CNP analogue and application thereof

Abstract

The invention discloses a CNP analogue and application thereof. According to the CNP analogue, a fatty acid side chain is coupled to a modified CNP peptide main chain, NPR-B agonist activity is reserved, the CNP analogue has a long half-life period, administration is more convenient, immunogenicity is smaller, irritation to an injection site is smaller, 50 nmol / kg of the CNP analogue is injected subcutaneously to a mouse, the effect of improving growth retardation caused by cartilage hypoplasia is better than that of drugs on the market, and the CNP analogue has a good application prospect. Moreover, the CNP analogue can also be used for treating various diseases which can be treated by CNP.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a CNP analogue and its application. BACKGROUND

[0002] Functional mutations in the FGFR3 gene cause achondroplasia (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD). These disorders are characterized by disproportionate rhizomelic dwarfism due to enhanced fibroblast growth factor receptor 3 (FGFR3) signaling, which ranges from mild (HCH) to severe (ACH) and lethal (TD). FGFR3 activity within the cartilage is an important regulator of skeletal development, acting through multiple intracellular signaling pathways, including the signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase (MAPK) pathways. Abnormal activation of FGFR3 affects the proliferation and terminal differentiation of the growth plate chondrocytes, as well as the synthesis of extracellular matrix, and is associated with increased phosphorylation levels of STAT and MAPK pathways.

[0003] The MAPK signaling pathway is modulated by C-type natriuretic peptide (CNP). Binding of CNP to its receptor NPR-B is able to inhibit downstream processes of FGFR3 signaling, thus promoting cartilage growth and normal development of the skeleton, as observed in mice and humans overexpressing CNP. Overproduction of CNP or continuous administration of CNP by intravenous injection (IV) can normalize the dwarfism symptoms in achondroplasia mice, suggesting that the use of CNP at supraphysiological levels is a strategy for treating ACH.

[0004] However, native CNP has a short half-life (about 2 minutes after intravenous injection), and its use in the pediatric population is challenging because of the need for continuous infusion. In addition, CNP is extensively inactivated in subcutaneous tissues, thus requiring intravenous injection. CNP has two degradation pathways in the body: receptor-mediated degradation and extracellular protease-mediated degradation. CNP is first degraded by the action of neutral endopeptidase (NEP), while it is removed from systemic circulation by the natriuretic peptide clearance receptor NPR-C. Upon binding to CNP, NPR-C deposits it in the lysosome, where CNP is further degraded.

[0005] Vosoritide is a modified recombinant human C-type natriuretic peptide (CNP) to which 17 amino acids have been added, resulting in a 39-amino acid analog. Vosoritide mimics the pharmacological activity of CNP at the growth plate and, due to its resistance to neutral endopeptidases, has a prolonged half-life, allowing for once-daily subcutaneous (SC) administration. However, vosoritide is a non-naturally occurring peptide and carries an increased risk of inducing an immune response compared to natural peptides. As described by Martz in "sFGFR for achondroplasia" (SciBx, Biocenture, October 2013), immune responses to vosoritide have been observed in animal studies, but these antibodies have not compromised the drug's pharmacological activity.

[0006] Vosoritide has a half-life of only 20 minutes, which is associated with a short duration of drug administration when administered daily. To achieve effective drug levels, the drug dose can be increased, but natriuretic peptides are a family of hormones that may affect blood volume and blood pressure. Increasing the dose may lead to adverse cardiovascular effects. Animal and human studies have shown that arterial blood pressure decreases and heart rate increases with increasing dose. For example, doses of vosoritide up to 15 μg / kg in healthy volunteers are associated with mild hypotension. Increasing the dose of drugs with CNP activity to increase drug exposure may result in unacceptable cardiovascular side effects. In summary, there is a need to develop more convenient or effective CNP treatments that reduce the risk of adverse effects. Summary of the Invention

[0007] To overcome the deficiencies in the prior art, the present invention provides CNP polypeptide analogs having NPRB agonist activity and their applications. Specifically,

[0008] The first aspect of the present invention provides a CNP polypeptide analog, wherein the CNP polypeptide analog comprises the following structure:

[0009] (x)LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 21),

[0010] Wherein, (x) comprises 1-4 amino acids, and the amino acids are selected from proline (Pro, P), glycine (Gly, G), serine (Ser, S) and alanine (Ala, A).

[0011] Preferably, (x) comprises 2-3 amino acids, and said (x) is Pro-Gly; Gly-Pro; Ser-Ser; Ala-Gly; Gly-Ala; Pro-Ser; Ser-Pro; Ala-Ser; Ser-Ala; Ala-Ala; Pro-Gly-Ser; Gly-Pro-Ser; Ser-Pro-Gly; Ala-Gly-Ser; Gly-Ala-Ser; Pro-Ser-Gly; Ser-Ala-Gly; Ala-Ser-Pro; Ser-Gly-Pro or Gly-Ser-Ala.

[0012] Preferably, the CNP polypeptide analogs include fatty acid modifications, and further preferably, the fatty acids include linear or branched, saturated or unsaturated C5-C30 carboxylic acids and natural fatty acids.

[0013] In a specific embodiment, the fatty acid is a C22 fatty acid.

[0014] Preferably, the fatty acid modification is located on lysine K. Further preferably, the fatty acid modification is located on the first lysine K at the N-terminus of SEQ ID NO: 21.

[0015] Preferably, the CNP polypeptide analog comprises the amino acid sequence of any one of SEQ ID NOs: 1-20.

[0016] Preferably, the CNP polypeptide analog further comprises other modifications. The modified CNP polypeptide analog has better properties than the corresponding unmodified CNP polypeptide analog, such as higher cell activity and stability, faster growth rate, lower immunogenicity and injection site irritation, and longer half-life.

[0017] Preferably, the modification includes but is not limited to amino acid mutation, amidation, acetylation, glycosylation, phosphorylation or addition of other functional components.

[0018] Preferably, the amino acid mutation includes but is not limited to amino acid deletion, insertion, inversion, duplication and substitution.

[0019] Specifically, some amino acid sequences of CNP polypeptides can be altered without significantly affecting the structure or function of the peptide. Such mutants include deletions, insertions, inversions, duplications, and substitutions selected according to general rules known in the art, thereby having little effect on activity. For example, Bowie et al. (1990), Science 247:1306-1310, the entire contents of which are incorporated herein by reference, provide guidance on how to make phenotypically silent amino acid substitutions, in which the authors identify two main approaches to studying the tolerance of altered amino acid sequences.

[0020] Preferably, the glycosylation includes glycosylation at any amino acid position, in the form of N-linked or O-linked glycosylation.

[0021] Preferably, the other functional components include but are not limited to other natriuretic peptide components, water-soluble polymers, serum albumin, transferrin, Fc fragments, unstructured polypeptides, polyamino acids, fibronectin, fibrinogen, zinc finger polypeptides, fibroblast growth factor 2 (FGF2), bone targeting components or fragments of any of the foregoing.

[0022] Preferably, the other natriuretic peptide components include but are not limited to natriuretic peptide precursor (NPPC), atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP).

[0023] Preferably, the water-soluble polymer includes but is not limited to polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), lipids, branched or unbranched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups and branched or unbranched C8-C30 alkyl groups.

[0024] Preferably, the unstructured polypeptides include, but are not limited to, XTEN and PAS polypeptides (eg, conformationally disordered polypeptide sequences composed of amino acids Pro, Ala, and / or Ser).

[0025] Preferably, the bone targeting component includes but is not limited to cartilage targeting peptides, bone proteins or other proteins derived from the bone targeting domain or its derivatives, and the proteins derived from the bone targeting domain or its derivatives include but are not limited to osteopontin, osteogenin or bone sialoglycoprotein.

[0026] The modifications described herein can occur at any position, including the N-terminus, C-terminus, or within the sequence.

[0027] The modification may be one or more, and the multiple modifications may be of the same or different modification types.

[0028] Preferably, the modification comprises directly or indirectly linking a modification component (such as other functional components) to the CNP polypeptide analog.

[0029] Further preferably, the indirect connection comprises the use of a linker or a non-naturally encoded amino acid linker. Exemplary linkers include, but are not limited to, small organic compounds, water-soluble polymers of various lengths (such as polyethylene glycol or polydextran) or polypeptides of various lengths.

[0030] The polypeptide analogs can be prepared by any method in the prior art, such as chemical synthesis, biosynthesis, etc.

[0031] The second aspect of the present invention provides a nucleic acid encoding the above-mentioned CNP polypeptide analog.

[0032] The third aspect of the present invention provides a product comprising the aforementioned CNP polypeptide analog and / or the aforementioned nucleic acid, wherein the product comprises a vector, a cell, a kit, a delivery system or a pharmaceutical composition.

[0033] Preferably, the vector comprises the above-mentioned nucleic acid.

[0034] Preferably, the vector can be any vector suitable for delivering the nucleic acid to the target tissue or target cell for expression.

[0035] Preferably, the vector is a viral vector. Preferably, the viral vector includes but is not limited to a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, a herpesvirus vector, and the like.

[0036] Preferably, the vector is a non-viral vector. Preferably, the non-viral vector includes but is not limited to any one or a combination of two or more of liposomes, lipid nanoparticles (LNP), polymers, proteins, aptamers, and N-acetylgalactosamine (GalNAc).

[0037] Preferably, the delivery system comprises the above-mentioned CNP polypeptide analog or the above-mentioned carrier.

[0038] Preferably, the delivery system comprises further modifications of the above-mentioned CNP polypeptide analogs. Further preferably, the modifications include but are not limited to fusion of penetrating peptides, responsive peptides or self-assembling oligomeric proteins.

[0039] More preferably, the penetrating peptide (CPP) includes but is not limited to protamine, Tat peptide, transportan peptide, penetratin peptide or oligoarginine peptide;

[0040] The responsive peptides include but are not limited to pH responsive peptides, enzyme responsive peptides, temperature responsive peptides or redox responsive peptides;

[0041] The oligomeric protein includes but is not limited to Helicobacter pylori ferritin, dioxotetrahydropterin synthase, C4b binding protein or dihydrosulfonyl acetyltransferase.

[0042] Preferably, the pharmaceutical composition comprises the CNP polypeptide analog or the delivery system, and pharmaceutically acceptable excipients.

[0043] In a fourth aspect, the present invention provides a method for preparing the aforementioned CNP polypeptide analog. The CNP polypeptide analog can be prepared by any method in the prior art, such as chemical synthesis and / or biosynthesis.

[0044] Preferably, the chemical synthesis method includes solid phase synthesis method, liquid phase synthesis method, etc., and the biosynthesis method includes fermentation method, gene recombination method, enzymatic hydrolysis method, etc.

[0045] Preferably, the preparation method comprises:

[0046] (1) synthesizing a crude product of a CNP polypeptide analogue using a chemical synthesis method;

[0047] (2) separating and purifying the crude product to obtain the CNP polypeptide analog.

[0048] Specifically, the CNP polypeptide analogs of the present invention can be prepared by standard peptide synthesis methods, for example, by standard solid-phase or liquid-phase methods, stepwise or by fragment assembly, and isolation and purification of the final peptide compound product, or by biosynthesis or any combination thereof. Preferably, the polypeptide analogs of the present invention can be synthesized by solid-phase or liquid-phase peptide synthesis methods.

[0049] In one embodiment, the preparation method comprises solid-phase peptide synthesis (SPPS). SPPS involves sequentially coupling the amino acids of the aforementioned peptide analogs to a resin to form a peptide chain. After sequence synthesis is complete, the N-terminal Fmoc protecting group is first deprotected (or after N-terminal modification), followed by deprotection of the side chain protecting groups, and the peptide is cleaved from the resin.

[0050] The fifth aspect of the present invention provides the use of the above-mentioned CNP polypeptide analog, the above-mentioned nucleic acid or the above-mentioned product in the preparation of a medicament for treating a disease that can be treated with CNP.

[0051] Preferably, the disease is selected from the group consisting of achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasia, thanatophoric dysplasia, osteogenesis imperfecta, hypochondrodysplasia punctata, homozygous achondroplasia, flexor dysostosis, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly, rhizoctonia punctata, Janssen's metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, osteodysplasia, distorted osteodysplasia, congenital short femur, Langer's limb metaphysis Dysplasia, Nievergelt-type mediomelia, Robinow syndrome, Reinhardt syndrome, acrometaphyseal dysplasia, peripheral skeletal dysplasia, Kniest dysplasia, fibrocartilage development, Roberts syndrome, acrometaphyseal dysplasia, phocomelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, spondyloepiphyseal dysplasia, neurofibromatosis, Legius syndrome, LEOPARD syndrome, Noonan syndrome syndrome, hereditary gingival fibromatosis, neurofibromatosis type 1, Legius syndrome, Cardiofaciocutaneous syndrome, Costello syndrome, SHOX deficiency, idiopathic short stature, growth hormone deficiency, osteoarthritis, cleidocranial dysplasia, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), digitiformis, brachydactyly, flexor digiti, polydactyly, syndactyly, Dyssegmental dysplasia, enchondroma, fibrous dysplasia, hereditary multiple exostoses, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Marfan syndrome, McCune-Albright syndrome, osteopetrosis, osteopetrosis fragility, ophthalmologic disorders, cancer associated with hyperactivation of FGFR3, or vascular smooth muscle disease.

[0052] Preferably, the ophthalmic disease includes but is not limited to glaucoma and / or elevated intraocular pressure.

[0053] Preferably, the cancer associated with overactivation of FGFR3 includes but is not limited to multiple myeloma, myeloproliferative syndrome, leukemia, plasma cell leukemia, lymphoma, glioblastoma, prostate cancer, bladder cancer or breast cancer.

[0054] Preferably, the vascular smooth muscle disease includes but is not limited to hypertension, restenosis, arteriosclerosis, acute decompensated heart failure, congestive heart failure, cardiac edema, hydronephrosis, hepatic edema, acute renal insufficiency and chronic renal insufficiency.

[0055] In an eighth aspect, the present application relates to a method of preventing and / or treating a disease, said method comprising administering to an individual suffering from a disease an effective amount of a CNP polypeptide analogue as described above, a nucleic acid as described above or a product as described above.

[0056] Preferably, the disease is selected from the group consisting of achondroplasia, hypochondroplasia, short stature, dwarfism, spondyloepiphyseal dysplasia, lethal developmental dysplasia, osteogenesis imperfecta, chondrodysplasia, point-like achondroplasia, homozygous achondroplasia, camptomelic dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short-rib polydactyly syndrome, rhizomelic point-like achondroplasia, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenita, bone dysplasia, twisted bone dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral bone dysplasia, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, mesomelic-acrodysostosis, micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, spondyloepiphyseal-metaphyseal dysplasia, neurofibromatosis, Legius syndrome, LEOPARD syndrome, Noonan syndrome, hereditary gingival fibromatosis, neurofibromatosis type 1, Legius syndrome, cardiofaciocutaneous syndrome, Costello syndrome, SHOX deficiency, idiopathic short stature, growth hormone deficiency, osteoarthritis, cranio-lacivarial dysostosis, craniosynostosis (e.g. Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome or Crouzonodermoskeletal syndrome), syndactyly, brachydactyly, camptodactyly, polydactyly, syndactyly, dyssegmental dysplasia, enchondromatosis, fibrous dysplasia, hereditary multiple exostoses, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Marfan syndrome, McCune-Albright syndrome, osteosclerosis, osteosclerotic brittle bones, an ophthalmic disease, a cancer associated with over-activation of FGFR3 or a vascular smooth muscle disease.

[0057] Preferably, the ophthalmic disease comprises, but is not limited to, glaucoma and / or elevated intraocular pressure.

[0058] Preferably, the cancer associated with overactivation of FGFR3 includes but is not limited to multiple myeloma, myeloproliferative syndrome, leukemia, plasma cell leukemia, lymphoma, glioblastoma, prostate cancer, bladder cancer or breast cancer.

[0059] Preferably, the vascular smooth muscle disease includes but is not limited to hypertension, restenosis, arteriosclerosis, acute decompensated heart failure, congestive heart failure, cardiac edema, hydronephrosis, hepatic edema, acute renal insufficiency and chronic renal insufficiency.

[0060] Preferably, the patient comprises a human or a non-human animal, such as a non-human mammal.

[0061] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating or reversing the progression or severity of a sign, symptom, disorder, condition or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions or disorders.

[0062] The "effective amount" of the present invention refers to the amount or dosage of the drug of the present invention that provides the desired treatment or prevention after administration to an individual or organ in a single or multiple doses.

[0063] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, as well as other specified components or steps that do not substantially affect them.

[0064] The "individual" described in the present invention can be a human or a non-human animal, and the non-human animal can be a non-human mammal such as a mouse, cow, sheep, rabbit, pig, monkey, etc.

[0065] Beneficial effects of the present invention:

[0066] The present invention performs amino acid optimization and fatty acid modification on the basis of CNP38 to synthesize and screen a new polypeptide with NPR-B agonist activity, a longer half-life, more convenient administration, less immunogenicity, less irritation at the injection site, and better improvement of growth retardation caused by achondroplasia. The present invention provides a highly active polypeptide that is more effective in improving growth retardation caused by achondroplasia than the marketed drug vosoritide and the Phase III clinical trial drug navepegritide, and is more convenient to administer and less toxic than vosoritide. DETAILED DESCRIPTION

[0067] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0068] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources.

[0069] Example 1: Design and synthesis of CNP polypeptide analogs

[0070] 1. Peptide sequence design

[0071] The target peptide amino acid sequence (SEQ ID NO: 24, CNP38 in CN107405409B) was obtained by retrieval:

[0072] LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO:22)

[0073] This sequence is completely consistent with the C-terminal 38 sequences of natural CNP53. On this basis, some amino acids were added and optimized, and the fatty acid side chain C22 was added to obtain the following sequence:

[0074] Table 1 Polypeptide sequences

[0075]

[0076] 2. Synthesis and purification of peptides

[0077] 2.1 Synthesis of peptides

[0078] Peptides are produced using solid-phase peptide synthesis (SPPS). SPPS involves sequentially coupling amino acids to a resin to form a peptide chain. After sequence synthesis, the N-terminal Fmoc protecting group is first deprotected (or after N-terminal modification), followed by deprotection of the side chain protecting groups, and the peptide is cleaved from the resin. The preparation process is as follows:

[0079] 1) Coupling the first amino acid: Take an appropriate amount of modified resin, add the prepared amino acid solution and coupling reagent to the resin, and react for a period of time;

[0080] 2) Removal of Fmoc: After adding Pip / DMF solution for a period of time, vacuum filtration was performed to remove the solvent;

[0081] 3) Washing: Add DMF to the resin (washing step) and remove the solvent by vacuum filtration;

[0082] 4) Resin test: Place the test reagents ninhydrin, N,N-dimethylformamide, and a small amount of resin in a test tube. Place the test tube in a metal bath for a few seconds and check to see if the resin changes color. If so, the Fmoc group has been successfully removed.

[0083] 5) Amino acid condensation: Add the prepared amino acid solution to the resin. Then add the coupling reagent, shake well for a while, and vacuum filter to remove the solvent.

[0084] 6) Repeat steps 2)-5) until the last amino acid is synthesized.

[0085] 2.2 Peptide purification

[0086] The purification process is as follows:

[0087] 1) Crude polypeptide: Based on the nature of the sequence, select appropriate reagents to completely dissolve the crude polypeptide.

[0088] 2) Set the purification gradient: Design the purification gradient based on the sequence properties.

[0089] 3) The crude polypeptide liquid sample is filtered through a filter membrane and injected into a high performance liquid chromatography machine, and the absorption peaks in the chromatogram are collected separately.

[0090] 4) Test the molecular weight and purity of the fraction to determine the target fraction.

[0091] The purified peptide was analyzed by HPLC and MS, and the results are as follows:

[0092] Table 2 Parameters after peptide purification

[0093]

[0094] The purity of each peptide was >95% and could be used for subsequent experiments.

[0095] Example 2 Functional testing of polypeptides

[0096] 1. Cell activity test

[0097] To evaluate the functional activity of CNP peptide analogs, a cell-based assay using NIH 3T3 cells (a mouse embryonic fibroblast cell line) was employed. NIH 3T3 cells express endogenous NPR-B on their surface, a receptor that is the primary target of CNP. Stimulation of NPR-B with CNP allows for the detection of the intracellular production of the second messenger cGMP, which is measured using a commercially available cGMP assay.

[0098] NIH 3T3 cells were routinely cultured in DMEM F-12 medium containing 5% FBS and 5mM glutamine at 37°C and 5% CO2. For each assay, 50,000 cells were resuspended in stimulation buffer (Dulbecco's PBS containing IBMX) and incubated with various concentrations of the peptides. CNPs were obtained by diluting them in PBS with 0.2% BSA. After incubation at 37°C and 5% CO2 for 30 minutes, the cells were lysed and cGMP levels were measured using a commercially available cGMP TR-FRET product (Cisbio, cGMP kit, catalog number 62GM2PEB).

[0099] The results are shown in Table 3, wherein the stimulatory effect of each polypeptide on cGMP production in NIH 3T3 cells is expressed as a percentage compared to the cGMP production value in the presence of 1 μM CNP22.

[0100] The results showed that SEQ ID NO: 16 had the highest NIH 3T3 cell stimulating activity.

[0101] Table 3 Cell activity test results

[0102]

[0103] 2. Neutral endopeptidase stability

[0104] Recombinant human NEP (2.5 μg / mL final concentration) and standard pentafluorophenol (PFP; 40 μg / mL final concentration) were added to digestion buffer (50 mM Tris-HCl, pH 7.4, 10 mM NaCl) at 100 μg CNP equivalents / mL for each peptide. The solution was incubated at 37°C and 500 rpm for up to 4 days, with samples taken at various time intervals. The reaction was terminated by a combination of reduction and heat denaturation, with the addition of TCEP ((tris(2-carboxyethyl)phosphine; 25 mM final concentration)) and incubation of the mixture at 95°C and 500 rpm for 5 min. The resulting reaction products were determined using HPLC-MS. The half-life of each peptide was calculated by the change in the ratio of the HPLC-UV peak areas of CNP and PFP over time. To compensate for variations in protease activity, CNP-38 or CNP-22 digestion was performed as a reference in each batch of measurements.

[0105] Table 4 Neutral endopeptidase stability test results

[0106]

[0107]

[0108] CNP22 neutral endopeptidase t 1 / 2The average value is 0.5h, CNP38 neutral endopeptidase t 1 / 2 The average is 13.4h.

[0109] Table 4 shows that the modified CNP polypeptides have longer neutral endopeptidase stability than CNP-22 and CNP-38, among which the six sequences of SEQ ID NO: 10, 13, 15, 16, 17, and 18 have longer half-lives, all greater than 96 h.

[0110] 3. Immunogenicity and injection site irritation

[0111] This study aimed to compare the immunogenicity and injection site irritation of SEQ ID NOs: 10, 16, and 18 against a control drug. Seven-week-old Balb / c mice, six per group, were enrolled. Before dosing, 50 μL of serum was collected from each mouse via tail vein bleeding to serve as a blank control. Mice were subsequently subcutaneously injected daily for 28 days with 50 nmol / kg of SEQ ID NOs: 10, 16, and 18, CNP22, vosoritide, and vehicle (30 mM acetate, pH 4, containing 5% sucrose and 1% benzyl alcohol). All animals were observed on the day of each dosing for the presence of erythema, hyperemia, edema, induration, ulceration, and pus discharge at the injection site. On day 45, orbital bleeding was collected from the mice, and serum was isolated after coagulation. Serum antibody titers were determined using a direct ELISA. The corresponding peptide was coated onto an ELISA plate. Mouse serum was diluted at a gradient of 1:50, 1:200, 1:1000, and 1:5000 and then added to the plate. A goat anti-mouse secondary antibody was used as the detection antibody. Serum samples from each mouse before administration served as negative controls. At the same dilution ratio, a test sample was considered positive (+) if its average absorbance OD value was greater than 2.1 times the average OD value of the negative control serum; otherwise, it was considered negative (-). The highest dilution yielding a positive result was the antibody titer.

[0112] The immunogenicity results are as follows:

[0113] Table 5 Immunogenicity results

[0114]

[0115]

[0116] Immunogenicity from small to large is: CNP22 <SEQ ID NO:16<SEQ ID NO:10<SEQ ID NO:18<沃索立肽。

[0117] The observed data on local irritation after administration are as follows:

[0118] Table 6 Results of local irritation after administration

[0119] serial number erythema swelling congestion induration ulcer solvent 1 / 6 0 / 6 0 / 6 0 / 6 0 / 6 CNP-22 1 / 6 1 / 6 1 / 6 0 / 6 0 / 6 Vosoritide 4 / 6 4 / 6 3 / 6 2 / 6 1 / 6 SEQ ID NO: 10 2 / 6 2 / 6 2 / 6 1 / 6 0 / 6 SEQ ID NO: 16 2 / 6 1 / 6 1 / 6 0 / 6 0 / 6 SEQ ID NO: 18 3 / 6 3 / 6 2 / 6 0 / 6 0 / 6

[0120] The order of local irritation from least to greatest is: CNP22 <SEQ ID NO:16<SEQ ID NO:10≈SEQ IDNO:18<沃索立肽。

[0121] 4. Growth Study of FVB Mice

[0122] This study aimed to compare the effects of subcutaneous administration of SEQ ID NO: 16 with a control drug on animal growth. Wild-type FVB male mice (nine per group) aged 21 to 22 days were subcutaneously injected with 50 nmol / kg of SEQ ID NO: 10Q3D, SEQ ID NO: 16Q3D, SEQ ID NO: 18Q3D, woxoritide Q3D, or vehicle (Q3D, 30 mM acetate pH 4 containing 5% sucrose and 1% benzyl alcohol) for 4 weeks. Various measures of growth plate expansion and bone growth were performed using digital X-ray and magnetic resonance imaging, as well as external measurements of limb and body length. By day 35, the following were observed compared to the vehicle control group:

[0123] Table 7 Growth results of FVB mice

[0124]

[0125]

[0126] The research conclusions showed that SEQ ID NO: 10, SEQ ID NO: 16 and SEQ ID NO: 18 were more effective in promoting the vertical and axial bone growth of animals than vosoridide, among which SEQ ID NO: 16 had the best growth-promoting effect.

[0127] Based on the above test results on cell activity, neutral endopeptidase stability, immunogenicity, injection site irritation and FVB mouse growth, all modified CNP polypeptides have good effects, among which SEQ ID NO: 16 has the best effect.

[0128] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and substance of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be readily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A CNP polypeptide analogue, characterized in that The CNP polypeptide analogue comprises the following structure: (x)LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 21), Wherein, (x) comprises 1-4 amino acids, and the amino acids are selected from proline, glycine, serine and alanine.

2. The CNP polypeptide analog according to claim 1, wherein The CNP polypeptide analogs include fatty acid modifications. Preferably, the fatty acids include linear or branched, saturated or unsaturated C5-C30 carboxylic acids and natural fatty acids.

3. The CNP polypeptide analog according to any one of claims 1-2, characterized in that The CNP polypeptide analogue comprises the amino acid sequence of any one of SEQ ID NOs: 1-20.

4. The CNP polypeptide analog according to any one of claims 1 to 3, characterized in that The CNP polypeptide analogs may also contain other modifications, including amino acid mutations, amidation, acetylation, glycosylation, phosphorylation or addition of other functional components; Preferably, the other functional components include other natriuretic peptide components, water-soluble polymers, serum albumin, transferrin, Fc fragments, unstructured polypeptides, polyamino acids, fibronectin, fibrinogen, zinc finger polypeptides, fibroblast growth factor 2, bone targeting components or fragments of any of the foregoing.

5. The CNP polypeptide analog according to any one of claims 1 to 4, characterized in that The modification occurs at any position of the N-terminus, C-terminus or internal site of the sequence.

6. A nucleic acid encoding the CNP polypeptide analogue according to any one of claims 1 to 5.

7. A product comprising the CNP polypeptide analogue according to any one of claims 1 to 5 and / or the nucleic acid according to claim 6, characterized in that: The products include vectors, cells, kits, delivery systems or pharmaceutical compositions.

8. The product according to claim 7, characterized in that The vector comprises the nucleic acid; The delivery system comprises the CNP polypeptide analog or the carrier. Preferably, the delivery system comprises further modification of the CNP polypeptide analog. Further preferably, the modification comprises fusion of a penetrating peptide, a responsive peptide or a self-assembling oligomeric protein. The pharmaceutical composition comprises the CNP polypeptide analog or the delivery system, and pharmaceutically acceptable excipients.

9. Use of the polypeptide according to any one of claims 1 to 5, the nucleic acid according to claim 6 or the product according to any one of claims 7 to 8 in the preparation of a medicament for treating a disease that can be treated with CNP, preferably, the disease is selected from achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasia, thanatophoric dysplasia, osteogenesis imperfecta, hypochondroplasia punctata, homozygous hypochondroplasia, flexor dysostosis, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, rhizoctonia punctata, Janssen's Metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, osteodysplasia, distorted osteodysplasia, congenital short femur, Lange-type mediophysial dysplasia, Nievergelt-type mediophysial dysplasia, Robinow syndrome, Reinhardt syndrome, acrometaphysis, peripheral bone development disorder, Kniest dysplasia, fibrocartilage development, Roberts syndrome, acrometaphysis dysplasia, brachymelia, Morquio syndrome, Kniest syndrome, epitrophic dysplasia, spondyloepiphyseal dysplasia, neuro fibromatosis, Legius syndrome, LEOPARD syndrome, Noonan syndrome, hereditary gingival fibromatosis, neurofibromatosis type 1, Legius syndrome, Cardiofaciocutaneous syndrome, Costello syndrome, SHOX deficiency, idiopathic short stature, growth hormone deficiency, osteoarthritis, cleidocranial dysplasia, craniosynostosis (eg, Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffe syndrome), r syndrome or Crouzonodermoskeletal syndrome), digitiformis, brachydactyly, flexed digits, polydactyly, syndactyly, Dyssegmental dysplasia, enchondroma, fibrous dysplasia, hereditary multiple exostoses, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Marfan syndrome, McCune-Albright syndrome, osteopetrosis, osteopetrosis fragility, ophthalmologic diseases, cancer associated with overactivation of FGFR3, or vascular smooth muscle disease.

10. The method for preparing the CNP polypeptide analogue according to any one of claims 1 to 5, characterized in that: The preparation method includes chemical synthesis and / or biological synthesis.

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