CN113577289A

CN113577289A - Application of RXFP1/3 inhibitor in preparation of medicine for preventing or treating adolescent idiopathic scoliosis

Info

Publication number: CN113577289A
Application number: CN202111086176.XA
Authority: CN
Inventors: 张莉
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-01-11
Filing date: 2021-01-11
Publication date: 2021-11-02
Also published as: CN112618719A; WO2022147897A1; CN112618719B

Abstract

The application discloses that the level of RLN3(Relaxin 3) in serum of an AIS patient is obviously higher than that of a normal control, and the incidence rate of scoliosis of a mouse animal model can be obviously changed by continuously injecting RLN3 or administering RXFP3(Relaxin Family Peptide Receptor 3) inhibitor, which indicates that RLN3 plays an important role in the incidence of AIS. Thus, the present application provides the use of an RXFP1/3 inhibitor for the manufacture of a medicament for the prevention and/or treatment of adolescent idiopathic scoliosis, as well as a pharmaceutical composition comprising an RXFP1/3 inhibitor for the prevention and/or treatment of adolescent idiopathic scoliosis, the use of an agent for detecting a level of RLN3 for the manufacture of a diagnostic agent for diagnosing a level of adolescent idiopathic scoliosis in a subject and a kit for diagnosing adolescent idiopathic scoliosis or a risk thereof.

Description

Application of RXFP1/3 inhibitor in preparation of medicine for preventing or treating adolescent idiopathic scoliosis

The application is a divisional application of Chinese patent application No. 202110029072.9, which has an application date of 2021, 1, 11 and is named as application of an RXFP1/3 inhibitor in preparing a medicament for preventing or treating adolescent idiopathic scoliosis.

Technical Field

The invention belongs to the field of biotechnology. More specifically, the present invention relates to methods and agents for the prevention and treatment of adolescent idiopathic scoliosis.

Background

Adolescent Idiopathic Scoliosis (AIS) is an important clinical disease, and the molecular regulatory mechanisms of its occurrence and development are currently receiving extensive attention: scoliosis is an ancient disease. The scoliosis is lateral curvature, measured by the Cobb method over 10 degrees, and excluding other secondary causes (e.g. congenital, degenerative, neuromuscular, etc.), called idiopathic scoliosis, with scoliosis occurring most frequently in adolescent age, called adolescent idiopathic scoliosis. Continuously progressing AIS patients are often accompanied by decreased lung function and persistent low back pain, even secondary cardiopulmonary dysfunction, requiring surgery to correct deformity. Scoliosis surgery is one of the more complex, risky, and most serious complications of spinal surgery, and is expensive. In view of the huge population base of China, the AIS has huge number of diseases, and heavy economic burden is brought to patients, families and society. The molecular control mechanism of the occurrence and development of AIS is an important scientific problem (Kikanloo SR, Tarpada SP, Cho W. ethology of advanced Idiopathic Sciolosis: A Literrure review. Asian Spine J.2019; 13(3):519-26.), the occurrence and development of which is currently considered to be a disease (Sun Pear, Lezong, He, Yang. finite element analysis in the study of Scoliosis biomechanics and progress [ J ]. Chinese tissue engineering study, 2019,23(32): ZongY, Zhigan Y, Qiangulin Z, Jingfeng L, Kai C, Yinguman Z, ChuanW, Dewei H, Xiadong Z, Ming L.522 Idention of translation Z, J.18. simulation of the same, J.18 J., jian Z, Haijian N, Changwei Y and Ming L.Positive negative correction in Lenke 1and 2AIS substrates after correction surgery prediction: a novel predictive index. BMC multisystem Disorders,2019,20: 405).

The etiology of AIS is not clear, and researchers have proposed many hypotheses such as the hypothesis of genetic abnormality, the hypothesis of neuroendocrine abnormality, the hypothesis of bone osteogenesis abnormality, the hypothesis of bone growth imbalance, etc., but none of the above hypotheses can completely explain the etiology of AIS (Kikanlooo SR, Tarpada SP, Cho W. ethology of Adolescent idiophatic Sciosis: A Literature review. Asian Spine J.2019; 13(3): 519-26.).

In addition to surgical correction of deformities, AIS therapy currently includes medical device therapy, for example, chinese patent application CN111053657A discloses a new scoliosis traction wheelchair, and CN111035495A discloses a walking scoliosis traction device. Research teams in Beijing coordination and hospitals found that Mesenchymal Stem Cells (MSC) of bone marrow of AIS patients show reduced osteogenic differentiation capacity, and proposed that NF90 (one of the most major protein isomers of interleukin enhancer binding factor 3 protein family) is used for preparing a biological preparation for regulating osteogenic differentiation of mesenchymal stem cells of bone marrow, and the application of NF90 in preparing a pharmaceutical composition for preventing and/or treating adolescent idiopathic scoliosis (CN 109568565A). Due to the high cost of surgery and the high risk and side effects, there remains an urgent need in the art for agents that can prevent, treat and prevent the exacerbation of AIS.

Disclosure of Invention

The applicant finds that the level of RLN3(Relaxin 3) in serum of AIS patients is significantly higher than that of normal controls, and the incidence of scoliosis in mouse animal models can be significantly changed by continuously injecting RLN3 or administering RXFP3(Relaxin Family Peptide Receptor 3) inhibitor, suggesting that RLN3 plays an important role in the incidence of AIS. The applicant determined the role of RLN3-RXFP1/3-ERK1/2 signaling axis in promoting the onset of AIS in spinal ligament fibroblasts. The applicant has thus proposed that the prevention or treatment of the occurrence and development of AIS can be achieved by inhibitors of the relevant molecules in the RLN3-RXFP1/3-ERK1/2 signaling axis, in particular RXFP1/3 inhibitors, thus completing the present invention.

Specifically, the applicant has achieved the present invention by the following means.

1. An application of RXFP1/3 inhibitor in preparing the medicines for preventing and/or treating adolescent idiopathic scoliosis is disclosed.

2. The use of item 1, wherein the RXFP3 inhibitor has an apparent equilibrium constant (pKi) of <9, preferably 5 to 9, for competitive binding to RXFP 3in an assay using the method described by Linda m.

3. The use of any of items 1 or 2, wherein the inhibitor is a small molecule compound such as a peptide, and an antibody or aptamer.

4. The use of any one of items 1 or 2, wherein the RXFP3 inhibitor is selected from R3, R3/I5, R3(B Δ 23-27) R/I5, R3(B1-22R) (or R3B 1-22R), R3B 1-22R acid, R3B 1-22R C10/22A, R3B 2-22R, R3B 3-22R, R3B 4-22R, R3B 5-22R, R3B 6-22R, R3B 1-22R dimer, INSL5, and a minimal relaxin 3-analog 3, preferably R3(B1-22) R, or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition for preventing and/or treating adolescent idiopathic scoliosis comprising an inhibitor of RXFP1/3 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of item 5, wherein the RXFP3 inhibitor has an apparent equilibrium constant (pKi) of <9, preferably 5 to 9, for competitive binding with RXFP 3in an assay using the method described by Linda m.

7. The pharmaceutical composition of clauses 5 or 6, wherein the inhibitor is a small molecule compound such as a peptide, and an antibody or aptamer.

8. The pharmaceutical composition of clauses 5 or 6, wherein the RXFP3 inhibitor is selected from R3, R3/I5, R3(B Δ 23-27) R/I5, R3(B1-22R) (or R3B 1-22R), R3B 1-22R acid, R3B 1-22R C10/22A, R3B 2-22R, R3B 3-22R, R3B 4-22R, R3B 5-22R, R3B 6-22R, R3B 1-22R dimer, INSL5, and a minimal relaxin 3-analog 3, preferably R3(B1-22) R, or a pharmaceutically acceptable salt thereof.

9. Use of an agent for detecting the level of RLN 3in the manufacture of a diagnostic agent for diagnosing the level of adolescent idiopathic scoliosis in a subject, wherein the diagnostic agent is for determining the level of RLN 3in a sample from the subject, wherein a level of RLN3 that is higher than the level of RLN 3in a sample from a healthy control group indicates that the subject has, or is at risk of having, adolescent idiopathic scoliosis.

10. The use of item 9, wherein the reagent for detecting the level of RLN3 is selected from the group consisting of a reagent for determining the level of RLN3 protein, such as an anti-RLN 3 antibody, a reagent for determining the level of mRNA encoding RLN3, such as a primer or a probe, and a nucleic acid, such as an aptamer, that specifically binds to RLN3 protein.

11. A kit for diagnosing adolescent idiopathic scoliosis or a risk thereof, wherein the kit comprises a diagnostic reagent for detecting a level of RLN 3.

12. The kit of item 11, wherein the reagent for detecting the level of RLN3 is selected from the group consisting of a reagent for determining the level of RLN3 protein, such as an anti-RLN 3 antibody, a reagent for determining the level of mRNA encoding RLN3, such as a primer or a probe, and a nucleic acid, such as an aptamer, that specifically binds to RLN3 protein.

As used herein, the term "individual", "subject" or "patient" includes, but is not limited to, humans and other primates (e.g., chimpanzees and other apes and monkey species). In some embodiments, the subject or patient is a human.

As used herein, "pharmaceutically acceptable" means that it avoids substantial toxic effects when used in conventional amounts for medical administration and is capable of being, or has been, approved by a government or regulatory agency licensed or listed in pharmacopeia or generally known pharmacopeia for use in animals, and more particularly in humans.

The pharmaceutically acceptable carriers described herein include various excipients, diluents and adjuvants that are not associated with significant side effects in pharmaceutical use, including but not limited to: purified water, physiological saline, buffer, glucose, water, glycerol, mannitol, ethanol, surfactants and salts such as sodium chloride, sodium EDTA and the like.

The term "pharmaceutically acceptable salt" as used herein refers to salts well known in the art and includes, but is not limited to: (1) salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or from compounds such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, acid addition salts with organic acids such as 1, 2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2,2,2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and hexadiene diacid; or (2) salts formed when an acidic proton present in the parent compound is substituted.

The RXFP1/3 inhibitors described herein represent inhibitors of RXFP1 and/or RXFP3 that are capable of binding to RXFP1 and/or RXFP3 and blocking the agonistic effects of relaxin 3(RLN3) on RXFP1 and/or RXFP 3. The RXFP1/3 inhibitors described herein include antagonists of RXFP 1/3. In one embodiment, the RXFP1/3 inhibitors described herein bind only to RXFP1 and/or RXFP3 but not to RXFP2 and/or RXFP 4.

The inventor of the application finds that the incidence rate of scoliosis in an experimental group is significantly smaller than that in a control group by blocking the binding of RXFP1 or RXFP3 and relaxin 3(RLN3) by a receptor RXFP1 or RXFP3 inhibitor in an animal experiment, and proposes that the occurrence and development of AIS can be prevented or treated by the application of an inhibitor of related molecules in an RLN3-RXFP1/3-ERK1/2 signal axis, particularly an inhibitor of RXFP1 or RXFP3(RXFP 1/3). In one embodiment, the RXFP3 inhibitor herein is an inhibitor that has an apparent equilibrium constant (pKi) of <9, preferably 5 to 9, for competitive binding to RXFP3 as determined using the method described by Linda m. In one embodiment, the RXFP3 inhibitors (or antagonists) herein include, but are not limited to, R3, R3/I5, R3(B Δ 23-27) R/I5, R3(B1-22R) (or R3B 1-22R), R1B 1-22R acid, R1B 1-22 1/22A, R1B 1-22R, R1B 1-22R, R1B 1-22R, R1B 1-22R, R1B 1-22R, R1B 1-22R dimer, INSL 1, minimized Relaxin 3-analogue 3 (miniaturized Relaxin-3 analog 1, etc.) (see Linda M.ugard-Kedstrom et al, Design, Synthesis, science of culture of Changewho Chemical engineering of culture of biological products 3665, Journal of Chemical engineering of biological FP 67, Journal of culture of the family of Chemical engineering, see FIGS. In a preferred embodiment of the invention, the RXFP3 inhibitor is R3(B1-22) R (the formula is Arg-Ala-Ala-Pro-Tyr-Gly-Val-Arg-Leu-Ser-Gly-Arg-Glu-Phe-Ile-Arg-Ala-Val-Ile-Phe-Thr-Ser-Arg-NH 2). Methods of screening for inhibitors of RXFP1 and/or RXFP3 are known in the art (see, e.g., Michelle L, Halls et al, International Union of Basic and Clinical Pharmacology.XCV.Recent Advances in the Understanding of the interpretation of the Pharmacology and Biological rules of relax Family Peptide Receptors 1-4, the Receptives for relax Family Peptides, Pharmacology Rev 67: 389-.

In some embodiments, the inhibitor of RXFP1 or RXFP3 of the invention is an antibody against RXFP1 or RXFP 3. The antibodies described herein may be monoclonal or polyclonal, preferably monoclonal. In one embodiment, the antibodies used in the present invention are monoclonal antibodies, including chimeric, humanized or human antibodies. In one embodiment, the antibody used in the invention is an antibody fragment, e.g., Fv, Fab, Fab ', scFv, diabody or F (ab')₂And (3) fragment. In another embodiment, the antibody is a full length antibody, e.g., an intact IgG1, IgG2, IgG3, or IgG4 antibody or other antibody class or isotype as defined herein. In some embodiments, the antibodies of the invention specifically bind to RXFP1 and/or RXFP3 and block the agonistic effect of blocking relaxin 3(RLN3) on RXFP1 and/or RXFP 3. The antibodies of the invention can be prepared by techniques well known in the art, such as hybridoma techniques and the like.

The pharmaceutical compositions of the present invention may include classical pharmaceutical formulations. The pharmaceutical composition according to the present invention may be administered by any conventional route as long as the target tissue is available by the route, for example, intravenously, intralesionally, intrathecally, intradermally, subcutaneously, intramuscularly, orally, and the like.

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, eliminating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis.

In some embodiments, the RXFP1 or RXFP3 inhibitors of the invention are aptamers. As used herein, "Aptamer" refers to a nucleic acid Aptamer (Aptamer), also called Aptamer, etc., which is a single-stranded RNA molecule or single-stranded DNA molecule, usually consisting of several tens to a hundred or more bases, and is a single-stranded oligonucleotide that can bind to a target substance with high specificity and high sensitivity, which is selected and isolated from a random library of artificially synthesized RNA or DNA molecules. While aptamers bind in a manner similar to antibodies, the unique properties of aptamers are superior to antibodies in many respects. Compared with the antibody, the aptamer can be obtained by in vitro screening, and has obvious advantages in the aspects of production cost, stability, modification difficulty, tissue penetrating power and the like. In general, aptamers are screened by the exponential enrichment of ligands by exponential evolution (SELEX). Taking a DNA aptamer as an example, SELEX basically comprises incubating a random library solution of DNA molecules containing a large-capacity random oligonucleotide sequence with a target molecule, separating the DNA molecules bound to the target molecule from the unbound DNA molecules by various separation means, amplifying the DNA molecules capable of binding by polymerase chain reaction, separating into single strands, generating a secondary library, and then performing the next round of screening, repeating the above rounds, selecting the enriched DNA molecules, and sequencing to obtain oligonucleotide molecules specifically recognizing the target molecule, i.e., nucleic acid aptamers. The large-capacity molecular library can cover various three-dimensional conformations, and theoretically can be screened aiming at any target molecule to obtain the aptamer specifically identified by the target molecule. In some embodiments, the aptamers of the invention are capable of specifically binding to RXFP1 and/or RXFP3 and blocking the agonistic effect of relaxin 3(RLN3) on RXFP1 and/or RXFP 3.

In some embodiments, the RXFP1 or RXFP3 inhibitors of the invention are small molecule compound inhibitors, such as peptides.

In some embodiments of the invention, the pharmaceutical composition further optionally comprises one or more other agents effective in treating AIS, which agents are well known to those skilled in the art. The pharmaceutical composition of the present invention may be administered in combination with other therapeutic means for the prevention and/or treatment of AIS.

The amount of RXFP1 or RXFP3 inhibitor for use in the pharmaceutical composition is an effective amount for the prevention and/or treatment of AIS. The effective amount can be determined by one skilled in the art according to methods well known in the art.

The pharmaceutical compositions of the invention may be administered to a subject, e.g., a human, in a suitable dosage.

Drawings

FIG. 1: serum Relaxin (RLN) levels in AIS patients were measured and suggested that serum RLN3 levels were significantly elevated in AIS patients. a-C, plasma RLN1, RLN2, and RLN3 levels were tested between AIS and control groups. D, AIS group joint mobility and RLN3 serological level correlation analysis.

FIG. 2 shows that the serologic level of RLN3 is obviously increased in a C57BL/6J mouse scoliosis animal model. A, X-ray examination picture of C57BL/6J mouse scoliosis animal model. B, three-dimensional CT examination picture of C57BL/6J mouse scoliosis animal model. C. Serological RLN3 levels in scoliotic mice (n ═ 67) and normal mice (n ═ 33) in a C57BL/6J mouse scoliosis animal model.

FIG. 3 shows that the activation of ligament fibroblast of AIS patients is obviously inhibited compared with the normal control; RLN3 inhibits the activation of human spinal ligament fibroblasts through RXFP3 receptor, and simultaneously promotes the expression of MMP2 and MMP9 of the human spinal ligament fibroblasts. A. Immunofluorescence images of α -SMA after TGF- β 1(10ng/mL) and RLN3(100ng/mL) treatment of human spinal ligament fibroblasts. B. CCK-8 experiments demonstrated that RLN3(100ng/mL) significantly inhibited the activation of TGF-beta 1(10ng/mL) on human spinal ligament fibroblasts,. p < 0.01. C. Hydroxyproline content of ligament fibroblasts after TGF β 1and RLN3 treatment. D-E, Western blot analysis confirmed that the expression of spinal ligament fibroblasts MMP2, MMP9 and RXFP3 is significantly increased after RLN3 treatment.

FIG. 4RLN3 elevates ERK1/2 related signal pathway levels through RXFP3, inhibiting TGF- β/SMAD2 signal pathways. A-B: effects of RLN3 on human spinal ligament fibroblast ERK 1/2-associated signaling pathways and TGF- β/SMAD-associated signaling pathways. Western Blot analysis confirmed that RLN3 promotes ERK1/2 phosphorylation and nNOS expression, and reduces phosphorylation of SMAD2 protein. Effect of C-D, R3(B1-22) R (RXFP3 receptor antagonist) on human spinal ligament fibroblast signaling pathway. Western-blot detection proves that R3(B1-22) R can obviously inhibit the activation of RLN3 on ERK1/2 and nNOS related signal channels.

FIG. 5: r3(B1-22) R can significantly reduce the incidence of scoliosis in a scoliosis mouse animal model. X-ray and CT images of C57BL/6J mouse scoliosis animal model after A-B, R3(B1-22) R treatment. C. R3(B1-22) changes in the incidence of scoliosis in R-treated C57BL/6J mouse scoliosis animal models.

Detailed Description

Relationship between AIS and ligament laxity

It has been shown that AIS is closely related to ligament laxity: (51%) of AIS patients with a 2-fold (21%) incidence of overactivity of the joints as compared to normal controls (Dariusz C, Tomasz K, Paulina P, et al. Joint hypermobility in the chip with interferometric surgery: SORT aware 2011 with [ J ]. Scoliosis,2011,6(1):22), several studies further support the results of this study (Hengwei F, Yang H, Qifei W, Weiqing T, Nali D, Ping Y, Junlin Y. Prevalance of interferometric surgery in chip schoolknowledge: a, position-base study. spine (Phila Pa 6), 2016,41(3): 259-64); (25%) 10 times higher than normal (1-2%) (Bozkurt S, Kayalar G, Tezel N, et al hypermobility Frequency in School Children: Relationship With Idiopathic Scolinis, Age, Sex and Musuloseltal schemes [ J ]. Arch Rheumatol,2019,34: 268. 273), With joint mobility higher than about 70% normal, two subsequent studies suggest that the incidence of AIS in ballet is even 27% and 30%, respectively (Longworth B, far R, Home D.prediction and precursors of surgery genetic diagnosis in adolescent tablets [ J ]. Phys et 95, physiology 1725: 20145); (iii) AIS incidence for athletes in prosodic operation is 27% (Tanchev, Panayot I, Dzherov, Assen D, Parushev, Anton D, et al. Scoliosis in Rhythmic Gymnasts [ J ] Spine,25(11): 1367-; (iv) clinical features of significant joint and arch standing collapse in AIS patients (white LF. adolescent idiophatic coliosis: The thermal Spine III: Is facial spiral The key; connective tissue abnormality-related diseases such as Marfan syndrome and Ehlers-Danlos syndrome spinal deformity are similar to the flexural abnormality of AIS (Giunda C, Baumann M, Fauth C, et al. A. co. of 17 tissues with Kyphoscolitic Ehlers-Danlos syndrome FKBP14: expansion of the clinical and pathological spectra and description of the natural history: [ J ] Genetics in Medicine,2018,20(1): 42-54). The Peter Newton of the pediatric medical center of san Diego, USA, found that AIS could be significantly corrected by anterior surgical tethering, suggesting that anterior Spinal ligament laxity may be closely related to AIS pathogenesis (Newton PO, Bartley CE, Bastrom TP, et al.

Relaxin

Relaxin (RLN) is a neuropeptide, a member of the insulin/Relaxin superfamily, widely distributedWherein human RLN comprises 7 members, most commonly RLN1, RLN2, RLN3, etc. (Kaftanovskaya EM, Ng HH, Soula M, et al]FASEB J., 2019, 33: 12435-12446). The biological function of the RLN1 molecule is unknown in humans and primates. RLN2 molecule is one of the earliest discovered members of the insulin/relaxin superfamily, with a molecular weight of 6KDa, secreted mainly by ovarian and other sexual organs in a pulsed manner, closely related to the change of estrogen level, and capable of relaxing pelvic ligament in lower rodents by anti-fibrotic action on fibroblasts to promote and protect the childbirth process (Unnemori E.Serelaxin in clinical severity: past, present and future) [ J]Br.J. Pharmacol, 2017,174: 921-932). Research shows that RLN2 can play an anti-fibrosis role in various organs, and is a very promising anti-fibrosis drug (Summers RJ, Recent progress in the understating of drainage family peptides and the same receptors) [ J]Br.J.Pharmacol, 2017,174: 915-. RLN3 has a molecular weight of 5kDa, comprises 2 peptide chains and 3 disulfide bonds, is secreted by cerebellar brainstem neuronuclear cells, is highly conserved among species, and is an ancestral molecule of RLN3, which is considered to be a member of the insulin/relaxin superfamily, in almost all vertebrates. RLN3 plays an important role in arousing, stimulating appetite, regulating stress and cognition, reducing anxiety and depression, regulating biological rhythms and other physiological processes (de)

C,Chometton S,Ma S,Pedersen LT,Timofeeva E,Cifani C,Gundlach AL.Effects of chronic silencing of relaxin-3production in nucleus incertus neurons on food intake,body weight,anxiety-like behaviour and limbic brain activity in female rats.Psychopharmacology(Berl).2020Jan 3.doi:10.1007/s00213-019-05439-1.[Epub ahead of print]) (ii) a It was found that RLN3 can also act on ligament fibroblasts to exert anti-fibrotic effects (Zhang X, Fu Y, Li H, Shen L, Chang Q, Pan L, Hong S, Yin X. H3relaxin inhibitors the collagen synthesis via ROS-and P2X7R-mediated NLRP3 inflamasome activation in cardiac fibrosis unit highucose.J Cell Mol Med.2018,22(3):1816 and 1825). RLN3 has an anti-fibrotic effect in diabetic cardiomyopathy rats (Zhang X, Pan L, Yang K, Fu Y, Liu Y, Chi J, Zhang X, Hong S, Ma X, Yin X. H3relaxin pro-technologies against myocardial inner surfaces in experimental cardiac muscular by inhibiting myocardial apoptosis, fibrosis and inflammation. cell physiology biochem.2017,43(4): 1311-. RLN3 also inhibited TGF-. beta.1-induced fibrosis of rat ventricular fibroblasts, myofibroblast differentiation and collagen deposition.

RXFP

The Receptor of RLN3 is a Relaxin Family Peptide Receptor (RXFP), including RXFP1, 3, 4. Although RXFP3 coupled to the G protein has the strongest affinity for RLN 3in each receptor (Ma S, Smith C M, Blasik A, et al distribution, physiology and pharmacology of relax-3/RXFP 3systems in blain [ J ]. British joural of pharmacology,2017,174(10):1034-1048), several studies suggest that RLN3 may also interfere with the normal fibrotic process of ligament tissue by inhibiting the differentiation of ligament fibroblasts into myofibroblasts via the RXFP1 downstream signaling pathway.

The effects and correlations of RLN in AIS have not been reported at present. The inventor tests the serum RLN1, RLN2 and RLN3 levels of AIS patients in earlier studies, and finds that the serum RLN3 level of the AIS patients is obviously higher than that of a normal control group, which indicates that RLN3 is related to the occurrence of AIS. The inventors speculate that RLN3 induces the pathogenesis of AIS by inhibiting fibrosis of fibroblasts in the perispinal ligaments, including reducing fibroblast proliferation, differentiation into myofibroblasts, deposition of collagen, etc., which in turn alters the properties of the ligaments. The inventor carries out the activity of receptor RXFP1 or RXFP3 of RLN3 for inhibiting RXFP1 or RXFP3 by using the inhibitor thereof in animal experiments, finds that the incidence rate of scoliosis in experimental groups is obviously lower than that of control groups, and proposes that the occurrence and development of AIS can be prevented or treated by the inhibitor of related molecules in RLN3-RXFP1/3-ERK1/2 signal axes, particularly RXFP1 and/or RXFP3(RXFP1/3) inhibitors. In one embodiment of the invention, the RXFP3 inhibitor is R3(B1-22) R.

Examples

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages are mass/volume percentages, parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1 determination of relaxin levels in AIS patients

Grouping of clinical Subjects

The study subjects were female AIS patients at the spinal surgery outpatient and institutional department of long-sea hospitals from 2016 to 2019, 4 months. AIS diagnosis was based on clinical history and X-ray examination. Non-idiopathic scoliosis such as connective tissue diseases (Marfan syndrome, Ehless-Danlos syndrome, etc.) is excluded. Supraspinal ligament tissue is obtained in AIS patients with posterior orthopedic internal fixation. The AIS patient serum specimen is collected from AIS patients in the department of living and outpatient clinics for braces or surgical treatment. Age-matched female patients with lumbar disc herniation (no scoliosis) who were seen at the outpatient clinic of spinal surgery in the Changhai hospital served as a control group. There were no significant statistical differences in body mass index, orgasmic age or Risser classification between the two groups. Table 1 summarizes the clinical characteristics of the subjects.

TABLE 1 summary of clinical characteristics of the subjects involved in the study

BMI, body mass index, VAS, visual pain score, CRP, C-reactive protein, ESR, blood sedimentation, JHM, joint mobility

The study was conducted according to the principles of the declaration of helsinki and was approved by the medical ethics committee of the shanghai long-sea hospital. Written informed consent was obtained from all participants prior to enrollment.

The experimental method comprises the following steps:

to determine the role of relaxin in AIS pathogenesis, we compared the serological levels of relaxin-1 (RLN1), relaxin-2 (RLN2) and relaxin-3 (RLN3) of AIS patients (n 30) and healthy controls (n 30). Patients with AIS were physically examined and their HJM joint activity scores were unequivocally determined. Determining whether there is a statistical difference between AIS and three RLN levels of a control group; it was clear whether there was a statistical correlation between AIS patient HJM activity score and RLN3 serological levels.

The experimental steps are as follows:

1. 10ml of venous blood was collected at 2 weeks after menstruation from AIS patients and normal control patients who met the selection criteria. The supernatant was centrifuged (horizontal/angular rotation high-speed refrigerated centrifuge Eppendorf 5810R,1000 rpm, 5 min).

2. Serum samples were analyzed for their serum levels by ELISA assay RLN1(EK1953, Human CHRDL1 ELISA Kit, Boster Biological Technology Co.), RLN2(EK1428, Human RLN2 ELISA Kit, Boster Biological Technology Co.), RLN3(EK1407Human Relaxin 3ELISA Kit, Boster Biological Technology Co), dual calibration curves, 3 replicate wells.

3. AIS patients were scored for joint mobility by the HJM scoring scale.

4. One-way anova was used to test whether there was a statistical difference in the serological levels of RLN1, RLN2, RLN 3in the AIS group and the control group. Correlation of HJM scores with RLN3 serological levels was determined by Pearson test. If there is a correlation, further linear correlation curve fitting is performed.

The experimental results are as follows:

the one-way anova results suggested no significant statistical difference between AIS and control groups in RLN1(76.69 ± 50.05ng/L vs.82.29 ± 44.13 ng/L; p ═ 0.696) or RLN2(9.72 ± 11.08ng/L vs.8.40 ± 7.71 ng/L; p ═ 0.649) (fig. 1A and 1B). However, we found that the serological level of RLN 3in the AIS group was significantly higher than that in the normal control group (84.94. + -. 64.48ng/L vs. 29.42. + -. 14.27 ng/L; p <0.001) (FIG. 1C). Meanwhile, the serum level of RLN3 of AIS patients is found to be positively correlated with the joint mobility score (JHM) (p is 0.004). Indicating that elevated levels of RLN3 may cause ligament relaxation. The correlation of RLN3 with ligament relaxation was further confirmed as shown by a linear correlation curve fit (fig. 1D).

Example 2 determination of relaxin levels in AIS scoliosis mouse animal models

1. Preparation of scoliosis mouse animal model

As reported in the literature (Machida, M.et al. Experimental sciences in a mammalian-specific C57BL/6J mice with a pinout molecular. journal of Pineal Research 41,1-7(2010)), C57BL/6J mice can be used to create a mouse scoliosis animal model (incidence rate 60-90%) by double forelimb excision without Pineal excision. Three weeks old C57BL/6J was obtained from Charles River laboratories (n ═ 100) and group housed in air-conditioning rooms (22 ± 2 ℃) with controlled lighting function (lighting starting at 07:00 to 19: 00). During the 4 months of modeling, the forelimb-removed mice were placed in purpose-built cages, with progressively increasing food and water placement heights to keep the mice from having to maintain a standing position to eat. Scoliosis can be diagnosed by X-ray examination of the anterior-posterior spine of mice with a Cobb angle >10 °. The degree of rotation of the vertebral body and the degree of rib deformity were assessed by spiral 3D-CT.

The second department of medical animal research, university of military medicine, approved the work of animals in this study. The experimental protocol was performed according to the guidelines for care and use of laboratory animals established by the national institutes of health.

Determination of relaxin 3(RLN3) levels in scoliosis mouse models and control mice

The relaxin3 level determination procedure was as follows:

the experimental steps are as follows:

1. c57BL/6J mouse animal model 3 weeks old was introduced and adapted to 1 week, 4 weeks old for removal of forelimb surgery.

2. An orbital bleed was performed in a 4-month-old C57BL/6J mouse animal model under 0.5% chloral hydrate anesthesia, and the blood was centrifuged at 1000R/min (horizontal/angular rotation high-speed refrigerated centrifuge Eppendorf 5810R) for 5min to obtain a supernatant.

3. Serum samples were analyzed for their serum levels of RLN3 by ELISA assay (Relaxin-3 kit SAB EK1460), single standard curve, double replicate wells.

4. And performing anteroposterior and lateral X-ray film examination and three-dimensional CT examination under the anesthesia state. And statistics are carried out on the incidence rate of the scoliosis.

3. One-way anova was used to test whether there was a statistical difference in the serological levels of RLN3 between the scoliotic and normal groups.

The experimental results are as follows:

examination of 4-month-old C57BL/6J mice by X-ray and three-dimensional CT reconstruction revealed that 67% (67/100) of the C57BL/6J mice developed scoliosis (FIGS. 2A and 2B). ELISA testing suggested that the serum RLN3 levels (8.9. + -. 0.7 ng/mL;) were significantly elevated (p <0.001) in scoliotic mice compared to normal C57BL/6J mice (7.0. + -. 0.6ng/mL) (FIG. 2C). It was further demonstrated that RLN3 is closely associated with AIS pathogenesis.

Example 3: effect of administration of an inhibitor of the RLN3 receptor RXFP3 on growth of ligament fibroblasts in scoliotic mice

The experimental method comprises the following steps:

1. mouse ligament fibroblasts were isolated from normal ligament tissue of the control group by collagenase I digestion. The digested cells were cultured in an incubator (37 ℃, 5% CO)₂) In Dulbecco's modified Eagle's Medium supplemented with fetal bovine serum (10%) and penicillin/streptomycin (1%). Ligament fibroblasts were identified by immunofluorescence of α -SMA. Subsequent cell experiments were performed on ligament fibroblasts at 3-5 passages. Ligament fibroblasts were stimulated for 48 hours with TGF-. beta.1 (10ng/mL), RLN3(100ng/mL) and R3(B1-22) R (0.1 umol/L).

2. Immunofluorescence assay: immunofluorescence assay was performed on ligament fibroblast α -SMA. An 8 μm frozen section of paraformaldehyde-fixed fibroblasts was incubated with phosphate buffer containing 0.3% Triton X-100 for 20 minutes at room temperature. Then, the cells were incubated with primary Antibody (Anti- α smoothen tissue Antibody (a-SMA) Antibody (monoclonal,1A4), BM0002, Boster Biological Technology Co.) overnight at 4 ℃ and then with fluorescein-labeled Secondary Antibody (Goat Anti-Mouse IgG (H + L) Secondary Antibody, Dylight 594, #35510, Invitrogen) for 1 hour at 37 ℃. The nuclei were stained with 4', 6-diamidino-2-phenylindole (dapi. beyotime) and then observed with a fluorescence microscope (Nikon, Japan).

3. Cell viability analysis: ligament fibroblast growth viability was measured using the Cell Counting Kit-8 assay (Beyotime, China). Ligament fibroblasts were seeded in 96-well plates (6 wells per group) and incubated overnight, followed by TGF- β and RLN3 treatment for 48 hours. After addition of 10. mu.L of Cell Counting Kit-8 solution, fibroblasts were incubated for 2 hours. Growth activity was measured using a microplate reader (BioTek, Germany) according to the instructions of the instruction manual.

4. Hydroxyproline detection: the hydroxyproline content in ligament fibroblasts was measured using the hydrolysate of the hydroxyproline assay kit (Sigma, MO, USA) according to the manufacturer's instructions. Briefly, the collected cells were hydrolyzed in 100L of concentrated hydrochloric acid (HCl, 12M) at 120 ℃ for 3 hours. After centrifugation, the supernatant (10 μ L) was transferred to a 96-well plate and then dried in an oven at 60 ℃. A chloramine T/oxidation buffer mixture and diluted DMAB reagent were added sequentially to each sample and standard well, followed by incubation at 60 ℃ for 90 minutes. The absorbance was measured at 560nm using a microplate reader (Beckman Coulter, Calif., USA).

5. Western Blot detection: electrophoretic separation of proteins was performed using 10% SDS-PAGE. A non-fat milk blocked polyvinylidene fluoride membrane was incubated with a primary antibody (ERK 1/2: CST Corp 4695; p-ERK 1/2: CST Corp 4376; nNOS: CST Corp 4231; smad 2: CST Corp 5339; psmad 2: CST Corp 18338; TGF-. beta.1: Abcam Corp ab 92486; MMP 2: Abcam Corp, ab 37150; MMP 9: Abcam Corp ab38898) overnight at 4 ℃ and then with a secondary antibody (HRP-labeled Goat Anti-rabbitIgG (H + L) Jackson 111-005-) for 2 hours at room temperature.

Visualization was achieved on the ChemiDoc MP system (Bio-Rad, PA, USA) using ECL Plus Western Blotting Substrate (Thermo Scientific, Wis., USA). The relative levels of protein were quantified based on band intensity values and analyzed using Image J software. Beta-actin served as an internal control.

The experimental results are as follows:

1. FIG. 3A shows immunofluorescence images of α -SMA after TGF- β 1(10ng/mL) and RLN3(100ng/mL) treatment of human spinal ligament fibroblasts. It can be seen that TGF-beta 1 can activate the expression of human spinal ligament fibroblast alpha-SMA, and RLN3 can inhibit the expression of alpha-SMA.

2. CCK-8 experiments demonstrated that RLN3(100ng/mL) significantly inhibited the activation of TGF-. beta.1 (10ng/mL) on human spinal ligament fibroblast growth viability (FIG. 3B).

3. Figure 3C shows the hydroxyproline content of ligament fibroblasts after TGF β 1and RLN3 treatment. Hydroxyproline significantly increased in TGF β 1 treated ligament fibroblasts, while hydroxyproline significantly decreased in RLN3 treated ligament fibroblasts. P < 0.01.

4. Western blot analysis confirmed that expression of spinal ligament fibroblasts MMP2, MMP9, and RXFP3 was significantly increased after treatment with RLN 3. Relative protein levels were evaluated from the grey values of the corresponding bands (fig. 3D-E). P < 0.01.

5. Western blot analysis proves that RLN3 promotes ERK1/2 phosphorylation and nNOS expression, and reduces phosphorylation of SMAD2 protein. Each group was set with 3 biological replicates. Relative protein levels were evaluated from the grey values of the corresponding bands (fig. 4A-B). P < 0.01.

6. Effect of R3(B1-22) R (a receptor RXFP3 antagonist) on human spinal ligament fibroblast signaling pathway: western-blot detection proves that R3(B1-22) R can obviously inhibit the activation of ERK1/2 and nNOS related signal channels by RLN3 (figure 4C-D).

The results show that the inhibitor (such as R3(B1-22) R) of the receptor RXFP3 of RLN3 can obviously inhibit the activation of the ERK1/2 and nNOS related signal pathways by RLN3, so that the inhibition of the activation of ligament fibroblasts by RLN3 is prevented, the activation of the ligament fibroblasts into myofibroblasts is promoted, and the physicochemical properties of ligaments are changed.

Example 4 Effect of administration of an inhibitor of the RLN3 receptor RXFP3 (R3(B1-22) R) on the incidence of scoliosis in a mouse animal model of scoliosis

Mouse animal models were prepared as in example 2.

The experimental steps are as follows:

1. c57BL/6J mouse animal model 3 weeks old was introduced and adapted to 1 week, 4 weeks old for removal of forelimb surgery. The 5-week-old patient began monthly surgery of 0.5% chloral hydrate anesthesia subcutaneous R3(B1-22) R osmotic pump implantation. Slow infusion of R3(B1-22) R (Phoenix Pharmaceuticals Inc, CA, USA, 036-142.5. mu.g/day), or physiological saline (NS) was implanted by subcutaneous osmotic pump (Alzet, Durect Corporation, CA, USA).

2. And performing anteroposterior and lateral X-ray film examination and three-dimensional CT examination under the anesthesia state. And statistics are carried out on the incidence rate of the scoliosis.

The inhibitor R3(B1-22) R was administered by subcutaneous osmotic pump for 16 weeks before spinal X-ray and CT examination in C57BL/6J mouse scoliosis animal model.

The experimental results are as follows:

x-ray and 3D-CT examination (FIGS. 5A and 5B) confirmed that the incidence of scoliosis in the R3(B1-22) R treated group was significantly lower in the C57BL/6J mouse scoliosis animal model than in the NS group (22.0% vs 62.0%, FIG. 5C). The above results indicate that inhibitors of the RLN3 receptor RXFP3 (e.g., R3(B1-22) R) can prevent or even reverse the development and progression of AIS.

It is clear that those skilled in the art will recognize that not only R3(B1-22) R is capable of reducing the incidence of scoliosis. Indeed, from the results of the above examples, one skilled in the art can expect inhibitors of the RLN3 receptor, in particular RXFP 1and RXFP3 receptors, such as small molecule compounds, antibodies, or aptamers, and the like. Without being bound by a particular theory, the inventors believe that the inhibitor of the receptors RXFP1 or RXFP3 prevents the inhibition of the activation of ligament fibroblasts by RLN3 through RLN3-RXFP1/3-ERK1/2 signaling axis by inhibiting or antagonizing the binding of the receptors RXFP 1and RXFP3 to their ligands RLN3, thereby promoting ligament fibroblast growth and preventing or even reversing the occurrence and development of AIS. Accordingly, a person skilled in the art can obtain (e.g., obtain commercially or prepare or screen according to known methods) various inhibitors of the RLN3 receptor, in particular inhibitors of the RXFP 1and RXFP3 receptor, and screen compounds suitable for administration to mammals, in particular humans, for the prevention and/or treatment of adolescent idiopathic scoliosis and prepare them as medicaments.

Statistical analysis method

All statistical analyses were performed using SPSS version 22.0. Qualitative data were compared using Fisher's exact test. Quantitative data were compared using a two-way ANOVA or Kruskal-Wallis test, if necessary. The relationship between RLN3 plasma levels and HJM points was analyzed by Pearson test. Comparisons of incidence of scoliosis were compared by the chi-square test. All p-values were two-sided, with p-values less than 0.05 considered statistically significant.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

2. The use according to claim 1, wherein the RXFP3 inhibitor has an apparent equilibrium constant (pKi) for competitive binding with RXFP 3in an assay using the method described by Linda m.

3. Use according to any one of claims 1 or 2, wherein the inhibitor is a small molecule compound such as a peptide, and an antibody or aptamer.

4. The use according to any one of claims 1 or 2, wherein the RXFP3 inhibitor is selected from R3, R3/I5, R3(B Δ 23-27) R/I5, R3(B1-22R) (or R3B 1-22R), R1B 1-22R acid, R1B 1-22 1/22A, R1B 1-22R dimer, INSL 1, and minimally relaxin 3-analogue 3, preferably R1 (B1-22) R, or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition of claim 5, wherein the RXFP3 inhibitor has an apparent equilibrium constant (pKi) of <9, preferably 5 to 9, for competitive binding with RXFP 3in an assay using the method described by Linda M.

7. The pharmaceutical composition of claim 5 or 6, wherein the inhibitor is a small molecule compound such as a peptide, and an antibody or aptamer.

8. The pharmaceutical composition according to claim 5 or 6, wherein the RXFP3 inhibitor is selected from R3, R3/I5, R3(B Δ 23-27) R/I5, R3(B1-22R) (or R3B 1-22R), R1B 1-22R acid, R1B 1-22A, R1B 1-22R, R1B 1-22R, R1B 1-22R, R1B 1-22R dimer, INSL 1, and a minimal relaxin 3-analogue 3, preferably R1 (B1-22) R, or a pharmaceutically acceptable salt thereof.

10. The use of claim 9, wherein the reagent for detecting the level of RLN3 is selected from the group consisting of a reagent for determining the level of RLN3 protein, such as an anti-RLN 3 antibody, a reagent for determining the level of mRNA encoding RLN3, such as a primer or probe, and a nucleic acid, such as an aptamer, that specifically binds to RLN3 protein.

12. The kit of claim 11, wherein the reagents for detecting the level of RLN3 are selected from the group consisting of reagents for determining the level of RLN3 protein, such as anti-RLN 3 antibodies, reagents for determining the level of mRNA encoding RLN3, such as primers or probes, and nucleic acids, such as aptamers, that specifically bind to RLN3 protein.