 1, wherein the p75ECD is administered by intravenous, intraperitoneal, intraventricular, intrathecal or intramuscular injection.
7. 7. The use of claim 4, wherein the p75ECD-Fc fusion AAV vector is at 1 x 10 ⁶ To 11X 10 ¹³ The dose between individual viral genes/kg body weight of the subject.
8. 8. The use of claim 4, wherein the p75ECD-Fc fusion AAV vector is at 1 x 10 ⁸ To 11X 10 ¹³ The dose between individual viral genes/kg body weight of the subject.
9. 9. The use of claim 5, wherein the p75ECD-Fc fusion recombinant peptide is administered at a dose between 0.1mg/kg subject body weight and 250mg/kg subject body weight.
10. 10. The use of claim 1, wherein the p75ECD is used separately, sequentially or simultaneously in combination with a second pharmacologically active compound.
11. 11. The use of claim 10, wherein the second pharmacological compound is selected from the group consisting of: opioid analgesics; a non-steroidal anti-inflammatory drug; steroid hormones; anti-rheumatoid drugs (DMARDs) to ameliorate disease; tumor necrosis factor alpha inhibitors; b cell inhibitors; t cell inhibitors; a cytokine inhibitor; a cytokine inhibitor; JAK1/JAK2 inhibitors; granulocyte-phagocyte colony stimulating factor (GM-CSF) receptor blockers.
12. 12. The use of claim 11, wherein the opioid analgesic is selected from the group consisting of morphine, heroin, hydromorphone, oxymorphone, levorphanol, methadone, pethidine, fentanyl, ***e, codeine, dihydrocodeine, oxycodone, hydrocodone, dextropropoxyphen, nalmefene, allylmorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine, or pentazocine;

    The non-steroidal anti-inflammatory drug is selected from aspirin, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, flubensal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, niflumiprofen, oxalazine, phenylbutazone, piroxicam, sulindac, tolmetin, or zomepirac acid;

    the disease modifying antirheumatic drugs (DMARDs) are selected from sulfasalazine, D-penicillamine, methotrexate, leflunomide, azathioprine, cyclosporine or hydroxychloroquine;

    the tumor necrosis factor alpha inhibitor is selected from infliximab, adalimumab, etanercept, golimumab or cetozagrel;

    the B cell inhibitor is rituximab or belimumab;

    the T cell inhibitor is selected from the group consisting of abacavir;

    the cytokine inhibitor is selected from tolizumab, western Lu Kum, cladazazumab, oxuzumab, sha Lilu mab or anakinra;

    the JAK1/JAK2 inhibitor is selected from the group consisting of baroretinib;

    the granulocyte-phagocyte colony-stimulating factor (GM-CSF) receptor blocker is selected from Ma Weili monoclonal antibodies.
13. 13. The use of claim 12, wherein the tumor necrosis factor alpha inhibitor is selected from etanercept.
14. 14. The use of claim 1, wherein the subject for which the medicament is for the treatment and/or prophylaxis of RA is a human.