Method for treating aortic aneurysm and aortic dissection by targeting IGF1R

Abstract

The invention relates to a novel method for preventing or treating aneurysm by using an IGF1R (Insulin-like Growth Factor 1 Receptor) inhibitor and application. The invention also relates to formulations comprising IGF1C suitable for use in this method and use. The invention also relates to a local minimally invasive delivery technique of a drug for preventing or treating aneurysm, in particular to a local minimally invasive delivery technique for delivering a drug comprising an IGF1R inhibitor of the invention.

Description

[0001] The present invention relates to a new method and use of applying an IGF1R inhibitor for preventing or treating aneurysms. The present invention also relates to a preparation containing IGF1C suitable for this method and use. The present invention also relates to a local minimally invasive drug delivery technique for preventing or treating aneurysms, in particular a local minimally invasive drug delivery technique for delivering the drug containing an IGF1R inhibitor of the present invention. Background of the invention:

[0003] Aortic aneurysms and aortic dissections are diseases that seriously threaten human health. Once the aneurysm ruptures in a patient, the fatality rate is as high as 90%. Even if the patient can survive and receive treatment in a timely manner, the mortality rate is still as high as 47% - 83%. Surgical operations (open surgery and endovascular procedures), as the most common methods for treating such diseases, have many disadvantages, such as high risk, high cost, inability to cure the root cause, and difficulty in popularization. At the same time, for small aneurysms (aneurysm diameter less than 5.5 cm), the risk of aneurysm rupture is less than the surgical risk, and only conservative observation can be carried out.

[0004] Compared with healthy people, the level of IGF1 in the serum of aneurysm patients is significantly increased. Some literature has proposed that IGF1 can be used as a biomarker for the occurrence of aneurysms (Eur J Vasc Endovasc Surg. 2011 Nov; 42(5):560 - 2).

[0005] At the same time, the AKT / mTOR signaling pathway is significantly activated in aneurysm tissues. Existing literature (Chronic mTOR activation induces a degradative smooth muscle cell phenotype, Guangxin Li et al., J Clin Invest. 2020; 130(3):1233 - 1251. https: / / doi.org / 10.1172 / JCI131048.) has shown that long - term activation of mTOR will stimulate vascular smooth muscle cells to transform into a degradative phenotype. And the transformation of smooth muscle cells into a degradative phenotype is the key cellular mechanism for inducing the occurrence of aortic aneurysms and aortic dissections. Therefore, it has been identified that mTOR - dependent phenotype regulation can be used as a therapeutic target against aortic aneurysms and aortic dissections.

[0006] There is a need in the art to develop new drugs targeting mTOR - dependent phenotype regulation, and also to develop a method for treating aortic aneurysms and / or aortic dissections in a non - surgical manner. Summary of the invention:

[0007] IGF1C is a polypeptide of the C domain of the IGF1 protein. Studies have proposed that IGF1C can act as a competitive inhibitor of IGF1 and inhibit the activation of IGF1R. The present invention has developed the function of the IGF1R inhibitor IGF1C in the treatment of aortic aneurysm and aortic dissection. As a protein widely present throughout the body, IGF1R plays a key role in life processes such as glucose metabolism, muscle function, and nerve signal transduction. Systemic inhibition of it can cause various side effects. The present invention has developed a method for locally delivering an IGF1R inhibitor by an interventional approach to treat aortic aneurysm and aortic dissection, reducing the side effects caused by systemic delivery.

[0008] The present invention discovers for the first time that an IGF1R inhibitor can effectively treat aneurysms or arterial dissections.

[0009] Therefore, on the one hand, the present invention relates to a method for treating aneurysms or arterial dissections, including administering an IGF1R inhibitor, or a drug or preparation containing the same, to an individual.

[0010] In some embodiments, the IGF1R inhibitor is selected from domain C of IGF1 (IGF1C) or a fragment thereof, an antibody against an insulin-like growth factor receptor (such as the receptor of IGF1), or an antigen-binding fragment thereof (such as an IGF1R antibody or an antigen-binding fragment thereof, such as cetuximab or an antigen-binding fragment thereof), or a small molecule inhibitor, such as Linsitinib or podophyllotoxin.

[0011] In some embodiments, the aneurysm or arterial dissection of the present invention is in the aorta or peripheral arteries.

[0012] In some embodiments, the aneurysm or arterial dissection of the present invention is in the abdominal or thoracic arteries.

[0013] In some embodiments, the aneurysm or arterial dissection is an aneurysm or arterial dissection of the abdominal aorta or thoracic aorta.

[0014] In some embodiments, the polypeptide of domain C of insulin-like growth factor (IGF1C) refers to domain C of IGF-1, corresponding to positions 30-41 of IGF-1. In some embodiments, IGF-1 is the protein shown under UniProtKB accession number P05019. In some embodiments, the IGF1C polypeptide contains the amino acid sequence GYGSSSRRAPQT (SEQ ID NO:1) or consists of the said amino acid sequence.

[0015] The IGF1C polypeptides described herein also encompass polypeptides having 1-8 amino acid differences compared to wild-type IGF1C. It is known in the art that the minimum active unit of IGF1C is SSSR (SEQ ID NO:2) (CN1263770C). Thus, other amino acids outside the minimum active unit can be mutated, such as substituted, deleted, or 1-10 amino acids can be added outside the minimum active unit, as long as the resulting IGF1C polypeptide still has substantially the same activity as wild-type IGF1C (e.g., the activity of binding to IGF1R). Accordingly, the present invention encompasses such IGF1C polypeptides that contain an amino acid sequence having 1-8 amino acid substitutions, insertions, or deletions compared to the amino acid sequence shown in SEQ ID NO:1 and contain SSSR.

[0016] In some embodiments, the amino acid at position 3 of IGF1C (e.g., G) can be substituted with A, or the R at position 8 can be substituted with A, or 1 amino acid (e.g., A) can be added after position 9. In some embodiments, the IGF1C polypeptide can contain the R8A substitution and an A added between positions 9-10. In some embodiments, the IGF1C polypeptide can contain G3A.

[0017] In some embodiments, the mutations (e.g., substitutions, deletions or additions) are conservative, i.e., they do not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the replacement of one amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, additional substitutions of amino acids can be made within groups of amino acids having similar side chains without departing from the invention. These include neutral hydrophobic amino acids: alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I), proline (Pro or P), tryptophan (Trp or W), phenylalanine (Phe or F) and methionine (Met or M). Similarly, neutral polar amino acids can be substituted for one another within their group of glycine (Gly or G), serine (Ser or S), threonine (Thr or T), tyrosine (Tyr or Y), cysteine (Cys or C), glutamine (Gln or Q) and asparagine (Asn or N). Basic amino acids are considered to include lysine (Lys or K), arginine (Arg or R) and histidine (His or H). Acidic amino acids are aspartic acid (Asp or D) and glutamic acid (Glu or E). Unless otherwise indicated or obvious from the context, amino acids referred to herein are to be considered L-amino acids. Standard amino acids can also be replaced with non-standard amino acids belonging to the same chemical class. As a non-limiting example, the basic side chain Lys can be replaced with a basic amino acid having a shorter side chain (ornithine, diaminobutyric acid or diaminopropionic acid). Lys can also be replaced with the neutral aliphatic isostere norleucine (Nle), which in turn can be replaced with an analogue having a shorter aliphatic side chain (aminobutyric acid or aminopropionic acid). In some embodiments, the term "conservative mutation" is used to refer to an amino acid modification that does not significantly affect or alter the activity of the parental polypeptide. For example, a variant after conservative modification retains at least 80%, 85%, 90%, 95%, 98%, 99% or higher, e.g., 100 - 110% or higher activity relative to the parental polypeptide.

[0018] In some embodiments, the present invention can also use active fragments of the IGF1C polypeptide, that is, fragments containing the minimal active expression unit SSSR, as long as they have the activity of the IGF1C polypeptide. The active fragments applicable to the present invention can be fragments of wild-type IGF1C containing SSSR or fragments of its variants. In some embodiments, the active fragments applicable to the present invention contain the amino acids SSSR (SEQ ID NO:2), SSSRR (SEQ ID NO:3), GSSSR (SEQ ID NO:4), GSSSRR (SEQ ID NO:5), ASSSRRAP (SEQ ID NO:6), GSSSRAAP (SEQ ID NO:7), GSSSRRAP (SEQ ID NO:8), GSSSRAAAP (SEQ ID NO:9), or GYGSSSRRAPQT (SEQ ID NO:1), or are composed of the said amino acid sequences.

[0019] In some embodiments, the IGF1C polypeptide or its active fragments of the present invention have one or more of the following activities:

[0020] (1) Bind to IGF1R;

[0021] (2) Competitively inhibit the binding of IGF1 to IGF1R;

[0022] (3) Inhibit the phosphorylation of IGF1R induced by IGF1;

[0023] (4) Inhibit IGF1-mediated smooth muscle phenotypic transformation, that is, from a contractile phenotype to a degradative phenotype (for example, inhibit the expression of MMP9 in smooth muscle cells);

[0024] (5) Inhibit aneurysms (such as the occurrence and development of aneurysms);

[0025] (6) Inhibit the degradation of elastic fibers in arteries.

[0026] In some embodiments, IGF1C is IGF1C bound to a chitosan molecule (CS-IGF1C). In some embodiments, the CS-IGF1C is obtained by the following methods. For example, IGF1C is covalently bound to a chitosan molecule through a chemical reaction; or (2) IGF1C is physically mixed and bound into chitosan. In some embodiments, the chemical reaction is a Click reaction and / or a condensation reaction.

[0027] In some embodiments, the insulin-like growth factor C domain polypeptide is obtained by covalently bonding to a chitosan molecule through a chemical reaction. Among them, CS-IGF-1C is synthesized by performing a Click chemical reaction on the azide of IGF-1C-N3 and the alkyne of alkynylated chitosan. In some embodiments, the conditions of the Click chemical reaction are that the feed molar ratio of IGF-1C-N3:CuSO4:sodium ascorbate is 1:0.2:0.4.

[0028] In some embodiments, CS-IGF-1C is obtained by synthesis through a Click reaction. The reaction includes dissolving alkynyl-substituted chitosan (CS), mixing it with IGF-1C-N3 obtained by connecting 6-azidohexanoic acid to the N-terminus of the C domain using a condensation reaction, and sodium ascorbate, and then performing dialysis and lyophilization after the Click reaction.

[0029] In some embodiments, the CS-IGF-1C is described in CN106667899B, which is incorporated herein by reference in its entirety.

[0030] In some embodiments, the IGF1R inhibitor can be an antibody or an antigen-binding fragment thereof that specifically binds to IGF1R. In some embodiments, the antibody contains 3 heavy-chain CDRs and 3 light-chain CDRs of cetuximab or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment contains the heavy-chain variable region and the light-chain variable region of cetuximab. In some embodiments, the antibody or its antigen-binding fragment contains the heavy chain and the light chain of cetuximab. In some embodiments, the antibody is cetuximab.

[0031] In some embodiments, the IGF1R inhibitor can be a small molecule inhibitor, such as a small molecule inhibitor that specifically binds to IGF1R. In some embodiments, the small molecule inhibitor is linsitinib or podophyllotoxin.

[0032] The present invention also relates to a drug or preparation containing an IGF1R inhibitor, and the drug or preparation is used for treating aneurysms or aortic dissections. In some embodiments, the drug or preparation further contains a pharmaceutical excipient, such as a pharmaceutical carrier known in the art.

[0033] As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, and the like. Pharmaceutical carriers suitable for use in the present invention include, for example, diluents, adjuvants, excipients, or carriers for administering a treatment, such as steroid liquids, such as water and oils, including those from petrochemical, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk, glycerol, propylene glycol, water, ethanol, and the like. If desired, the composition may also contain small amounts of wetting agents or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, hair tonics, capsules, powders, sustained release formulations, and the like. In some embodiments, the pharmaceutical carrier is a hydrogel, such as a chitosan hydrogel (CN106667899A) or a gelatin hydrogel. For the use and applications of pharmaceutical excipients, see also "Handbook of Pharmaceutical Excipients", 8th Edition, R.C. Rowe, P.J. Seskey, and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0034] In some embodiments, the present invention relates to a hydrogel comprising an IGF1R inhibitor. In some embodiments, the present invention relates to a hydrogel comprising an IGF1C polypeptide or an active fragment thereof.

[0035] Materials suitable for preparing the hydrogels of the present invention are selected from chitosan hydrogels, alginate hydrogels, hyaluronic acid hydrogels, PLGA hydrogels, PEG hydrogels, and gelatin hydrogels. In some embodiments, the gelatin hydrogel is gelatin methacrylate GelMa. In some embodiments, the hydrogel is GelMa60.

[0036] In some embodiments, the hydrogel comprising an IGF1C polypeptide or an active fragment thereof is prepared by physically mixing the IGF1C polypeptide or an active fragment thereof with the hydrogel. In some embodiments, the hydrogel comprising an IGF1C polypeptide or an active fragment thereof is prepared by functionalizing the IGF1C polypeptide or an active fragment thereof, such as by chemically binding it to a hydrogel material.

[0037] In some embodiments, the hydrogel comprising the IGF1C polypeptide or its active fragment is obtained by dissolving the IGF1C polypeptide or its active fragment in a GelMa hydrogel solution. In some embodiments, the concentration of the IGF1C polypeptide or its active fragment is between greater than 0.5 - 5 mg / ml, such as approximately 1, 2, or 3 mg / ml. In some embodiments, the concentration of GelMa in the GelMa hydrogel solution is between approximately 5% - 15%, such as approximately 10%. In some embodiments, the GelMa is GelMa60. In some embodiments, after dissolution, it further includes gelation of the hydrogel by excitation with light having a wavelength of 400 - 450 nm (EFL light source).

[0038] Thus, in some embodiments, the hydrogel comprising the IGF1C polypeptide or its active fragment comprises the IGF1C polypeptide and GelMa, such as GelMa60.

[0039] In some embodiments, the hydrogel containing the IGF1C polypeptide or its active fragment of the present invention is obtained as follows:

[0040] (1) Prepare a GelMa hydrogel solution, wherein the concentration is between 5% - 15%, such as approximately 10%,

[0041] (2) Dissolve the IGF1C polypeptide or its active fragment in the GelMa hydrogel solution at a certain concentration, such as the concentration is approximately 1 mg / ml; and

[0042] (3) Stir evenly.

[0043] In some embodiments, the hydrogel is gelled by excitation with light having a wavelength of approximately 400 - 450 nm (such as approximately 405 nm, such as EFL light source) (irradiation for approximately 0.5 - 1.5 minutes, such as approximately 1 minute). In some embodiments, the hydrogel is gelled by laser excitation before administration.

[0044] In some embodiments, the hydrogel comprising the IGF1C polypeptide or its active fragment is an injectable hydrogel.

[0045] In some embodiments, the hydrogel comprising the IGF1C polypeptide or its active fragment is a chitosan hydrogel, such as the chitosan hydrogel described in CN106667899B (incorporated herein in its entirety).

[0046] The IGF1R inhibitor of the present invention can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0047] The effective amount of the IGF1R inhibitor of the present invention in treating a disorder or disease can be determined by standard clinical techniques based on this specification. The exact dose used in the formulation depends on the route of administration and the severity of the condition, and should be determined according to the judgment of the practitioner and the circumstances of each subject.

[0048] In one embodiment, the drug or formulation containing the IGF1R inhibitor of the present invention is locally delivered to the lesion site (such as at an aneurysm or arterial dissection). In some embodiments, the drug or formulation of the present invention, such as a hydrogel, is delivered to the lesion site by laparocentesis. In some embodiments, the lesion site is the adventitia of the diseased aortic blood vessel. In some embodiments, the laparocentesis is performed under ultrasound guidance.

[0049] In some embodiments, the drug or formulation is administered by laparocentesis under ultrasound guidance (such as by injection), for example, locally, such as directly delivered to the lesion site such as at an aneurysm or arterial dissection, or the adventitia of the diseased aortic blood vessel.

[0050] In some embodiments, the drug or formulation containing the IGF1R inhibitor of the present invention is a hydrogel containing the IGF1R inhibitor. In some embodiments, after being delivered to the lesion site, a light source is applied to irradiate the hydrogel to form a gel.

[0051] In one embodiment, the drug or formulation containing the IGF1R inhibitor of the present invention is administered by injection.

[0052] In some embodiments, the drug or formulation of the present invention is administered by local sustained release.

[0053] In some embodiments, the drug or formulation of the present invention is administered minimally invasively.

[0054] In some embodiments, the administration of the drug or formulation of the present invention can reduce the side effects of systemic delivery.

[0055] In some embodiments, the ultrasound-mediated method includes the following steps:

[0056] (1) The ultrasound instrument detects the location of the artery (such as the aorta or peripheral artery, such as in the abdomen or chest, such as the abdominal aorta) (such as in the abdominal - high penetration mode, obtained by moving the probe).

[0057] (2) In front of the position where the probe is located, insert the puncture needle (for example, at an angle of about 50 - 70°, such as about 60°);

[0058] (3) Locate the puncture needle in the ultrasound image;

[0059] (4) Under the guidance of the ultrasound image, guide the puncture needle to the aneurysm site;

[0060] (5) Inject a drug or preparation (such as a hydrogel containing IGF1C or a fragment thereof);

[0061] (6) Induce gelation of the laser (such as a laser of about 400 - 450 nM) optical fiber through the puncture needle;

[0062] (7) Withdraw the puncture needle and disinfect the local area (such as applying povidone iodine).

[0063] In some embodiments, the present invention also relates to a kit, which comprises the drug or preparation of the present invention, and one or more other therapeutic agents, such as drugs for adjuvant treatment of aneurysms or aortic dissections.

[0064] The present invention also provides a drug combination or a drug combination product, which comprises the IGF1 polypeptide or a fragment thereof or a pharmaceutically acceptable salt thereof of the present invention. Optionally, the drug combination or the drug combination product further comprises one or more other therapeutic agents.

[0065] The present invention also provides a kit containing the drug combination, for example, the kit contains in the same package:

[0066] - A first container containing a pharmaceutical composition comprising the drug of the present invention or a pharmaceutically acceptable salt thereof;

[0067] - Optionally, a second container further containing a pharmaceutical composition comprising one or more other therapeutic agents (in some embodiments, two or more other therapeutic agents are in the same container or in different containers respectively).

[0068] The present invention also relates to an IGF1R inhibitor or a drug or preparation containing the same, which is used for the treatment of diseases, for example, for the uses of the present invention, such as for preventing or treating aneurysms or aortic dissections.

[0069] The present invention also relates to the use of an IGF1R inhibitor in the preparation of a drug or preparation, and the drug or preparation is used for the treatment of diseases, for example, for the uses of the present invention, such as for preventing or treating aneurysms or aortic dissections.

[0070] Definitions:

[0071] It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0072] The term "about", when used in conjunction with a numerical value, means a numerical value that encompasses a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0073] As used herein, the term "and / or" means any one of the alternatives, or two or more or all of the alternatives.

[0074] As used herein, the term "comprising" or "including" means including the stated elements, integers, or steps, but not excluding any other elements, integers, or steps. In this context, when the term "comprising" or "including" is used, unless otherwise specified, the case consisting of the stated elements, integers, or steps is also covered. For example, when referring to a protein "comprising" a specific sequence, it is also intended to cover a protein consisting of that specific sequence.

[0075] Insulin-like growth factors are important growth factors that regulate the growth of human cells, including insulin-like growth factor-1 (hereinafter referred to as "IGF-1") and insulin-like growth factor-2 (hereinafter referred to as "IGF-2"). The mature form of IGF-1 (UniProtKB entry P05019) is a 70-amino acid polypeptide that shares a large segment of sequence identity and high structural homology with IGF-2 and insulin. The IGF-1 polypeptide chain can contain 4 domains, namely B (amino acid residues 1-29), C (30-41), A (42-62), and D (63-70). Domain A and domain B are structural homologs of the B and A chains of insulin, respectively. Domain C is similar to the connecting peptide of proinsulin, while domain D has no counterpart in insulin. IGF1 receptors include: insulin receptor (IR), insulin-like growth factor 2 receptor (IGF2R), hybrid receptor (IR / IGF1R), and other insulin receptor-related receptors (IRR). Among them, IGF1 has the strongest affinity for the IGF1R receptor. IGF1R is located on the cell surface and is a tetrameric tyrosine kinase receptor. After binding to the ligand, it activates downstream signaling pathways such as Ras-raf-MAPK and PI3K-AKT-mTOR to play a role in promoting cell proliferation and regulating cell phenotype. IGF1 / IGF1R is a key target for the treatment of diseases such as cancer and glaucoma. Drugs targeting this target have been developed and have achieved good results in clinical treatment.

[0076] In this text, an amino acid mutation can be an amino acid substitution, deletion, insertion, or addition. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of a polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. In some embodiments, preferred amino acid mutations are amino acid substitutions, such as single amino acid substitutions, or combinations of several amino acid substitutions.

[0077] In this text, when referring to the amino acid position to be mutated, it is determined by reference to the amino acid sequence shown in SEQ ID NO:1. The corresponding amino acid positions on polypeptides with other amino acid sequences can be identified by aligning the amino acid sequences with SEQ ID NO:1. For example, when referring to "G3", it refers to the G at the 3rd position of SEQ ID NO:1, or the amino acid residue at the corresponding position on the amino acid sequence of other variant polypeptides after alignment.

[0078] In this text, when referring to mutations of the IGF1C polypeptide, single amino acid substitutions are described in the following manner: [original amino acid residue / position / substituted amino acid residue] or [position / substituted amino acid residue]. For example, the substitution of glycine (or the corresponding other amino acid) at position 3 with alanine can be represented as G3A or as 3A. When there are multiple optional amino acid substitution ways (such as A, R) at a given position (such as position 3), the amino acid substitution can be represented as: 3A / R or G3 / A / R. Accordingly, the single amino acid substitutions can be linked together by the plus sign "+" or "-".

[0079] The terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include but are not limited to domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0080] The term "treatment" includes administering a composition or a heterologous polypeptide to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease, relieve symptoms, or prevent or inhibit the further progression of a disease, condition, or disorder. The term "prevention" includes the inhibition of the occurrence or development of a disease or disorder or the symptoms of a specific disease or disorder.

[0081] The term "pharmaceutical excipient" refers to diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), excipients, carriers, or stabilizers, etc., that are administered together with the active substance.

[0082] The term "pharmaceutical composition" refers to a composition that exists in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0083] The term "effective amount" refers to such an amount or dose of a polypeptide, composition, or combination of the present invention that, when administered to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prophylaxis.

[0084] "Therapeutically effective amount" refers to an amount that, at the required dose and for the required period of time, effectively achieves the desired therapeutic result. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the IGF1C polypeptide, composition, or combination are less than the therapeutic beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits or improves a measurable parameter by at least about 40%, even more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0085] "Prophylactically effective amount" refers to an amount that, at the required dose and for the required period of time, effectively achieves the desired prophylactic result. Generally, since prophylactic doses are used in a subject before or at an earlier stage of a disease, a prophylactically effective amount will be less than a therapeutically effective amount.

[0086] As used herein, the term "IGF1R inhibitor" refers to a compound that targets, reduces, or inhibits at least one activity of the insulin growth factor-1 receptor, including antibodies, proteins, nucleic acids, or small molecule inhibitors. In some embodiments, the IGF1R inhibitor has one or more of the following activities: inhibiting the binding of IGF1 to IGF1R, inhibiting IGF1-induced phosphorylation of IGF1R, and inhibiting one or more other activities of IGF1R.

[0087] "GelMA" described in this article is a photosensitive hydrogel material - gelatin methacrylate anhydride, and its advantages include: having cell adhesion sites and matrix metalloproteinase hydrolysis sites in its structure, so it can well support cell proliferation and migration; the presence of methacrylate anhydride groups makes it photosensitive and can rapidly crosslink under ultraviolet light irradiation; its physical and chemical properties can be flexibly adjusted; it can be fabricated into structural units with unique morphological characteristics through many microfabrication processes, such as bioprinting, photolithography technology, self-assembly, microfluidics technology, etc. GelMA hydrogel has been proven to have unique advantages in tissue regeneration such as bone, cartilage, myocardium, blood vessels, etc., and has also achieved good results in fields such as basic cell research, cell signal transduction, drug / gene controlled release, and biological sensing. These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows immediately) and in the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application can be combined in various embodiments of the present invention. The following examples further illustrate the present invention. However, it should be understood that the examples are described in an illustrative rather than limiting manner, and those skilled in the art can make various modifications. Brief Description of the Drawings:

[0088] Figure 1 It shows that IGF1C inhibits the binding of IGF1 to smooth muscle cells.

[0089] Figure 2 It shows that IGF1C inhibits the binding of IGF1 to IGF1R.

[0090] Figure 3 It shows that IGF1C inhibits IGF1-induced phosphorylation of IGF1R.

[0091] Figure 4 It shows the cell image of IGF1C inhibiting IGF1-mediated phosphorylation of IGF1R.

[0092] Figure 5 It shows that IGF1C inhibits IGF1-mediated MMP9 expression.

[0093] Figure 6 It shows that IGF1C inhibits AngⅡ-induced aneurysm formation.

[0094] Figure 7 It shows that IGF1C inhibits extracellular matrix degradation.

[0095] Figure 8 It shows that IGF1C inhibits AngⅡ-induced phenotypic transformation of smooth muscle cells.

[0096] Figure 9 It shows that IGF1C inhibits CaCl2-induced aneurysm formation.

[0097] Figure 10 It shows that cetuximab inhibits CaCl2-induced aneurysm formation.

[0098] Figure 11 It shows that lestaurtinib (OSI-906) inhibits CaCl2-induced aneurysm formation.

[0099] Figure 12 It shows that picropodophyllotoxin (AXL1717) inhibits CaCl2-induced aneurysm formation.

[0100] Figure 13 It shows that the IGF1R inhibitor prevents CaCl2-induced elastic lamina degradation.

[0101] Figure 14 It shows that the IGF1R inhibitor prevents CaCl2-induced smooth muscle cell phenotypic transformation.

[0102] Figure 15 It shows that the IGF1C-loaded hydrogel inhibits CaCl2-induced aneurysm formation.

[0103] Figure 16 It shows that the IGF1C-loaded hydrogel is coated on the outer wall of the aneurysm blood vessel.

[0104] Figure 17 It shows that the IGF1C-loaded hydrogel inhibits aneurysm formation induced by rat xenotransplantation.

[0105] Figure 18 The figure shows that the IGF1C-functionalized hydrogel inhibits aneurysm formation induced by rat xenotransplantation.

[0106] Figure 19 It shows that the IGF1C-loaded hydrogel inhibits the occurrence of abdominal aortic aneurysm in Bama pigs.

[0107] Figure 20 It shows the ultrasound-guided delivery of the hydrogel to the abdominal aorta.

[0108] Figure 21 It shows the CT image of the distribution of the hydrogel in the abdominal aorta.

[0109] Figure 22 It shows the ultrasound-guided delivery of the IGF1C-loaded hydrogel to transplant and expand the abdominal aortic aneurysm in Bama pigs. Example:

[0110] Example 1. Flow cytometry detection of IGF1C inhibiting the binding of IGF1 to smooth muscle cells

[0111] Experimental method: 1×10 per well 6The density of human aortic smooth muscle cells (purchased from Procell) was seeded into 6-well plates. The cells were cultured in smooth muscle cell complete medium (SMCM, Sciencell) at 37 °C and 5% CO2. When the density reached 90%, after washing with PBS, the smooth muscle cells were digested with 0.5 mL of trypsin (0.25%) without EDTA. After the cells detached from the bottom of the culture plate, 1 mL of complete medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm at 23 °C. The supernatant was discarded, and the cells were resuspended with PBS and centrifuged again at 1000 rpm at 23 °C. The cells were resuspended with 1 mL of serum-free medium and the cell suspension was gently mixed.

[0112] The pre-prepared synthetic IGF1C polypeptide (SEQ ID NO: 1 GYGSSSRRAPQT, concentration 100 μg / mL) stock solution (GenScript) was added to make the final concentration of the polypeptide 0, 10, and 100 ng / mL respectively. After incubation for 30 minutes, the recombinant IGF1-His (IG1-H5245, ACROBiosystems) solution was added to make its final concentration 1 μg / mL. After 30 minutes, the cells were washed with PBS. After blocking the cells with 4% BSA for 30 minutes, the His antibody (ab245114) was incubated. After secondary antibody binding, the proportion of cells binding to exogenous IGF1-His was detected by flow cytometry.

[0113] The results are shown in Figure 1 , IGF1C effectively inhibited the binding of IGF1 to the cells. As the concentration of IGF1C increased, the proportion of cells binding to exogenous IGF1 decreased.

[0114] Example 2. Immunoprecipitation (COIP) assay for detecting the experiment of IGF1C inhibiting the binding of IGF1 to IGF1R

[0115] Experimental method: At a density of 1×10 per well 6The density of human aortic smooth muscle cells (purchased from Punosai Biotechnology Co., Ltd.) was seeded into 6-well plates. The cells were cultured in smooth muscle cell complete medium (SMCM, Sciencell) at 37 °C and 5% CO2. After the density reached 70%, the medium was replaced with fresh serum-free medium. After the cells were starved in serum-free medium for 24 hours, the fresh medium was replaced again, and the pre-prepared IGF1C stock solution was added respectively to make the final concentration of the polypeptide 0, 10, 100 ng / mL. After 30 minutes, recombinant IGF1-His (IG1-H5245, ACROBiosystems) solution was added to make the final concentration 1 μg / mL for treatment. After incubation at 37 °C for 30 minutes, the cells were washed twice with pre-cooled PBS. Total cellular proteins were extracted and the concentration was measured using the BCA method (PC0020, Solarbio).

[0116] The protein homogenate of the above total cellular proteins was used as the Input sample, and the levels of IGF1R and β-actin proteins in the sample were detected by WB. IGF1R antibody (Cell signalling technology, catalog number: A0243 rabbit source, concentration based on titer confirmation) was added to the remaining protein homogenate, and a CoIP kit (88804, Pierce TM ) was used to collect the IGF1R antibody-labeled proteins. Through the CoIP experiment, the competitive binding of IGF1C to IGF1R was detected. The results are shown in Figure 2 .

[0117] Immunoprecipitation (IP) was applied to detect IGF1R, and immunoblotting (IB) was applied to detect IGF1-His. As the concentration of IGF1C increased, the IGF1-His bound to IGF1R gradually decreased.

[0118] The expressions of IGF1R and β-actin (internal reference) were detected by Western blotting in Input, showing that the expression of IGF1R did not change.

[0119] Therefore, IGF1C competes with IGF1 for binding to IGF1R, and the introduction of IGF1C interferes with the binding of IGF1 and IGF1R.

[0120] Example 3. Immunoblotting (IB) experiment to detect the inhibition of IGF1-induced IGF1R phosphorylation by IGF1C

[0121] Experimental method: At 1×10 per dish 6Seed human aortic smooth muscle cells into confocal dishes at a density of. Culture them normally in smooth muscle complete medium (SMCM, Sciencell). After the density reaches 70%, replace with fresh serum-free medium. After 24 hours, replace the fresh medium again and add the pre-prepared IGF1C stock solution to make its final concentrations 0, 10, and 100 ng / mL respectively. After 30 minutes, stimulate with 25 ng / mL of recombinant IGF1 protein (Novoprotein, C032). Collect proteins at four time points: 0, 20, 40, and 60 minutes. Detect the level of phosphorylated IGF1R (pIGF1R) by western blot (Cell signalling technology, catalog number: A0243 rabbit source, concentration confirmed based on titer). The results are shown in Figure 3 , the binding of IGF1C to IGF1R does not cause phosphorylation of the receptor. After IGF1 stimulation, the phosphorylation level of IGF1R gradually increases, reaches a peak at 40 minutes, and then gradually declines. Pretreatment with IGF1C significantly inhibits the phosphorylation level of IGF1R.

[0122] Example 4: Detection of IGF1C inhibition of IGF1-induced IGF1R phosphorylation by immunofluorescence staining

[0123] Experimental method: Seed human aortic smooth muscle cells into confocal dishes at a density of 1×10 per dish 6 . Culture them normally in smooth muscle complete medium (SMCM, Sciencell). After the density reaches 70%, replace with fresh serum-free medium. After 24 hours, replace the fresh medium again and add the pre-prepared IGF1C stock solution to make its final concentration 100 ng / mL. After 30 minutes, stimulate with 25 ng / mL of recombinant IGF1 protein (Novoprotein, C032).

[0124] After 40 minutes, wash the cells with PBS. Fix with 4% paraformaldehyde solution for 30 minutes. Wash twice with PBS. Perform immunofluorescence staining of the cells with an antibody against phosphorylated IGF1R protein (AF3123, Affinity Biosciences). Observe the phosphorylation degree of IGF1R protein on the cell surface using a fluorescence upright microscope (Axio Imager Z1, ZEISS). The results are shown in Figure 4, the control group was smooth muscle cells not subjected to any stimulation; the IGF1C group was smooth muscle cells pretreated only with IGF1C; the IGF1 group was smooth muscle cells stimulated only with IGF1; the IGF1 + IGF1C group was smooth muscle cells stimulated with IGF1 after pretreatment with IGF1C. The results showed that the binding of IGF1C to IGF1R did not cause phosphorylation of the receptor. At the same time, IGF1C competitively bound to IGF1R, thereby inhibiting the phosphorylation of IGF1R induced by IGF1.

[0125] Example 5, IGF1C inhibits IGF1-mediated phenotypic transformation of smooth muscle cells

[0126] IGF1 can induce phenotypic transformation of smooth muscle cells, from a contractile phenotype to a degrading phenotype, manifested by an increase in MMP9 expression. This example demonstrated that IGF1C can inhibit the phenotypic transformation of smooth muscle cells induced by IGF1.

[0127] Experimental method: Human aortic smooth muscle cells were seeded into confocal dishes at a density of 1×10 5 . They were cultured normally in smooth muscle complete medium (SMCM, Sciencell). After the density reached 50%, the medium was replaced with fresh serum-free medium. After 24 hours, they were treated with 10 ng / ml of IGF1C. After 24 hours, the medium was replaced with fresh serum-free medium, and the cells were stimulated with 25 ng / mL of recombinant IGF1 protein (Novoprotein, C032). The cells were washed twice with pre-cooled PBS. Fixed with 4% paraformaldehyde solution for 30 minutes. Washed twice with PBS. The cells were immunofluorescently stained with an antibody against MMP-9 (ab228402, Abclonal). And the expression level of MMP-9 protein in the cells was observed using a fluorescence upright microscope (Axio Imager Z1, ZEISS). The results are shown in Figure 5 , the Control group was smooth muscle cells not subjected to any stimulation; the IGF1C group was smooth muscle cells pretreated only with IGF1C (10 ng / ml); the IGF1 group was smooth muscle cells stimulated only with IGF1 (25 ng / mL); the IGF1 + IGF1C group was smooth muscle cells stimulated with IGF1 after pretreatment with IGF1C.

[0128] Figure 5 The results showed that MM9 was expressed in smooth muscle cells treated only with IGF1C, while the expression of MMP9 was significantly reduced in the IGF1 + IGF1C group treated with IGF1C later. The results indicated that IGF1C inhibited IGF1-mediated MMP9 expression.

[0129] Example 6, IGF1C inhibits Ang II-induced aneurysm formation

[0130] In this example, an aneurysm model induced by AngII was constructed to investigate the therapeutic effect of IGF1C as an inhibitor of IGF1R in this abdominal aortic aneurysm model.

[0131] Experimental method: Twenty 10-week-old male ApoE− / − C57BL / 6 mice were purchased from Vital River Laboratories and housed in a stable environment for 3 days before modeling. An aneurysm model was constructed using an osmotic pump (model 2004; Alzet, Cupertino) to slowly release AngII (HY-13948, MedChemexpress).

[0132] Specifically as follows: Mice were fasted and weighed 36 h before surgery; the amount of AngII required for 28 days of modeling was calculated based on body weight and loaded into the pump, with the release rate of AngII being (1000 ng / kg / min); the mice were anesthetized with 4% chloral hydrate (8 μl / g), placed prone on the experimental table, and fixed; the hair on the neck was removed with depilatory cream and disinfected with iodophor; an approximately 1-cm surgical incision was made in the neck skin using ophthalmic scissors. Then, the skin and muscle on the back were bluntly dissected using a needle holder, and the osmotic pump containing AngII was implanted subcutaneously; the wound was sutured with 5-0 sutures, and after applying iodophor again, the mice were placed in a clean cage; they were fed with regular feed, and the 20 model mice were randomly divided into two groups.

[0133] Saline group: 100 μl of normal saline was intraperitoneally injected every two days; IGF1C treatment group: 100 μl of a 1 mg / ml IGF1C (SEQ ID NO:1) solution was intraperitoneally injected every two days. Samples were taken after 28 days.

[0134] The results are shown in Figure 6 , Figure 6 A is a photograph of a representative mouse. Figure 6 B is the average value of all mice (the average maximum diameter of aneurysms in the saline group of 20 mice and the average maximum diameter of aneurysms in the IGF1C group). The results showed that the supra-renal aorta in the saline group (Saline) was significantly enlarged, while the addition of IGF1C significantly inhibited the occurrence of aneurysms.

[0135] Example 7. IGF1C inhibits the degradation of arterial elastic lamina induced by AngII

[0136] Experimental method: Van Gieson's staining kit (DC0058, Legend) was used for VVG staining to evaluate the degradation of elastic fibers. Van Gieson's staining kit (DC0058, Legend) was used for VVG staining to evaluate the degradation of elastic fibers.

[0137] The samples of the suprarenal abdominal aorta of the saline group and the IGF1C group in Example 6 were frozen sectioned, left at room temperature for 10 min, and rinsed with running water for 5 min; Verhoeff dye A, Verhoeff dye B and Verhoeff dye C were prepared in a ratio of 5:2:2; the sections were placed in the dye for 30 min; quickly rinsed with running water for 5 min; differentiated with differentiation solution and examined under a microscope until the elastic fibers were black and the background was gray; rinsed with running water for 5 min; deiodinated with 95% ethanol for 1 s; stained with Lichun red for 10 s, and the staining solution was discarded; dehydrated with anhydrous ethanol for 1 s; transparentized with xylene twice, each time for 3 min; sealed with neutral gum, and photographed and observed under an upright optical microscope (Leica DFC420C).

[0138] Results Figure 7 . Figure 7 A is a photograph of a representative mouse. Figure 7 B is the average value of all mice (average value of elastic fiber degradation level). The results showed that AngII treatment induced the rupture and degradation of elastic fibers (black) in the vascular wall, while the addition of IGF1C inhibited the degradation of elastic fibers, and the vascular wall contained relatively continuous and complete elastic fibers.

[0139] Example 8: IGF1C inhibits AngⅡ-induced smooth muscle cell phenotypic transformation

[0140] Experimental method: CNN1 and α-SMA, MMP9 and α-SMA were co-stained to examine the number of contractile and degradative smooth muscle cells, respectively.

[0141] The samples of the suprarenal abdominal aorta in the saline group and the IGF1C group in Example 6 were frozen and fixed with ice acetone for 10 minutes; after drying, they were washed twice in PBS; 0.1% Triton was used to break the membrane for 9 minutes, and PBS was used to wash twice; goat serum was used to block at room temperature for 30 minutes; antibodies to CNN1 (abclonal, A3734) and α-SMA (abcam, ab7817) (or MMP9 (abclonal, A0289) and α-SMA (abcam, ab7817)) were added, respectively, overnight at 4°C, and secondary antibody staining was performed after washing 6 times with PBS. Finally, DAPI resin (Southerntech) was used to seal the slides. The captured images were observed using a fluorescent advanced upright microscope (Axio Imager Z1, ZEISS).

[0142] Results Figure 8 , Figure 8 A is an image of a representative mouse. Figure 8B is the average value of all mouse statistics (the average number of MMP9 and α-SMA double-positive cells and the average number of CNN1 and α-SMA double-positive cells). After AngII treatment, the number of control group (saline group) induced degradation phenotype smooth muscle cells (showing MMP9 and α-SMA double-positive) increased significantly, while the number of contractile smooth muscle cells (showing CNN1 and α-SMA double-positive) decreased significantly. IGF1C effectively inhibited the phenotypic transformation of smooth muscle cells, making the smooth muscle cells show a contractile phenotype.

[0143] Example 9: IGF1C inhibits CaCl2-induced aneurysm formation

[0144] In this example, a CaCl2-induced aneurysm model was constructed to investigate the therapeutic effect of IGF1C as an inhibitor of IGF1R in this model.

[0145] Experimental method: Twelve 10-week-old male C57BL / 6 mice were purchased from Vital River Laboratories and housed in a stable environment for 3 days before modeling. A CaCl2 (Sigma, SLCG2286)-induced aneurysm model was constructed.

[0146] Specifically as follows: The mice were anesthetized with 4% chloral hydrate (8 μl / g), and the mice were glued to the experimental table in the supine position; the abdominal hair was removed with depilatory cream and disinfected with iodophor; an abdominal skin incision about 1 cm long was made with an ophthalmic scissors, and then the abdominal aorta was dissected free with microsurgical forceps. A 1×1 cm 2 gauze was soaked in 0.5 M CaCl2 solution for 5 minutes. Subsequently, the soaked gauze was covered on the dissected and exposed abdominal aorta and incubated for 15 minutes. After removing the gauze, the incubation site was washed with PBS. After replacing the internal organs, the wound was sutured with 5-0 suture, and the mice were placed in a clean cage after applying iodophor again; they were fed with normal feed. The 12 model mice were randomly divided into two groups.

[0147] Saline group: 100 μl of normal saline was intraperitoneally injected every two days; IGF1C treatment group: 100 μl of 1 mg / ml IGF1C solution was intraperitoneally injected every two days. Samples were taken after 28 days. The aorta was dissected and observed under a stereomicroscope.

[0148] The results are shown in Figure 9 , Figure 9 A shows representative observation images, Figure 9 B shows the average value of the maximum diameter of aneurysms in all mice in the saline group and the IGF1C group. The results showed that the infrarenal abdominal aorta of mice in the saline group was significantly enlarged, while IGF1C significantly inhibited the formation of aneurysms.

[0149] Example 10: Cetuximab inhibits CaCl2-induced aneurysm formation

[0150] In this example, a CaCl2-induced aneurysm model was constructed to investigate the therapeutic effect of cetuximab as an antibody inhibitor of IGF1R in this abdominal aortic aneurysm model.

[0151] Experimental method: Six 10-week-old male C57BL / 6 mice were purchased from Vital River Laboratories and housed in a stable environment for 3 days before modeling. A CaCl2 (Sigma, SLCG2286)-induced aneurysm model was constructed.

[0152] Specifically as follows: The mice were anesthetized with 4% chloral hydrate (8 μl / g) and glued to the experimental table in a supine position; the abdominal hair was removed with depilatory cream and disinfected with iodophor; an abdominal skin incision about 1 cm long was made with an ophthalmic scissors, and then the abdominal aorta was dissected free with microsurgical forceps. A 1×1 cm 2 gauze was soaked in 0.5 M CaCl2 solution for 5 minutes. Subsequently, the soaked gauze was placed over the dissected and exposed abdominal aorta and incubated for 15 minutes. After removing the gauze, the incubation site was washed with PBS. After replacing the viscera, the wound was sutured with 5-0 suture, and the mice were placed in a clean cage after applying iodophor again; they were fed with normal feed,

[0153] Every two days, 100 μl of a 200 μg / mL cetuximab solution was injected intraperitoneally. After 28 days, the samples were taken. The aorta was dissected and observed under a stereomicroscope.

[0154] The results are shown in Figure 10 , Figure 10 A shows a representative image, Figure 10 B shows the mean of the maximum values of the mouse aortas in the saline-treated group and the cetuximab-treated group. Compared with the saline group in Example 8, cetuximab effectively inhibited the dilation of the infrarenal abdominal aorta.

[0155] Example 11, Linsitinib (OSI-906) inhibits CaCl2-induced aneurysm formation

[0156] In this example, a CaCl2-induced aneurysm model was constructed to investigate the therapeutic effect of linsitinib as a small molecule inhibitor of IGF1R in the abdominal aortic aneurysm model.

[0157] Experimental method: Twelve 10-week-old male C57BL / 6 mice were purchased from Vital River Laboratories and housed in a stable environment for 3 days before modeling. A CaCl2 (Sigma, SLCG2286)-induced aneurysm model was constructed.

[0158] The specific steps are as follows: Anesthetize the mice with 4% chloral hydrate (8 μL / g), and stick the mice on the experimental table in the supine position; remove the abdominal hair with depilatory cream, and apply iodophor for disinfection; use ophthalmic scissors to make an incision about 1 cm long on the abdominal skin, and then use micro forceps to free the abdominal aorta. Soak a 1×1 cm 2 gauze in 0.5 M CaCl2 solution for 5 minutes. Then cover the freed and exposed abdominal aorta with the soaked gauze and incubate for 15 minutes. After removing the gauze, wash the incubation site with PBS. After replacing the internal organs, suture the wound with 5-0 suture thread, apply iodophor again, and place the mice in a clean cage; feed with normal feed and collect samples after 28 days. Randomly divide 12 model mice into two groups.

[0159] Solvent control group: Intraperitoneally inject 100 μL of the solvent without drug (DMSO: PEG300: Tween80: saline = 2:8:1:9) every two days; Linsitinib treatment group: Dissolve linsitinib drug in the mixed solvent (DMSO: PEG300: Tween80: saline = 2:8:1:9) at a concentration of 6.25 mg / mL. Intraperitoneally inject 100 μL of linsitinib solution every two days.

[0160] Collect samples after 28 days. Dissect the aorta and observe under a stereomicroscope.

[0161] The results are shown in Figure 11 , Figure 11 A shows representative images, Figure 11 B shows the average value of the maximum values of the aortas of mice in the solvent control treatment group and the linsitinib treatment group. Compared with the solvent control group, linsitinib effectively inhibited the dilation of the infrarenal abdominal aorta.

[0162] Example 12: Picropodophyllotoxin (AXL1717) inhibits CaCl2-induced aneurysm formation

[0163] In this example, a CaCl2-induced aneurysm model was constructed to investigate the therapeutic effect of picropodophyllotoxin as a small molecule inhibitor of IGF1R in the abdominal aortic aneurysm model.

[0164] Experimental method: Purchase 12 male C57BL / 6 mice at 10 weeks old from Vital River Laboratories, and house the mice in a stable environment for 3 days before modeling. Use CaCl2 (Sigma, SLCG2286) to construct an aneurysm model.

[0165] The specific steps are as follows: Anesthetize the mice with 4% chloral hydrate (8 μL / g), and stick the mice on the experimental table in the supine position; remove the abdominal hair with depilatory cream, and apply iodophor for disinfection; use ophthalmic scissors to make an incision about 1 cm long on the abdominal skin, and then use micro forceps to free the abdominal aorta. Soak a 1×1 cm2 The gauze was soaked in a 0.5 M CaCl2 solution for 5 minutes. Subsequently, the wet gauze was covered over the free and exposed abdominal aorta and incubated for 15 minutes. After removing the gauze, the incubation site was washed with PBS. After replacing the viscera, the wound was sutured with 5-0 sutures, and iodophor was applied again before placing the mice in a clean cage; they were fed with normal feed and sacrificed after 28 days. Twelve model mice were randomly divided into two groups.

[0166] Solvent control group: 100 μL of the drug-free solvent (DMSO:PEG300:Tween80:saline = 2:8:1:9) was intraperitoneally injected every two days;

[0167] Picropodophyllotoxin treatment group: The picropodophyllotoxin drug was dissolved in a mixed solvent (DMSO:PEG300:Tween80:saline = 2:8:1:9) at a concentration of 11.25 mg / mL. 100 μL of the picropodophyllotoxin solution was intraperitoneally injected every two days.

[0168] After 28 days, samples were taken. The aorta was dissected and observed under a stereomicroscope.

[0169] The results are shown in Figure 12 , Figure 12 A shows representative images, Figure 12 B shows the mean of the maximum values of the mouse aortas in the solvent control treatment group and the picropodophyllotoxin treatment group. Compared with the solvent control group, picropodophyllotoxin effectively inhibited the dilation of the infrarenal abdominal aorta.

[0170] Example 13. IGF1R inhibitor prevents CaCl2-induced elastic lamina degradation

[0171] Experimental method: VVG staining was performed using a Van Gieson's staining kit (DC0058, Legend) to evaluate elastic fiber degradation. Frozen sections were prepared from the infrarenal abdominal aorta samples of the saline group, solvent control group, IGF1C treatment group, cetuximab treatment group, linitinib treatment group, and picropodophyllotoxin treatment group in Examples 9, 10, 11, and 12, allowed to stand at room temperature for 10 min, and rinsed with running water for 5 min.

[0172] Prepare Verhoeff's stain by mixing Verhoeff's stain A, Verhoeff's stain B, and Verhoeff's stain C in a ratio of 5:2:2. Immerse the sections in the stain for 30 min, then quickly rinse with running water for 5 min. Differentiate with the differentiating solution and examine the differentiation under the microscope until the elastic fibers turn black and the background is gray. Rinse with running water for 5 min, deiodize with 95% ethanol for 1 s, stain with ponceau S for 10 s, and discard the staining solution. Dehydrate with absolute ethanol for 1 s, clear with xylene twice, 3 min each time. Mount with neutral balsam and observe under an upright optical microscope (Leica DFC420C) by taking pictures.

[0173] The results are shown in Figure 13 , Figure 13 A is a representative observation image. Figure 13 B is the average value of the degradation level of elastic fibers in each group of mice. The results showed that the elastic fibers (black) in the infrarenal abdominal aortic vascular wall of mice in the saline group and the solvent control group were broken and degraded, while the degradation of elastic fibers was inhibited in the IGF1C-treated, cetuximab-treated, linitinib-treated, and podophyllotoxin-treated groups, and the vascular wall contained relatively continuous and intact elastic fibers.

[0174] Example 14: IGF1R inhibitor prevents CaCl2-induced phenotypic transformation of smooth muscle cells

[0175] Experimental method: Co-stain CNN1 and α-SMA, and MMP9 and α-SMA respectively to examine the number of contractile and degradative smooth muscle cells.

[0176] For the infrarenal abdominal aortic samples of mice in the saline group, solvent control group, IGF1C-treated group, cetuximab-treated group, linitinib-treated group, and podophyllotoxin-treated group in Examples 9, 10, 11, and 12, prepare frozen sections and fix with ice-cold acetone for 10 min. After air-drying, wash twice in PBS. Permeabilize with 0.1% Triton for 9 min and wash twice in PBS. Block with goat serum at room temperature for 30 min. Add antibodies against CNN1 (abclonal, A3734) and α-SMA (abcam, ab7817) (or MMP9 (abclonal, A0289) and α-SMA (abcam, ab7817)) respectively, incubate overnight at 4 °C, and then stain with secondary antibodies (Thermofisher, A-11001) (Thermofisher, A-11012) after washing six times with PBS.

[0177] Finally, mount with DAPI resin (Southerntech). Examine the captured images using a fluorescence upright microscope (Axio Imager Z1, ZEISS).

[0178] The results are shown in Figure 14 ,Figure 14 A is the representative image of each group, Figure 14 B is the average number of double-positive cells of CNN1 and α-SMA in mice of each group, Figure 14 C is the average number of double-positive cells of MMP9 and α-SMA in mice of each group. The results showed that the number of subrenal abdominal aortic degrading phenotypic smooth muscle cells (showing double-positivity for MMP9 and α-SMA) in the saline group and the solvent control group of mice increased significantly, while the number of contractile smooth muscle cells (showing double-positivity for CNN1 and α-SMA) decreased significantly. Treatment with IGF1C, cetuximab, linitinib, and podophyllotoxin effectively inhibited the phenotypic transformation of smooth muscle cells, making the smooth muscle cells show a contractile phenotype.

[0179] Example 15. Preparation of IGF1C-loaded hydrogel

[0180] To avoid the side effects brought by the IGF1R inhibitor to the system, this study further constructed an IGF1C-loaded hydrogel for local delivery of IGF1C.

[0181] Experimental method: Commercial GelMa hydrogel (GelMa60) was purchased from EFL Company (Hangzhou), and a 10% solution was prepared. An appropriate amount of IGF1C powder was dissolved in the GelMa hydrogel solution at a concentration of 1 mg / ml. After stirring evenly, the hydrogel was gelled (IGF1C / GelMa) by light excitation at a wavelength of 400 - 450 nm (EFL light source).

[0182] Example 16. Inhibition of CaCl2-induced aneurysm formation by IGF1C-loaded hydrogel

[0183] In this example, a CaCl2-induced aneurysm model was constructed to investigate the therapeutic effect of the IGF1C-functionalized hydrogel in the abdominal aortic aneurysm model.

[0184] Experimental method: Six 10-week-old male C57BL / 6 mice were purchased from Vital River Laboratories. The mice were housed in a stable environment for 3 days before modeling. An aneurysm model was constructed using CaCl2 (Sigma, SLCG2286).

[0185] Specifically as follows: The mice were anesthetized with 4% chloral hydrate (8 μL / g), and the mice were adhered to the experimental table in the supine position; the abdominal hair was removed with depilatory cream and disinfected with iodophor; an abdominal skin incision about 1 cm long was made with an ophthalmic scissors, and then the abdominal aorta was dissected free using micro forceps. A 1×1 cm 2The gauze was soaked in 0.5 M CaCl2 solution for 5 minutes. Subsequently, the wet gauze was covered over the free and exposed abdominal aorta and incubated for 15 minutes. After removing the gauze, the incubation site was washed with PBS. The IGF1C-loaded hydrogel solution prepared in Example 15 was applied to the outer surface of the abdominal aorta, and the hydrogel was gelled by irradiating with light at a wavelength of 400 - 450 nm (EFL light source) for 1 minute. After the viscera were repositioned, the wound was sutured with 5-0 suture thread, and after applying povidone iodine again, the mice were placed in a clean cage; they were fed with normal feed and sacrificed after 28 days. The aorta was dissected and observed under a stereomicroscope.

[0186] The results are shown in Figure 15 , Figure 15 A is a representative image, Figure 15 B is the average value of the maximum diameter of the aorta in the saline group and the IGF1C / GelMa group. The results showed that compared with the saline group (Example 9), the IGF1C-functionalized hydrogel effectively inhibited the dilation of the infrarenal abdominal aorta.

[0187] Example 17. The IGF1C-loaded hydrogel inhibits the occurrence of aneurysms induced by xenotransplantation in rats

[0188] Eighteen 16-week-old male guinea pigs were purchased from Vital River and their abdominal aortas were obtained. At 37 °C, the abdominal aortas were gently shaken in 0.1% SDS solution for 18 hours for decellularization treatment. Subsequently, the decellularized arteries were washed 5 times with PBS and stored at 4 °C in PBS to obtain decellularized arteries.

[0189] Eighteen 12-week-old male Sprague-Dawley rats were purchased from Vital River, and the decellularized arteries of guinea pigs were implanted into the abdominal aortas of rats as vascular grafts in a sterile environment to construct a xenotransplantation model.

[0190] The specific steps are as follows: The rats were fasted 24 hours before surgery, anesthetized by intraperitoneal injection of 10% (w / v) chloral hydrate (3.3 mL / kg body weight), the abdominal hair was shaved off and the rats were fixed in the supine position on the operating table. Systemic heparinization of the blood was achieved by injecting sodium heparin solution (100 U.I / kg dose) via the caudal vein of the rats, and the skin was disinfected with iodophor. The abdominal skin and muscle layer were incised along the midline of the abdomen with scissors, and the viscera were carefully wrapped with sterile medical gauze and pushed to both sides of the abdominal cavity; the abdominal aorta was exposed, and the abdominal aorta and inferior vena cava below the kidney were bluntly dissected, about 1.2 cm in length, and the small arterial branches were ligated with 9-0 nylon suture; the artery was clamped at both ends and the abdominal aorta was transected; the vascular cross-section was rinsed with 50 U / mL sodium heparin solution, and the connective tissue outside the blood vessel was moderately removed. The acellular abdominal aorta of guinea pigs was cut into 1.0 cm segments, and the acellular artery was sutured to the autologous blood vessel of the rat by interrupted suture with 9-0 nylon suture with a needle at eight points. The proximal end was anastomosed first, and the distal end was anastomosed finally. After the suture at both ends was completed, the artery clamp was slowly removed to restore blood flow, and the total ischemic time of the whole operation was ensured not to exceed 45 min. The abdominal cavity was rinsed with 320 U / mL gentamicin solution.

[0191] The model rats were randomly divided into three groups.

[0192] Normal saline group: After vascular suture, normal saline was applied on the vascular surface as a control.

[0193] Hydrogel group: After vascular suture, the unloaded hydrogel solution was applied on the outer surface of the implanted xenogeneic acellular artery, and the hydrogel was irradiated with light with a wavelength of 400 - 450 nm (EFL light source) for 1 minute to form a gel.

[0194] IGF1C treatment group: After vascular suture, the hydrogel solution loaded with IGF1C was applied on the outer surface of the implanted xenogeneic acellular artery, and the hydrogel was irradiated with light with a wavelength of 400 - 450 nm (EFL light source) for 1 minute to form a gel.

[0195] Finally, the abdominal cavity of the rats was closed. After the rats recovered their activity for 12 hours, their diet was restored. After 28 days, the degree of vascular dilation was observed by various methods such as Doppler ultrasound, stereoscopic observation, and CT.

[0196] As Figure 16 shown (representative photo), the hydrogel can be applied on the outer wall of the transplanted abdominal aorta. As Figure 17 shown (representative photo), 4 weeks after transplantation of the xenogeneic abdominal aorta, it was observed that the IGF1C hydrogel could significantly inhibit the dilation and rupture of aneurysms.

[0197] Example 18. Preparation of IGF1C-functionalized hydrogel

[0198] Dissolve alkynyl-substituted -CS with deionized water. Add azide-modified IGF1C, CuSO4, and sodium ascorbate in a molar ratio of 1:0.2:0.4. React at 37 °C for 24 h. Dialyze with deionized water for 3 days, and obtain the CS-IGF-1C material by freeze-drying. Add the CS-IGF1C material into the GelMa hydrogel solution prepared in Example 15 to make its final concentration 2 mg / mL. After stirring evenly, excite the hydrogel to gel with light at a wavelength of 400 - 450 nm (EFL light source).

[0199] Example 19. Inhibition of Aneurysm Formation Induced by Xenotransplantation in Rats by IGF1C-Functionalized Hydrogel

[0200] Construct an aneurysm model induced by xenotransplantation in rats according to Example 21. Randomly divide the model rats into three groups.

[0201] Normal saline group: After vascular suture, apply normal saline on the vascular surface as a control.

[0202] GelMa / CS hydrogel group: After vascular suture, apply the GelMa / CS (the chitosan component is chitosan hydrochloride without modified IGF1C) hydrogel solution on the outer surface of the implanted xenogeneic acellular artery, and irradiate the hydrogel with light at a wavelength of 400 - 450 nm (EFL light source) for 1 minute to gel.

[0203] GelMa / CS-IGF1C treatment group: After vascular suture, apply the IGF1C-functionalized hydrogel (GelMa / CS-IGF1C) solution on the outer surface of the implanted xenogeneic acellular artery, and irradiate the hydrogel with light at a wavelength of 400 - 450 nm (EFL light source) for 1 minute to gel.

[0204] Finally, close the abdominal cavity of the rats. After the rats resume activity for 12 hours, resume the rats' diet. After 28 days, observe the degree of vascular dilation by stereoscopy. As Figure 18 (Representative example) shows that the IGF1C-functionalized hydrogel (GelMa / CS-IGF1C) can significantly inhibit the dilation and rupture of aneurysms.

[0205] Example 20. Inhibition of the Occurrence of Abdominal Aortic Aneurysm in Bama Pigs by IGF1C-Loaded Hydrogel

[0206] In this example, an aneurysm model of abdominal aorta in Bama pigs induced by xenotransplantation was constructed, and the therapeutic effect of the IGF1C-functionalized hydrogel in a large animal abdominal aortic aneurysm model was investigated.

[0207] Experimental method: 14 Bama pigs weighing 25 kg - 35 kg were purchased from Tianjin Bainong Biotechnology Company. Fresh goat abdominal aorta was obtained from the slaughterhouse. The abdominal aorta was immersed in a 0.5% SDS solution (prepared with Tris-EDTA buffer) and gently shaken for 24 hours. The abdominal aorta was washed with saline and then immersed in 0.2 mg / mL DNase I and shaken at 37°C for 24 hours. After washing the decellularized abdominal aorta with normal saline, it was immersed in fresh normal saline and stored at 4°C.

[0208] The Bama pigs were induced to anesthesia with a combination of rompun and midazolam. Then, anesthesia was induced with isoflurane. Electrocardiogram monitoring and blood oxygen monitoring were continuously carried out. After the Bama pigs had stable breathing, the abdomen was disinfected and laparotomy was performed in a sterile environment. An abdominal retractor was used to keep the abdomen open. The internal organs were removed and wrapped with a moist sterile gauze. The abdominal aorta was separated with blunt forceps. Lidocaine injection was administered into the adventitia of the abdominal aorta to prevent spasm. A 4-cm decellularized sheep abdominal aorta was transplanted into the Bama pig abdominal aorta using 6-0 suture (Mersilk) by interrupted suture. The proximal end was anastomosed first, and the distal end was anastomosed last. After the suturing at both ends was completed, the artery clamp was slowly removed to restore blood flow.

[0209] The model Bama pigs were randomly divided into two groups.

[0210] Normal saline group: After vascular suture, normal saline was applied on the vascular surface as a control.

[0211] Hydrogel group: After vascular suture, an unloaded hydrogel solution was applied on the outer surface of the implanted xenogeneic decellularized artery, and the hydrogel was irradiated with light at a wavelength of 400 - 450 nm (EFL light source) for 1 minute to form a gel.

[0212] IGF1C treatment group: After vascular suture, an IGF1C-functionalized hydrogel solution was applied on the outer surface of the implanted xenogeneic decellularized artery, and the hydrogel was irradiated with light at a wavelength of 400 - 450 nm (EFL light source) for 1 minute to form a gel.

[0213] Finally, the abdominal cavity of the Bama pigs was closed. Two months later, the degree of vascular dilation was examined by CT. The parameters were as follows: voltage = 120 kV, tomographic scan thickness = 5 mm, pitch = 0.992, reconstruction thickness = 1.25 mm, detector collimation width = 80 mm, rotation speed = 0.5.

[0214] As Figure 19 (Schematic representation of representative pictures) shown, 8 weeks after xenotransplantation of the abdominal aorta, obvious aneurysmal structures occurred in the transplanted arteries in the normal saline group. The IGF1C hydrogel could significantly inhibit the dilation of aneurysms.

[0215] Example 21: Ultrasound-guided hydrogel delivery to the abdominal aorta

[0216] Experimental method: One Bama pig weighing 25 kg - 35 kg was purchased from Tianjin Bainong Biology Company. After anesthesia with isoflurane, the back was disinfected and prepared for skin incision. Turn on the ultrasound machine (Mindray, M9), adjust it to the abdominal - high - penetration mode, place the C5 - 1s probe on the mid - line of the back, and slowly move it to obtain the location of the abdominal aorta. In front of the position where the probe is located, insert a puncture needle at an angle of 60°.

[0217] Locate the puncture needle in the ultrasound image and guide the puncture needle to the abdominal aortic aneurysm site. During the needle - inserting process, avoid organs such as the kidneys and do not penetrate the peritoneum. Mix iohexol in the hydrogel solution prepared in Example 15 (final concentration is 46%), and inject it through the puncture needle into the adventitia of the abdominal aorta. Subsequently, insert the optical fiber of the 405 - nm laser through the puncture needle, reach the glue solution site, irradiate for 2 minutes, and induce gelation. Finally, pull out the puncture needle and disinfect the local area with iodophor.

[0218] On the second day after the operation, a plain CT scan (GE Healthcare, Waukesha, WI, USA) was performed. The parameters are as follows: voltage = 120 kV, tomographic scan thickness = 5 mm, pitch = 0.992, reconstruction thickness = 1.25 mm, detector collimation width = 80 mm, rotation speed = 0.5.

[0219] The ultrasound - guided process is as Figure 20 shown. As Figure 20 (Representative picture illustration) As can be seen in the right - hand figure, the puncture needle can penetrate through the skin and reach the location of the abdominal aorta, delivering the hydrogel encapsulating IGF1C to the abdominal aorta site.

[0220] The distribution of the hydrogel at the abdominal aorta site is as Figure 21 (Representative picture illustration) shown. The CT results show that the hydrogel can be distributed at the abdominal aortic aneurysm site.

[0221] Example 22: Ultrasound - guided delivery of IGF1C - loaded hydrogel inhibits the dilation of abdominal aortic aneurysm in Bama pigs

[0222] The present invention further develops an ultrasound - guided drug delivery method. The hydrogel loaded with IGF1C is locally delivered to the abdominal aorta site with the aid of ultrasound technology.

[0223] Experimental method: Six Bama pigs weighing 25 kg - 35 kg were purchased from Tianjin Bainong Biology Company. According to the method in Example 23, an abdominal aortic aneurysm model of Bama pigs was constructed, and no drug stimulation was performed on the transplanted xenogeneic blood vessels after the operation.

[0224] Two weeks later, the model Bama pigs were divided into two groups: the normal saline group and the ultrasound-guided hydrogel delivery treatment group. After anesthesia with isoflurane, the back was disinfected and prepared for skin incision. The ultrasound machine (Mindray, M9) was turned on and adjusted to the abdominal - high penetration mode. The C5-1s probe was placed on the midline of the back and slowly moved to obtain the location of the abdominal aorta. In front of the position where the probe was located, a puncture needle was inserted at an angle of 60°. The puncture needle was found in the ultrasound image and guided to the abdominal aortic aneurysm site. During the needle insertion process, organs such as the kidneys should be avoided and the peritoneum should not be penetrated. In the normal saline group, 2 mL of normal saline was injected. In the ultrasound-guided hydrogel delivery treatment group, the hydrogel solution loaded with IGF1C prepared in Example 15 was injected into the adventitia of the abdominal aorta. Subsequently, the optical fiber of the 405 nm laser was passed through the puncture needle to reach the glue solution site and irradiated for 2 minutes to induce gelation. Finally, the puncture needle was withdrawn and the local area was disinfected with iodophor. The puncture treatment was performed once every two weeks.

[0225] As Figure 22 (Schematic representation of representative pictures) shown, ultrasound-guided local delivery of IGF1C-functionalized hydrogel can significantly inhibit the expansion of abdominal aortic aneurysm.

Claims

1. A method for preventing or treating an aneurysm or arterial dissection, which comprises administering an IGF1R inhibitor or a drug or preparation comprising the same.

2. The method of claim 1, wherein the aneurysm is selected from an aortic aneurysm and a peripheral aneurysm, or the arterial dissection is selected from an aortic dissection or a peripheral arterial dissection.

3. The method of claim 1 or 2, wherein the aneurysm or arterial dissection is selected from an abdominal or thoracic aneurysm or arterial dissection.

4. The method of any one of claims 1-3, wherein the aneurysm is selected from an abdominal aortic aneurysm or a thoracic aortic aneurysm, or the arterial dissection is selected from an abdominal aortic dissection or a thoracic aortic dissection.

5. The method of any one of claims 1-4, wherein the IGF1R inhibitor is an IGF1C polypeptide or an active fragment thereof.

6. The method of any one of claims 1-5, wherein the IGF1C polypeptide comprises GYGSSSRRAPQT, or comprises an amino acid sequence having 1-8 amino acid substitutions, insertions or deletions compared with GYGSSSRRAPQT and comprising SSSR, or consists of the amino acid sequence.

7. The method of claim 6, wherein the IGF1C polypeptide comprises SSSR, SSSRR, GSSSR, GSSSRR, ASSSRRAP, GSSSRAAP, GSSSRRAP, GSSSRAAAP or GYGSSSRRAPQT, or consists of the amino acid sequence.

8. The method of any one of claims 1-4, wherein the IGF1R inhibitor is an IGF1R antibody or an antigen-binding fragment thereof, for example, it comprises 3 heavy chain CDRs and 3 light chain CDRs of cetuximab; or the heavy chain variable region and the light chain variable region of cetuximab; or the heavy chain and the light chain of cetuximab; Preferably, the IGF1R antibody is cetuximab.

9. The method of any one of claims 1-4, wherein the IGF1R inhibitor is a small molecule inhibitor, for example, a small molecule inhibitor that specifically binds to IGF1R, such as linsitinib or podophyllotoxin.

10. The method of any one of claims 1-9, wherein the drug or preparation is a hydrogel comprising an IGF1R inhibitor such as an IGF1C polypeptide or an active fragment thereof, and the hydrogel material is selected from, for example, chitosan hydrogel, alginate hydrogel, hyaluronic acid hydrogel, PLGA hydrogel, PEG hydrogel, gelatin hydrogel. Preferably, the gelatin hydrogel is methacrylated gelatin GelMa, such as GelMa60.

11. The method of claim 10, wherein the hydrogel is prepared by the following method (1) Physical mixing, for example, mixing an IGF1C polypeptide or an active fragment thereof with GelMa; (2) Functional modification, for example, covalently bonding an IGF1C polypeptide or an active fragment thereof with chitosan through a chemical reaction.

12. The method of claim 11, wherein the hydrogel is prepared by the following method: (1) Prepare a GelMa hydrogel solution with a concentration between 5% and 15%, for example, at approximately 10%. (2) Dissolve the IGF1C polypeptide or its active fragment in the GelMa hydrogel solution at a certain concentration, such as at approximately 1 mg / ml; and (3) Stir evenly to obtain a hydrogel solution. And optionally, (4) Irradiate the hydrogel with light at a wavelength of approximately 400 - 450 nm (such as approximately 405 nm, such as an EFL light source) to form a gel.

13. The method according to any one of claims 1 - 12, wherein the administration is to locally deliver the drug or formulation to the lesion.

14. The method of claim 13, wherein the local delivery is performed by puncturing the abdominal cavity under ultrasound guidance to deliver to the lesion. Preferably, the lesion is an aneurysm or an arterial dissection, or the adventitia of the diseased aortic blood vessel.

15. The method of claim 14, wherein the drug or formulation is administered under ultrasound guidance by the following method: (1) Use an ultrasound device to detect the location of the artery (such as the aorta or a peripheral artery, such as in the abdomen or chest, such as the abdominal aorta) (for example, in the abdominal - high - penetration mode, obtained by moving the probe). (2) Insert a puncture needle in front of the location of the probe (such as at approximately 50 - 70°, such as at approximately 60°). (3) Locate the puncture needle in the ultrasound image. (4) Use the ultrasound image to guide the puncture needle to the aneurysm site. (5) Inject the drug or formulation (such as a hydrogel containing IGF1C or its fragment). (6) Pass a laser (such as a laser with a wavelength of approximately 400 - 450 nm, such as a laser with a wavelength of approximately 405 nm) optical fiber through the puncture needle to initiate gel formation. (7) Withdraw the puncture needle and disinfect the local area (such as applying iodophor).

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