Method for treating aortic aneurysm and aortic dissection by targeting IGF1r

Abstract

The present invention relates to a novel method for preventing or treating an aneurysm by using an IGF1R inhibitor and use. The present invention also relates to a formulation comprising IGF1C suitable for the method and use. The present invention further relates to a local minimally invasive delivery technique of drugs for preventing or treating an aneurysm, and in particular, to a local minimally invasive delivery technique for delivering a medicament comprising an IGF1R inhibitor according to the present invention.

Description

A method for treating aortic aneurysm and aortic dissection by targeting IGF1R

[0001] The present invention relates to a novel method and use for preventing or treating aneurysms using an IGF1R inhibitor. The present invention also relates to a formulation containing IGF1C suitable for use in the method and use. The present invention also relates to a minimally invasive local drug delivery technology for preventing or treating aneurysms, particularly a minimally invasive local drug delivery technology for delivering a drug containing an IGF1R inhibitor according to the present invention.

[0002] Background of the invention:

[0003] Aortic aneurysms and aortic dissections are serious threats to human health, with approximately 200,000 new cases reported in my country each year, resulting in an annual economic burden exceeding 10 billion RMB. Once a patient's aneurysm ruptures, the mortality rate is as high as 90%. Even for those who survive and receive timely treatment, the mortality rate remains as high as 47% to 83%. Surgical procedures (open and endovascular) are the most common treatments for these conditions, but they have numerous drawbacks, including high risk, high cost, inability to cure the underlying cause, and limited accessibility. Furthermore, for small aneurysms (less than 5.5 cm in diameter), the risk of rupture is lower than that of surgery, necessitating conservative observation.

[0004] Compared with healthy subjects, serum IGF1 levels in patients with aneurysms are significantly elevated. Some literature suggests that IGF1 can be used as a biomarker for aneurysm development (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 aneurysmal tissue. 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.) shows that long-term activation of mTOR stimulates the transformation of vascular smooth muscle cells into a degradative phenotype. The transformation of smooth muscle cells to a degradative phenotype is a key cellular mechanism that induces the occurrence of aortic aneurysms and aortic dissections. Therefore, the identification of mTOR-dependent phenotypic regulation can serve as a therapeutic target for combating aortic aneurysms and aortic dissections.

[0006] There is a need in the art to develop new drugs that modulate mTOR-dependent phenotypes, and it is also desirable to develop a method for treating aortic aneurysm and / or aortic dissection in a non-surgical manner.

[0007] Summary of the invention:

[0008] IGF1C is a C-domain polypeptide of the IGF1 protein. Studies have suggested that IGF1C can act as a competitive inhibitor of IGF1, inhibiting the activation of IGF1R. The present invention explores the function of the IGF1R inhibitor IGF1C in the treatment of aortic aneurysms and aortic dissections. IGF1R, a protein ubiquitous throughout the body, plays a key role in life processes such as glucose metabolism, muscle function, and neural signaling. Systemic inhibition of IGF1R can cause various side effects. The present invention develops a method for the local delivery of an IGF1R inhibitor for the treatment of aortic aneurysms and aortic dissections through interventional delivery, reducing the side effects associated with systemic delivery.

[0009] The present invention finds for the first time that IGF1R inhibitors can effectively treat aneurysms or arterial dissections.

[0010] Therefore, one aspect of the present invention relates to a method for treating an aneurysm or arterial dissection, comprising administering to a subject an IGF1R inhibitor, or a medicament or formulation comprising the same.

[0011] In some embodiments, the IGF1R inhibitor is selected from domain C of IGF1 (IGF1C) or a fragment thereof, an antibody or antigen-binding fragment thereof to an insulin-like growth factor receptor (e.g., a receptor for IGF1) (e.g., an IGF1R antibody or antigen-binding fragment thereof, such as cixitumumab or an antigen-binding fragment thereof), or a small molecule inhibitor, such as linsitinib) or picropodophyllotoxin.

[0012] In some embodiments, the aneurysm or arterial dissection of the present invention is in the aorta or a peripheral artery.

[0013] In some embodiments, the aneurysm or arterial dissection of the present invention is in an abdominal or thoracic artery.

[0014] In some embodiments, the aneurysm or arterial dissection is an aneurysm or arterial dissection of the abdominal aorta or thoracic aorta.

[0015] In some embodiments, the insulin-like growth factor domain C (IGF1C) polypeptide refers to domain C of IGF-1, corresponding to positions 30-41 of IGF-1. In some embodiments, IGF-1 is the protein set forth under UniProtKB accession number P05019. In some embodiments, the IGF1C polypeptide comprises or consists of the amino acid sequence GYGSSSRRAPQT (SEQ ID NO: 1).

[0016] 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 minimal active unit of IGF1C is SSSR (SEQ ID NO: 2) (CN1263770C). Therefore, other amino acids outside the minimal active unit can be mutated, such as substituted, deleted, or 1-10 amino acids can be added outside the minimal active unit, as long as the resulting IGF1C polypeptide still has substantially the same activity as wild-type IGF1C (e.g., IGF1R binding activity). Therefore, the present invention encompasses IGF1C polypeptides comprising an amino acid sequence having 1-8 amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in SEQ ID NO: 1 and comprising SSSR.

[0017] In some embodiments, the amino acid at position 3 of IGF1C (e.g., G) can be substituted with A, or R at position 8 can be substituted with A, or an amino acid (e.g., A) can be added after position 9. In some embodiments, the IGF1C polypeptide can comprise an R8A substitution and an addition of A between positions 9 and 10. In some embodiments, the IGF1C polypeptide can comprise G3A.

[0018] In some embodiments, the mutation (e.g., substitution, deletion, or addition) is conservative, meaning that it does not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the substitution of an 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 within a group of amino acids with similar side chains can be made without departing from the present invention. These include the 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 each other 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 (Glu 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 noted or apparent from the context, the amino acids mentioned herein should be considered to be L-amino acids. Standard amino acids can also be replaced by non-standard amino acids belonging to the same chemical class. As a non-limiting example, the basic side chain Lys can be replaced by basic amino acids with shorter side chain lengths (ornithine, diaminobutyric acid or diaminopropionic acid). Lys can also be replaced by neutral aliphatic isosteric norleucine (Nle), which in turn is replaced by analogs containing shorter aliphatic side chains (aminobutyric acid or aminopropionic acid). In some embodiments, the term "conservative mutation" is used to refer to amino acid modifications that do not significantly affect or change the activity of the parent polypeptide. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, of an activity relative to the parent polypeptide.

[0019] In some embodiments, the present invention also includes active fragments of IGF1C polypeptides, i.e., fragments comprising the minimal active expression unit SSSR, as long as they exhibit the activity of an IGF1C polypeptide. Active fragments suitable for use in the present invention can be fragments of wild-type IGF1C comprising SSSR, or fragments of variants thereof. In some embodiments, active fragments suitable for use in the present invention comprise, or consist of, the amino acid sequences 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).

[0020] In some embodiments, the IGF1C polypeptide of the present invention or an active fragment thereof has one or more of the following activities:

[0021] (1) Binding to IGF1R;

[0022] (2) competitively inhibiting the binding of IGF1 to IGF1R;

[0023] (3) inhibiting IGF1-induced phosphorylation of IGF1R;

[0024] (4) inhibiting IGF1-mediated smooth muscle phenotypic transformation, i.e., from contractile phenotype to degradative phenotype (e.g., inhibiting the expression of MMP9 in smooth muscle cells);

[0025] (5) inhibiting aneurysms (e.g., the occurrence and development of aneurysms);

[0026] (6) Inhibit the degradation of elastic fibers in arteries.

[0027] In some embodiments, IGF1C is IGF1C bound to a chitosan molecule (CS-IGF1C). In some embodiments, the CS-IGF1C is obtained by, for example, covalently binding IGF1C to a chitosan molecule via a chemical reaction; or, (2) binding IGF1C to chitosan via physical mixing. In some embodiments, the chemical reaction is a click reaction and / or a condensation reaction.

[0028] In some embodiments, the insulin-like growth factor C domain polypeptide is covalently bound to a chitosan molecule via a chemical reaction, wherein CS-IGF-1C is synthesized by a click chemistry reaction of the azide of IGF-1C-N3 and the alkyne of alkynylated chitosan. In some embodiments, the click chemistry reaction is performed under conditions where the feed molar ratio of IGF-1C-N3:CuSO4:sodium ascorbate is 1:0.2:0.4.

[0029] In some embodiments, CS-IGF-1C is synthesized by a Click reaction, which comprises dissolving alkynyl-substituted chitosan (CS), connecting 6-azidohexanoic acid to the N-terminus of the C domain by a condensation reaction to obtain IGF-1C-N3, and sodium ascorbate, performing a Click reaction, and then dialyzing and freeze-drying.

[0030] In some embodiments, the CS-IGF-1C is described in CN106667899B, which is incorporated herein by reference in its entirety.

[0031] In some embodiments, the IGF1R inhibitor can be an antibody or antigen-binding fragment thereof that specifically binds to IGF1R. In some embodiments, the antibody comprises three heavy chain CDRs and three light chain CDRs of ciuxitumumab or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain variable region and light chain variable region of ciuxitumumab. In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain and light chain of ciuxitumumab. In some embodiments, the antibody is ciuxitumumab.

[0032] 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 lincitinib or picropodophyllotoxin.

[0033] The present invention also relates to a drug or formulation comprising an IGF1R inhibitor, wherein the drug or formulation is used to treat aneurysms or arterial dissections. In some embodiments, the drug or formulation further comprises a pharmaceutical excipient, such as a pharmaceutical carrier known in the art.

[0034] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic agents and absorption delaying agents that are physiologically compatible. Pharmaceutical carriers suitable for the present invention include diluents, adjuvants, excipients, or vehicles for administering treatment, such as steroidal 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, etc. 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, etc. If necessary, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. These compositions can take the form of solutions, suspensions, emulsions, tablets, hair tonics, capsules, powders, sustained-release formulations, etc. In some embodiments, the pharmaceutical carrier is a hydrogel, such as chitosan hydrogel (CN106667899A) or gelatin hydrogel. For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0035] 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.

[0036] 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 methacrylic anhydride-treated gelatin (GelMa). In some embodiments, the hydrogel is GelMa60.

[0037] In some embodiments, a hydrogel comprising an IGF1C polypeptide or an active fragment thereof is prepared by physically mixing the IGF1C polypeptide or an active fragment thereof with a hydrogel. In some embodiments, a hydrogel comprising multiple IGF1C strands or active fragments thereof is prepared by functionalizing the IGF1C polypeptide or an active fragment thereof, such as by chemically binding the polypeptide or an active fragment thereof to a hydrogel material.

[0038] 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 greater than 0.5-5 mg / ml, for example, about 1, 2 or 3 mg / ml. In some embodiments, the concentration of GelMa in the GelMa hydrogel solution is between about 5%-15%, for example, about 10%. In some embodiments, the GelMa is GelMa60. In some embodiments, after dissolution, the method further comprises exciting the hydrogel to gelation with light of 400-450 nm wavelength (EFL light source).

[0039] Therefore, in some embodiments, the hydrogel comprising an IGF1C polypeptide or an active fragment thereof comprises an IGF1C polypeptide and GelMa, such as GelMa60.

[0040] In some embodiments, the hydrogel containing the IGF1C polypeptide or an active fragment thereof of the present invention is obtained as follows:

[0041] (1) Prepare a GelMa hydrogel solution with a concentration between 5% and 15%, for example, about 10%.

[0042] (2) dissolving the IGF1C polypeptide or its active fragment in the GelMa hydrogel solution at a certain concentration, for example, about 1 mg / ml; and

[0043] (3) Stir evenly.

[0044] In some embodiments, the hydrogel is stimulated to gel at a wavelength of about 400-450 nm (e.g., about 405 nm, such as an EFL light source) (e.g., irradiation for about 0.5-1.5 minutes, such as about 1 minute). In some embodiments, the hydrogel is stimulated to gel by laser application before application.

[0045] In some embodiments, the hydrogel comprising the IGF1C polypeptide or an active fragment thereof is an injectable hydrogel.

[0046] In some embodiments, the hydrogel comprising the IGF1C polypeptide or an active fragment thereof is a chitosan hydrogel, such as the chitosan hydrogel described in CN106667899B (incorporated herein in its entirety).

[0047] The IGF1R inhibitors 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, phosphorus, acetic acid, oxalic acid, tartaric acid, etc., and those formed with 16 free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0048] The amount of the IGF1R inhibitor of the invention effective in treating a condition or disease can be determined by standard clinical techniques based on the present disclosure. The precise dose to be employed in the formulation depends on the route of administration and the severity of the condition, and should be decided according to the judgment of the practitioner and each subject's circumstances.

[0049] In one embodiment, the drug or formulation comprising an IGF1R inhibitor of the present invention is locally delivered to a lesion (e.g., 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 by peritoneal puncture. In some embodiments, the lesion is a diseased aortic adventitia. In some embodiments, the peritoneal puncture is performed under ultrasound guidance.

[0050] In some embodiments, the drug or formulation is administered (eg, injected) via abdominal puncture under ultrasound guidance, for example, locally, for example, directly delivered to a lesion such as an aneurysm or arterial dissection, or to the diseased aortic adventitia.

[0051] In some embodiments, the drug or formulation comprising an IGF1R inhibitor of the present invention is a hydrogel comprising an IGF1R inhibitor. In some embodiments, after being delivered to the lesion, a light source is applied to irradiate the hydrogel to form a gel.

[0052] In one embodiment, the medicament or formulation of the invention comprising an IGF1R inhibitor is administered by injection.

[0053] In some embodiments, the medicament or formulation of the present invention is administered for local sustained release.

[0054] In some embodiments, the medicament or formulation of the present invention is minimally invasive to administer.

[0055] In some embodiments, administration of a medicament or formulation of the present invention can reduce the side effects of systemic delivery.

[0056] In some embodiments, the ultrasound-mediated approach comprises the following steps:

[0057] (1) The ultrasound device detects the location of an artery (e.g., the aorta or a peripheral artery, such as in the abdomen or chest, such as the abdominal aorta) (e.g., in the abdomen-high penetration mode, by moving the probe);

[0058] (2) inserting the puncture needle in front of the probe (e.g., at about 50-70°, e.g., at about 60°);

[0059] (3) Find the puncture needle in the ultrasound image;

[0060] (4) Ultrasound images guide the puncture needle to the aneurysm site;

[0061] (5) injectable drugs or preparations (e.g., hydrogels containing IGF1C or its fragments);

[0062] (6) Passing a laser (e.g., approximately 400-450 nm laser) through a puncture needle to induce gelation;

[0063] (7) Remove the puncture needle and disinfect the area (e.g., using iodine tincture).

[0064] In some embodiments, the present invention also relates to a kit comprising the drug or formulation of the present invention and one or more other therapeutic agents, such as drugs for assisting in the treatment of aneurysms or arterial dissections.

[0065] The present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising the IGF1 polypeptide or fragment thereof or a pharmaceutically acceptable salt thereof of the present invention. Optionally, the pharmaceutical combination or pharmaceutical combination product further comprises one or more other therapeutic agents.

[0066] The present invention also provides a complete kit comprising the drug combination, for example, the complete kit comprises in the same package:

[0067] - a first container containing a pharmaceutical composition of the present invention or a pharmaceutically acceptable salt thereof;

[0068] - Optionally, a second container further comprising a pharmaceutical composition comprising one or more additional therapeutic agents (in some embodiments, the two or more additional therapeutic agents are in the same container, or in separate containers).

[0069] The present invention also relates to an IGF1R inhibitor or a medicament or formulation comprising the same, which is used for treating a disease, such as for the use of the present invention, such as preventing or treating aneurysm or arterial dissection.

[0070] The present invention also relates to the use of an IGF1R inhibitor in the preparation of a medicament or formulation for treating a disease, such as for the use of the present invention, such as for preventing or treating an aneurysm or an arterial dissection.

[0071] definition:

[0072] 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 terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0073] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within 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.

[0074] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0075] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but not to exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context also encompasses consisting of the recited elements, integers, or steps. For example, when reference is made to a protein "comprising" a particular sequence, it is intended to encompass a protein consisting of that particular sequence.

[0076] 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 stretch of sequence identity and high structural homology with IGF-2 and insulin. The IGF-1 polypeptide chain can contain four 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 insulin B and A chains, respectively, domain C is similar to the connecting peptide of proinsulin, and the binding domain is similar to the connecting peptide of proinsulin. Domain D has no counterpart in insulin. IGF1 receptors include the insulin receptor (IR), insulin-like growth factor 2 receptor (IGF2R), hybrid receptors (IR / IGF1R), and other insulin receptor-related receptors (IRRs). IGF1 has the strongest affinity for the IGF1R receptor. IGF1R is a tetrameric tyrosine kinase receptor located on the cell surface. Upon ligand binding, it activates downstream signaling pathways such as Ras-raf-MAPK and PI3K-AKT-mTOR, promoting cell proliferation and regulating cell phenotype. IGF1 / IGF1R is a key target for the treatment of cancer and diseases such as glaucoma. Drugs targeting this target are already under development and have achieved good results in clinical treatment.

[0077] In this article, amino acid mutations can be amino acid substitutions, deletions, insertions and additions. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of the 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.

[0078] Reference herein to amino acid positions to be mutated is made by reference to the amino acid sequence set forth in SEQ ID NO: 1. Corresponding amino acid positions in polypeptides having alternative amino acid sequences can be identified by alignment with SEQ ID NO: 1. For example, reference to "G3" refers to G at position 3 of SEQ ID NO: 1, or to the amino acid residue at the corresponding position in the amino acid sequence of another variant polypeptide.

[0079] When referring to mutations in an IGF1C polypeptide herein, single amino acid substitutions are described as follows: [original amino acid residue / position / substituted amino acid residue] or [position / substituted amino acid residue]. For example, a substitution of glycine (or the corresponding other amino acid) at position 3 with alanine can be represented as G3A or 3A. When multiple alternative amino acid substitutions (e.g., A, R) are possible at a given position (e.g., position 3), the amino acid substitution can be represented as 3A / R or G3 / A / R. Accordingly, single amino acid substitutions can be linked by a plus sign (+) or a minus sign (-).

[0080] The terms "individual" or "subject" are used interchangeably and refer to mammals. 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.

[0081] The term "treating" includes administering a composition or hybrid polypeptide to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease, to alleviate symptoms, or to arrest or inhibit further development of a disease, condition, or disorder. The term "preventing" includes inhibiting the onset or development of a disease or disorder or symptoms of a particular disease or disorder.

[0082] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0083] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0084] The term "effective amount" refers to that amount or dosage of a polypeptide or composition or combination of the present invention which, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0085] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also one in which any toxic or detrimental effects of the IGF1C polypeptide, composition, or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.

[0086] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0087] 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.

[0088] "GelMA," described herein, is a photosensitive hydrogel material—methacrylic anhydride-modified gelatin. Its advantages include: structurally possessing cell adhesion sites and matrix metalloproteinase hydrolysis sites, thus effectively supporting cell proliferation and migration; the presence of methacrylic anhydride groups imparts photosensitivity, enabling rapid crosslinking under ultraviolet light; flexible and tunable physical and chemical properties; and the ability to fabricate structural units with unique morphological characteristics through a variety of microfabrication processes, such as bioprinting, photolithography, self-assembly, and microfluidics. GelMA hydrogels have demonstrated unique advantages in the regeneration of tissues such as bone, cartilage, myocardium, and blood vessels, and have also achieved promising results in areas such as basic cell research, cell signaling, controlled drug / gene release, and biosensing. These and other aspects and embodiments of the present invention are described in the accompanying drawings (followed by a brief description of the drawings) and the following detailed description, and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may 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 presented by way of illustration and not limitation, and that various modifications are possible by those skilled in the art. Description of the drawings:

[0089] Figure 1 shows that IGF1C inhibits the binding of IGF1 to smooth muscle cells.

[0090] FIG2 shows that IGF1C inhibits the binding of IGF1 to IGF1R.

[0091] FIG3 shows that IGF1C inhibits IGF1-induced IGF1R phosphorylation.

[0092] FIG4 shows cellular images showing that IGF1C inhibits IGF1-mediated IGF1R phosphorylation.

[0093] FIG5 shows that IGF1C inhibits IGF1-mediated MMP9 expression.

[0094] FIG6 shows that IGF1C inhibits AngII-induced aneurysmogenesis.

[0095] FIG7 shows that IGF1C inhibits extracellular matrix degradation.

[0096] FIG8 shows that IGF1C inhibits AngII-induced smooth muscle cell phenotypic transformation.

[0097] FIG9 shows that IGF1C inhibits CaCl2-induced aneurysmogenesis.

[0098] FIG10 shows that cilutumumab inhibits CaCl2-induced aneurysmogenesis.

[0099] FIG11 shows that linsitinib (OSI-906) inhibits CaCl2-induced aneurysmogenesis.

[0100] FIG12 shows that picropodophyllotoxin (AXL1717) inhibits CaCl2-induced aneurysmogenesis.

[0101] FIG. 13 shows that IGF1R inhibitors prevent CaCl 2 -induced elastic lamina degradation.

[0102] FIG. 14 shows that IGF1R inhibitors prevent CaCl 2 -induced phenotypic conversion of smooth muscle cells.

[0103] FIG15 shows that IGF1C-loaded hydrogels inhibited CaCl2-induced aneurysmogenesis.

[0104] FIG16 shows that the IGF1C-loaded hydrogel is coated on the outer wall of the aneurysm vessel.

[0105] FIG17 shows that IGF1C-loaded hydrogels inhibit the occurrence of xenograft-induced aneurysms in rats.

[0106] FIG18 shows that IGF1C functionalized modified hydrogel inhibits the occurrence of xenograft-induced aneurysms in rats.

[0107] FIG19 shows that IGF1C-loaded hydrogel inhibits the occurrence of abdominal aortic aneurysm in Bama pigs.

[0108] FIG. 20 shows ultrasound-guided hydrogel delivery to the abdominal aorta.

[0109] FIG21 shows a CT image of the distribution of the hydrogel in the abdominal aorta.

[0110] FIG22 shows the expansion of abdominal aortic aneurysms in Bama pigs transplanted with IGF1C-loaded hydrogels under ultrasound guidance. Example:

[0111] Example 1: Flow cytometry analysis of IGF1C inhibition of IGF1 binding to smooth muscle cells

[0112] Experimental method: 1×10 6 Human aortic smooth muscle cells (purchased from Pronocell Biotech) were seeded into 6-well plates at a density of 100 μg / L. The cells were cultured in smooth muscle cell complete medium (SMCM, Sciencell) at 37°C and 5% CO2. After the cells reached a density of 90%, they were washed with PBS and digested with 0.5 mL of EDTA-free trypsin (0.25%). After the cells were freed from the bottom of the culture plate, 1 mL of complete medium was added to terminate the digestion. The cells were centrifuged at 23°C and 1000 rpm. The supernatant was discarded, the cells were resuspended in PBS, and the cells were centrifuged again at 23°C and 1000 rpm. The cells were resuspended in 1 mL of serum-free medium and the cell suspension was gently mixed.

[0113] Pre-configured synthetic IGF1C peptide (SEQ ID NO: 1 GYGSSSRRAPQT, concentration is 100μg / mL) stock solution (Nanjing GenScript) was added to make the final peptide concentrations of 0, 10 and 100ng / mL, respectively. After incubation for 30 minutes, recombinant IGF1-His (IG1-H5245, ACROBiosystems) solution was added to make the final concentration of 1μg / mL. After 30 minutes, the cells were washed with PBS. After blocking the cells with 4% BSA for 30 minutes, His antibody (ab245114) was incubated. After the secondary antibody was bound, the proportion of cells bound to exogenous IGF1-His was detected by flow cytometry.

[0114] The results are shown in Figure 1. IGF1C effectively inhibited the binding of IGF1 to cells. As the concentration of IGF1C increased, the proportion of cells binding to exogenous IGF1 decreased.

[0115] Example 2: Co-immunoprecipitation (COIP) assay to detect the inhibition of IGF1C on the binding of IGF1 to IGF1R

[0116] Experimental method: 1×10 6 Human aortic smooth muscle cells (purchased from Procell Biotech) were seeded into 6-well plates at a density of 100 μg / mL. The cells were cultured in complete smooth muscle cell culture medium (SMCM, Sciencell) at 37°C and 5% CO2. When the cells reached a density of 70%, the medium was replaced with fresh serum-free medium. After starvation culture for 24 hours in serum-free medium, the medium was replaced with fresh medium and pre-prepared IGF1C stock solution was added to give final peptide concentrations of 0, 10, and 100 ng / mL, respectively. After 30 minutes, recombinant IGF1-His (IG1-H5245, ACROBiosystems) solution was added to a final concentration of 1 μg / mL. After incubation at 37°C for 30 minutes, the cells were washed twice with pre-chilled PBS. Total protein was extracted and the concentration was measured using the BCA assay (PC0020, Solarbio).

[0117] The protein homogenate of the total cell protein was used as the input sample, and the levels of IGF1R and β-actin protein in the sample were detected by WB. IGF1R antibody (Cell signaling technology, cat. no.: A0243 rabbit, concentration confirmed based on titer) was added to the remaining protein homogenate, and the CoIP kit (88804, Pierce TM ) IGF1R antibody-labeled protein was collected. A CoIP assay was performed to detect competitive binding of IGF1C to IGF1R. The results are shown in Figure 2.

[0118] IGF1R was detected by immunoprecipitation (IP) and IGF1-His was detected by immunoblotting (IB). As the concentration of IGF1C increased, the amount of IGF1-His bound to IGF1R gradually decreased.

[0119] The expression of IGF1R and β-actin (internal control) was detected by Western blotting in the input, which showed that the expression of IGF1R did not change.

[0120] Therefore, IGF1C competes for the binding of IGF1 to IGF1R, and the introduction of IGF1C interferes with the binding of IGF1 and IGF1R.

[0121] Example 3: Immunoblotting (IB) assay to detect the inhibitory effect of IGF1C on IGF1R phosphorylation induced by IGF1

[0122] Experimental method: 1×10 6 Human aortic smooth muscle cells were seeded onto confocal microplates at a density of 100 μg / mL. Cultured in complete smooth muscle culture medium (SMCM, Sciencell) at normal concentrations, the cells were replaced with fresh serum-free medium after reaching 70% density. After 24 hours, the medium was replaced with fresh medium and a pre-prepared IGF1C stock solution was added to final concentrations of 0, 10, and 100 ng / mL. Thirty minutes later, the cells were stimulated with 25 ng / mL of recombinant IGF1 protein (Novoprotein, C032). Protein was collected at time points of 0, 20, 40, and 60 minutes. Phosphorylated IGF1R (pIGF1R) levels were assessed by western blot (Cell Signaling Technology, Cat. No. A0243 rabbit source, concentration determined based on titer). The results are shown in Figure 3. Binding of IGF1C to IGF1R does not induce receptor phosphorylation. Following IGF1 stimulation, IGF1R phosphorylation levels gradually increased, peaking at 40 minutes before gradually diminishing. Pretreatment with IGF1C significantly inhibited the phosphorylation level of IGF1R.

[0123] Example 4: Immunofluorescence staining to detect the inhibition of IGF1C on IGF1R phosphorylation induced by IGF1

[0124] Experimental method: 1×10 6Human aortic smooth muscle cells were seeded onto confocal microplates at a density of 100 μg / mL. Cultured normally in smooth muscle complete culture medium (SMCM, Sciencell) until the cells reached a density of 70%. Fresh serum-free medium was then replaced 24 hours later. Fresh medium was then added to the cells, along with a pre-prepared IGF1C stock solution to a final concentration of 100 ng / mL. Thirty minutes later, cells were stimulated with 25 ng / mL of recombinant IGF1 protein (Novoprotein, C032).

[0125] After 40 minutes, the cells were washed with PBS and fixed with 4% paraformaldehyde solution for 30 minutes. The cells were washed twice with PBS. Immunofluorescence staining was performed using an antibody against phosphorylated IGF1R protein (AF3123, Affinity Biosciences). The phosphorylation of IGF1R protein on the cell surface was observed using an advanced fluorescence upright microscope (Axio Imager Z1, ZEISS). The results are shown in Figure 4. The control group is smooth muscle cells that were not stimulated; the IGF1C group is smooth muscle cells pretreated with IGF1C alone; the IGF1 group is smooth muscle cells stimulated with IGF1 alone; and the IGF1+IGF1C group is smooth muscle cells pretreated with IGF1C and then stimulated with IGF1. The results indicate that the binding of IGF1C to IGF1R does not cause receptor phosphorylation. At the same time, IGF1C inhibits IGF1-induced IGF1R receptor phosphorylation by competitively binding to IGF1R.

[0126] Example 5: IGF1C inhibits IGF1-mediated smooth muscle cell phenotypic transformation

[0127] IGF1 induces phenotypic transformation of smooth muscle cells from a contractile phenotype to a degradative phenotype, which is manifested by increased expression of MMP9. This example demonstrates that IGF1C can inhibit the phenotypic transformation of smooth muscle cells induced by IGF1.

[0128] Experimental method: 1×10 5Human aortic smooth muscle cells were seeded onto confocal microplates at a density of 50%. Cultured normally in smooth muscle complete medium (SMCM, Sciencell) until the density reached 50%, the medium was replaced with fresh serum-free medium. After 24 hours, the cells were treated with 10 ng / ml IGF1C. After 24 hours, the medium was replaced with fresh serum-free medium and stimulated with 25 ng / mL recombinant IGF1 protein (Novoprotein, C032). The cells were washed twice with pre-chilled PBS. Fixation was performed with 4% paraformaldehyde solution for 30 minutes. Wash twice with PBS. Immunofluorescence staining of the cells was performed using an antibody against MMP-9 (ab228402, Abclonal). The expression level of MMP-9 protein in the cells was observed using an advanced fluorescence upright microscope (Axio Imager Z1, ZEISS). The results are shown in Figure 5. The Control group is smooth muscle cells without any stimulation; the IGF1C group is smooth muscle cells pretreated with IGF1C (10 ng / ml) alone; the IGF1 group is smooth muscle cells stimulated with IGF1 (25 ng / mL) alone; and the IGF1+IGF1C group is smooth muscle cells pretreated with IGF1C and then stimulated with IGF1.

[0129] Figure 5 shows that MMP9 expression was observed in smooth muscle cells treated with IGF1C alone, whereas MMP9 expression was significantly reduced in the IGF1+IGF1C group, which subsequently received IGF1C. This result indicates that IGF1C inhibits IGF1-mediated MMP9 expression.

[0130] Example 6: IGF1C inhibits Ang II-induced aneurysm development

[0131] In this example, an AngII-induced aneurysm model was constructed, and the therapeutic effect of IGF1C as an IGF1R inhibitor in this abdominal aortic aneurysm model was investigated.

[0132] Experimental Methods: Twenty 10-week-old ApoE- / - C57BL / 6 male mice were purchased from Vital River and housed in a stable environment for 3 days before modeling. An aneurysm model was established using an osmotic pump (model 2004; Alzet, Cupertino) with sustained release of Ang II (HY-13948, MedChemexpress).

[0133] The procedure was as follows: 36 hours before surgery, mice were fasted and weighed. The required AngII dosage for 28 days of modeling was calculated based on body weight and loaded with a pump to achieve an AngII release rate of 1000 ng / kg / min. Mice were anesthetized with 4% chloral hydrate (8 μl / g) and placed on a laboratory table in the prone position. Neck hair was removed with depilatory cream and disinfected with iodine. A 1-cm surgical incision was made in the neck skin using ophthalmic scissors. Then, the dorsal skin and muscle were bluntly dissected using a needle holder, and the osmotic pump containing AngII was implanted subcutaneously. The wound was closed with 5-0 sutures, and the mice were again coated with iodine before being placed in a clean cage. Twenty model mice were fed a standard diet and randomly divided into two groups.

[0134] The normal saline group received intraperitoneal injection of 100 μl of normal saline every two days; the IGF1C-treated group received intraperitoneal injection of 100 μl of 1 mg / ml IGF1C (SEQ ID NO: 1) solution every two days.

[0135] The results are shown in Figure 6. Figure 6A shows a photograph of a representative mouse. Figure 6B shows the average of all mice (the average maximum diameter of aneurysms in the saline group and the IGF1C group, respectively). The results showed that the suprarenal aorta in the saline group experienced significant enlargement, while the addition of IGF1C significantly inhibited aneurysm development.

[0136] Example 7: IGF1C inhibits Ang II-induced arterial elastic lamina degradation

[0137] Experimental method: Using Van Gieson's staining kit (DC0058, Legend) was used to perform VVG staining to evaluate the degradation of elastic fibers.

[0138] The suprarenal abdominal aorta samples of the saline group and the IGF1C group in Example 6 were frozen sectioned, allowed to stand at room temperature for 10 minutes, and rinsed with running water for 5 minutes. Verhoeff stain A, Verhoeff stain B, and Verhoeff stain C were prepared in a ratio of 5:2:2. The sections were placed in the stain for 30 minutes. The sections were rapidly rinsed with running water for 5 minutes. The sections were differentiated with differentiation solution and the differentiation was examined microscopically until the elastic fibers appeared black and the background was gray. The sections were rinsed with running water for 5 minutes. The sections were deiodinated with 95% ethanol for 1 second. The sections were stained with Lichun red for 10 seconds, and the staining solution was discarded. The sections were dehydrated with anhydrous ethanol for 1 second. The sections were cleared with xylene twice, each for 3 minutes. The sections were mounted with neutral gum and photographed under an upright optical microscope (Leica DFC420C).

[0139] The results are shown in Figure 7. Figure 7A shows photographs of representative mice. Figure 7B shows the average value for all mice (average level of elastic fiber degradation). The results show that AngII treatment induced the fragmentation and degradation of elastic fibers (black) in the vascular wall, while the addition of IGF1C inhibited elastic fiber degradation, resulting in relatively continuous and intact elastic fibers in the vascular wall.

[0140] Example 8: IGF1C inhibits Ang II-induced smooth muscle cell phenotypic transformation

[0141] Experimental methods: CNN1 and α-SMA, MMP9 and α-SMA were co-stained to investigate the number of contractile and degradative smooth muscle cells, respectively.

[0142] Suprarenal abdominal aorta samples from the saline and IGF1C groups described in Example 6 were frozen and fixed with ice-cold acetone for 10 minutes. After air drying, the sections were washed twice in PBS. The membranes were permeabilized with 0.1% Triton for 9 minutes and washed twice in PBS. The sections were blocked with goat serum (source unknown) for 30 minutes at room temperature. Antibodies against CNN1 (abclonal, A3734) and α-SMA (abcam, ab7817) (or MMP9 (abclonal, A0289) and α-SMA (abcam, ab7817)) were added, incubated overnight at 4°C, and washed six times in PBS before secondary antibody staining. The sections were mounted with DAPI resin (Southerntech). Images were captured using an advanced fluorescence upright microscope (Axio Imager Z1, ZEISS).

[0143] The results are shown in Figure 8, where Figure 8A is an image of a representative mouse. Figure 8B is the average value of all mice (the 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 control group (saline group) induced a significant increase in the number of degradative phenotype smooth muscle cells (expressed as MMP9 and α-SMA double-positive), while the contractile smooth muscle cells (expressed as CNN1 and α-SMA double-positive) were significantly reduced. IGF1C effectively inhibited the phenotypic transformation of smooth muscle cells, causing the smooth muscle cells to exhibit a contractile phenotype.

[0144] Example 9: IGF1C inhibits CaCl2-induced aneurysm development

[0145] In this example, a CaCl2-induced aneurysm model was constructed and the therapeutic effect of IGF1C as an IGF1R inhibitor in this model was investigated.

[0146] Experimental Methods: Twelve 10-week-old C57BL / 6 male mice were purchased from Vital River and housed in a stable environment for 3 days before modeling. An aneurysm model was established using CaCl2 (Sigma, SLCG2286).

[0147] The details are as follows: 4% chloral hydrate anesthetized mice (8ul / g), and the mice were placed in supine position on the experimental table; the abdominal hair was removed with depilatory cream and iodine was applied for disinfection; an incision of about 1 cm long was made in the abdominal skin with ophthalmic scissors, and then the abdominal aorta was freed using microtweezers. 2 Soak gauze in 0.5M CaCl₂ solution for 5 minutes. Then, place the soaked gauze over the freed and exposed abdominal aorta and incubate for 15 minutes. After removing the gauze, wash the incubation area with PBS. After viscera are replaced, suture the wound with 5-0 sutures. Apply iodine again, and place the mouse in a clean cage; feed it a standard diet. Twelve model mice were randomly divided into two groups.

[0148] The saline group received 100 μL of saline intraperitoneally every two days; the IGF1C-treated group received 100 μL of a 1 mg / mL IGF1C solution intraperitoneally every two days. Samples were collected 28 days later. The aorta was dissected and observed under a stereomicroscope.

[0149] The results are shown in Figure 9. Figure 9A shows representative observation images, and Figure 9B shows the average maximum diameter of aneurysms for all mice in the saline and IGF1C groups. The results showed that the infrarenal abdominal aorta of mice in the saline group exhibited significant enlargement, while IGF1C significantly suppressed aneurysm development.

[0150] Example 10: Citumumab inhibits CaCl2-induced aneurysm development

[0151] In this example, a CaCl2-induced aneurysm model was constructed to investigate the therapeutic effect of cilutumumab, an antibody inhibitor of IGF1R, in this abdominal aortic aneurysm model.

[0152] Experimental Methods: Six 10-week-old C57BL / 6 male mice were purchased from Vital River and housed in a stable environment for 3 days before modeling. An aneurysm model was established using CaCl2 (Sigma, SLCG2286).

[0153] The details are as follows: 4% chloral hydrate anesthetized mice (8ul / g), and the mice were placed in supine position on the experimental table; the abdominal hair was removed with depilatory cream and iodine was applied for disinfection; an incision of about 1 cm long was made in the abdominal skin with ophthalmic scissors, and then the abdominal aorta was freed using microtweezers. 2Soak the gauze in 0.5M CaCl2 solution for 5 minutes. Then cover the free and exposed abdominal aorta with the soaked gauze and incubate for 15 minutes. After removing the gauze, wash the incubation site with PBS. After the internal organs are put back in place, suture the wound with 5-0 sutures, apply iodine again, and place the mouse in a clean cage; feed it with normal feed.

[0154] Every two days, 100 μL of 200 μg / mL sitomumab solution was injected intraperitoneally. Samples were collected 28 days later. The aorta was dissected and observed under a stereomicroscope.

[0155] The results are shown in Figure 10, where Figure 10A shows representative images and Figure 10B shows the average of the maximum values ​​of the aorta of mice in the saline-treated and cilutumumab-treated groups. Compared with the saline group in Example 8, cilutumumab effectively inhibited the enlargement of the infrarenal abdominal aorta.

[0156] Example 11: Linsitinib (OSI-906) inhibits CaCl2-induced aneurysm development

[0157] In this example, a CaCl2-induced aneurysm model was constructed to investigate the therapeutic effect of lincitinib as a small molecule inhibitor of IGF1R in the abdominal aortic aneurysm model.

[0158] Experimental Methods: Twelve 10-week-old C57BL / 6 male mice were purchased from Vital River and housed in a stable environment for 3 days before modeling. An aneurysm model was established using CaCl2 (Sigma, SLCG2286).

[0159] The details are as follows: 4% chloral hydrate anesthetized mice (8uL / g), and the mice were placed in supine position on the experimental table; the abdominal hair was removed with depilatory cream and iodine was applied for disinfection; an incision of about 1 cm long was made in the abdominal skin with ophthalmic scissors, and then the abdominal aorta was freed using micro forceps. 2 Soak gauze in 0.5M CaCl₂ solution for 5 minutes. Then, place the soaked gauze over the freed and exposed abdominal aorta and incubate for 15 minutes. After removing the gauze, wash the incubation area with PBS. After viscera are restored, suture the wound with 5-0 sutures, apply iodine again, and place the mouse in a clean cage. Feed the mouse with a standard diet and sample the mouse after 28 days. Twelve model mice were randomly divided into two groups.

[0160] The solvent control group received an intraperitoneal injection of 100 μL of a drug-free solvent (DMSO:PEG300:Tween80:saline = 2:8:1:9) every two days. The linsitinib-treated group received an intraperitoneal injection of 100 μL of the linsitinib solution, dissolved in a mixed solvent (DMSO:PEG300:Tween80:saline = 2:8:1:9) at a concentration of 6.25 mg / mL.

[0161] After 28 days, the aorta was removed and observed under a stereomicroscope.

[0162] The results are shown in Figure 11 , where Figure 11A shows representative images and Figure 11B shows the average of the maximum values ​​of the aorta of mice in the vehicle control group and the linsitinib-treated group. Compared with the vehicle control group, linsitinib effectively inhibited the dilatation of the infrarenal abdominal aorta.

[0163] Example 12: Picropodophyllotoxin (AXL1717) inhibits CaCl2-induced aneurysm formation

[0164] 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.

[0165] Experimental Methods: Twelve 10-week-old C57BL / 6 male mice were purchased from Vital River and housed in a stable environment for 3 days before modeling. An aneurysm model was established using CaCl2 (Sigma, SLCG2286).

[0166] The details are as follows: 4% chloral hydrate anesthetized mice (8uL / g), and the mice were placed in supine position on the experimental table; the abdominal hair was removed with depilatory cream and iodine was applied for disinfection; an incision of about 1 cm long was made in the abdominal skin with ophthalmic scissors, and then the abdominal aorta was freed using micro forceps. 2 Soak gauze in 0.5M CaCl₂ solution for 5 minutes. Then, place the soaked gauze over the freed and exposed abdominal aorta and incubate for 15 minutes. After removing the gauze, wash the incubation area with PBS. After viscera are restored, suture the wound with 5-0 sutures, apply iodine again, and place the mouse in a clean cage. Feed the mouse with a standard diet and sample the mouse after 28 days. Twelve model mice were randomly divided into two groups.

[0167] Solvent control group: 100 μL of solvent (DMSO: PEG300: Tween80: saline = 2:8:1:9) without drug was injected intraperitoneally every two days;

[0168] Picropodophyllotoxin treatment group: Picropodophyllotoxin 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 picropodophyllotoxin solution was intraperitoneally injected every two days.

[0169] After 28 days, the aorta was removed and observed under a stereomicroscope.

[0170] The results are shown in Figure 12 , where Figure 12A shows representative images and Figure 12B shows the average of the maximum values ​​of the aorta of mice in the solvent control group and the picropodophyllotoxin-treated group. Compared with the solvent control group, picropodophyllotoxin effectively inhibited the dilatation of the infrarenal abdominal aorta.

[0171] Example 13: IGF1R inhibitors prevent CaCl2-induced elastic lamina degradation

[0172] Experimental method: Using Van Gieson's staining kit (DC0058, Legend) was used to evaluate elastic fiber degradation. Frozen sections of infrarenal abdominal aorta samples from the saline, solvent, IGF1C, cixitumumab, linsitinib, and picropodophyllotoxin-treated mice in Examples 9, 10, 11, and 12 were placed at room temperature for 10 minutes and then rinsed with running water for 5 minutes.

[0173] Verhoeff stain A, Verhoeff stain B, and Verhoeff stain C were prepared in a ratio of 5:2:2; sections were placed in the stain for 30 minutes; rapidly rinsed with running water for 5 minutes; differentiated with differentiation solution and examined microscopically until the elastic fibers appeared black and the background was gray; rinsed with running water for 5 minutes; deiodinated with 95% ethanol for 1 second; stained with Lichun red for 10 seconds, and the stain was discarded; dehydrated with anhydrous ethanol for 1 second; cleared with xylene twice for 3 minutes each time; mounted with neutral gum, and photographed under an upright optical microscope (Leica DFC420C).

[0174] The results are shown in Figure 13. Figure 13A shows a representative observation image, and Figure 13B shows the average level of elastic fiber degradation for each group of mice. The results showed that elastic fibers (black) in the infrarenal abdominal aorta wall of mice in the saline and solvent control groups were broken and degraded. However, treatment with IGF1C, cilutumumab, linsitinib, and picropodophyllotoxin inhibited elastic fiber degradation, resulting in relatively continuous and intact elastic fibers in the vessel wall.

[0175] Example 14: IGF1R inhibitors prevent CaCl2-induced smooth muscle cell phenotypic transformation

[0176] Experimental methods: CNN1 and α-SMA, MMP9 and α-SMA were co-stained to examine the number of contractile and degradative smooth muscle cells respectively.

[0177] The infrarenal abdominal aorta samples of the saline group, solvent control group, IGF1C-treated group, cilutumumab-treated group, linsitinib-treated group, and picropodophyllotoxin-treated group of mice in Examples 9, 10, 11, and 12 were frozen sectioned and fixed with ice acetone for 10 minutes; after drying, they were washed twice in PBS; the membranes were permeabilized with 0.1% Triton for 9 minutes and washed twice with PBS; goat serum was blocked 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, and the sections were incubated at 4°C overnight. After washing 6 times with PBS, the sections were stained with secondary antibodies (Thermofisher, A-11001) (Thermofisher, A-11012).

[0178] Finally, the slides were mounted with DAPI resin (Southerntech) and images were captured using a fluorescence microscope (Axio Imager Z1, ZEISS).

[0179] The results are shown in Figure 14, Figure 14A is a representative image of each group, Figure 14B is the average number of CNN1 and α-SMA double-positive cells in each group of mice, and Figure 14C is the average number of MMP9 and α-SMA double-positive cells in each group of mice. The results showed that the number of degraded phenotype smooth muscle cells (expressed as MMP9 and α-SMA double positive) in the infrarenal abdominal aorta of mice in the saline group and the solvent control group was significantly increased, while the contractile smooth muscle cells (expressed as CNN1 and α-SMA double positive) were significantly reduced. IGF1C treatment, citrullumab treatment, linsitinib treatment and picropodophyllotoxin treatment effectively inhibited the phenotypic transformation of smooth muscle cells, causing the smooth muscle cells to exhibit a contractile phenotype.

[0180] Example 15: Preparation of IGF1C-loaded hydrogel

[0181] To avoid the side effects of systemic IGF1R inhibitors, this study further constructed an IGF1C-loaded hydrogel for local delivery of IGF1C.

[0182] Experimental Method: Commercial GelMa hydrogel (GelMa60) was purchased from EFL (Hangzhou). A 10% solution was prepared and an appropriate amount of IGF1C powder was dissolved in the GelMa hydrogel solution at a concentration of 1 mg / ml. After stirring, the solution was stimulated by 400-450 nm wavelength light (EFL light source) to form a gel (IGF1C / GelMa).

[0183] Example 16: IGF1C-loaded hydrogel inhibits CaCl2-induced aneurysm formation

[0184] In this example, a CaCl2-induced aneurysm model was constructed to investigate the therapeutic effect of IGF1C-functionalized hydrogel in the abdominal aortic aneurysm model.

[0185] Experimental Methods: Six 10-week-old C57BL / 6 male mice were purchased from Vital River and housed in a stable environment for 3 days before modeling. An aneurysm model was established using CaCl2 (Sigma, SLCG2286).

[0186] The details are as follows: 4% chloral hydrate anesthetized mice (8uL / g), and the mice were placed in supine position on the experimental table; the abdominal hair was removed with depilatory cream and iodine was applied for disinfection; an incision of about 1 cm long was made in the abdominal skin with ophthalmic scissors, and then the abdominal aorta was freed using micro forceps. 2 Soak the gauze in 0.5M CaCl2 solution for 5 minutes. Then cover the free and exposed abdominal aorta with the soaked gauze and incubate for 15 minutes. After removing the gauze, wash the incubation site with PBS. Apply the IGF1C-loaded hydrogel solution prepared in Example 15 to the outer surface of the abdominal aorta, and irradiate the hydrogel with 400-450nm wavelength light (EFL light source) for 1 minute to form a gel. After the internal organs are put back in place, suture the wound with 5-0 sutures, apply iodine tincture again, and place the mouse in a clean cage; feed it with ordinary feed, and collect the sample after 28 days. Peel off the aorta and observe it under a stereo microscope.

[0187] The results are shown in Figure 15 , where Figure 15A is a representative image and Figure 15B is the average maximum diameter of the aorta in the saline and IGF1C / GelMa groups. The results show that compared to the saline group (Example 9), the IGF1C-functionalized hydrogel effectively inhibited the enlargement of the infrarenal abdominal aorta.

[0188] Example 17: IGF1C-loaded hydrogel inhibits aneurysm formation induced by xenografts in rats

[0189] Eighteen 16-week-old male guinea pigs were purchased from Vital River and the abdominal aortas were harvested. The aortas were gently shaken in a 0.1% SDS solution at 37°C for 18 hours to decellularize. The decellularized arteries were then washed five times with PBS and stored in PBS at 4°C.

[0190] Eighteen 12-week-old male SD rats were purchased from Vital River. Guinea pig decellularized arteries were implanted as vascular grafts into the abdominal aorta of the rats in a sterile environment to establish a xenotransplantation model.

[0191] The details are as follows: rats were fasted for 24 hours before surgery, anesthetized with intraperitoneal injection of 10% (w / v) chloral hydrate (3.3 mL / kg body weight), their abdominal hair was shaved, and they were fixed in a supine position on the operating table. Heparin sodium solution (100 U.I / kg dose) was injected through the rat tail vein to achieve systemic heparinization of the blood, and the skin was disinfected with iodine. Using scissors, the abdominal skin and muscle layers were cut open along the midline. The organs were carefully wrapped with sterile medical gauze and moved to the sides of the abdominal cavity. The abdominal aorta was exposed, and the infrarenal abdominal aorta and vein were bluntly dissected, approximately 1.2 cm in length. Small arterial branches were ligated with 9-0 nylon sutures. The arterial clamps were clamped at both ends, and the abdominal aorta was transected. The vessel section was irrigated with 50 U / mL sodium heparin solution, and extravascular connective tissue was removed as needed. The guinea pig decellularized infrarenal artery was cut into 1.0 cm segments. The decellularized artery was then anastomosed to the rat autologous vessel using an eight-point interrupted suture pattern using 9-0 nylon sutures with a needle, first at the proximal end and then at the distal end. After suturing the two ends, the arterial clamps were slowly removed to restore blood flow. The total ischemic time during the entire procedure was maintained at no more than 45 minutes. The abdominal cavity was then irrigated with 320 U / mL gentamicin solution.

[0192] The model rats were randomly divided into three groups.

[0193] Normal saline group: After vascular suture, normal saline was applied on the surface of the blood vessels as a control.

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

[0195] IGF1C treatment group: After vascular suture, the IGF1C-loaded hydrogel solution was coated on the outer surface of the implanted xenogeneic decellularized artery, and the hydrogel was irradiated with 400-450 nm wavelength light (EFL light source) for 1 minute to form a gel.

[0196] Finally, the rat's abdominal cavity was closed. After the rat had recovered for 12 hours, its diet was resumed. After 28 days, the degree of vascular dilatation was observed using various methods, including Doppler ultrasound, stereoscopic observation, and CT.

[0197] As shown in Figure 16 (representative photos), the hydrogel can be coated on the outer wall of the transplanted abdominal aorta. As shown in Figure 17 (representative photos), IGF1C hydrogel was observed to significantly inhibit aneurysm expansion and rupture 4 weeks after xenograft abdominal aorta transplantation.

[0198] Example 18: Preparation of IGF1C functionalized hydrogel

[0199] Dissolve the alkynyl-substituted -CS in deionized water. Add azide-modified IGF1C, CuSO4, and sodium ascorbate at a molar ratio of 1:0.2:0.4. Incubate at 37°C for 24 hours. Dialyze against deionized water for 3 days and lyophilize to obtain the CS-IGF-1C material. Add the CS-IGF1C material to the GelMa hydrogel solution prepared in Example 15 to a final concentration of 2 mg / mL. After stirring, stimulate the hydrogel to form a gel using light of 400-450 nm (EFL light source).

[0200] Example 19: IGF1C functionalized hydrogel inhibits aneurysm formation induced by xenografts in rats

[0201] A rat xenograft-induced aneurysm model was constructed as described in Example 21. The model rats were randomly divided into three groups.

[0202] Normal saline group: After vascular suture, normal saline was applied on the surface of the blood vessels as a control.

[0203] GelMa / CS hydrogel group: After vascular suture, GelMa / CS (chitosan component is chitosan hydrochloride without IGF1C modification) hydrogel solution was applied to the outer surface of the implanted xenogeneic decellularized artery, and the hydrogel was irradiated with 400-450 nm wavelength light (EFL light source) for 1 minute to form gel.

[0204] GelMa / CS-IGF1C treatment group: After vascular suture, the IGF1C functionalized modified hydrogel (GelMa / CS-IGF1C) solution was coated on the outer surface of the implanted xenogeneic decellularized artery, and the hydrogel was irradiated with 400-450 nm wavelength light (EFL light source) for 1 minute to form a gel.

[0205] Finally, the rats' abdominal cavities were closed. After 12 hours of recovery, the rats were allowed to resume their diet. After 28 days, the degree of vascular dilation was observed stereologically. As shown in Figure 18 (representative example), the IGF1C-functionalized hydrogel (GelMa / CS-IGF1C) significantly inhibited aneurysm dilation and rupture.

[0206] Example 20: IGF1C-loaded hydrogel inhibits the occurrence of abdominal aortic aneurysm in Bama pigs

[0207] In this example, a xenotransplantation-induced abdominal aortic aneurysm model of Bama pigs was constructed, and the therapeutic effect of IGF1C functionalized hydrogel in the abdominal aortic aneurysm model of large animals was investigated.

[0208] Experimental Methods: 14 Bama pigs weighing 25-35 kg were purchased from Tianjin Bainong Biological Company. Fresh goat abdominal aortas were obtained from a slaughterhouse. The abdominal aortas were immersed in 0.5% SDS solution (prepared with Tris-EDTA buffer) and gently shaken for 24 hours. The abdominal aortas were washed with saline and then immersed in 0.2 mg / mL DNase I and shaken at 37°C for 24 hours. The decellularized abdominal aortas were washed with saline and then immersed in fresh saline and stored at 4°C.

[0209] Lumianning and midazolam were used to induce anesthesia in Bama pigs. Anesthesia was then induced with isoflurane. ECG monitoring and blood oxygen monitoring were carried out. After the Bama pigs' breathing stabilized, the abdomen was disinfected and opened in a sterile environment. The abdomen was kept open using abdominal separation clamps. The internal organs were removed and wrapped with moist sterile gauze. Blunt forceps were used to separate the abdominal aorta. Lidocaine injection was injected into the adventitia of the abdominal aorta to prevent spasm. Using 6-0 sutures (mousse), the 4cm decellularized sheep abdominal aorta was transplanted into the Bama pig abdominal aorta using interrupted sutures. The proximal end was anastomosed first, and the distal end was anastomosed last. After the sutures at both ends were completed, the arterial clamp was slowly removed to restore blood flow.

[0210] Model Bama pigs were randomly divided into two groups.

[0211] Normal saline group: After vascular suture, normal saline was applied on the surface of the blood vessels as a control.

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

[0213] IGF1C treatment group: After vascular suture, the IGF1C functionalized modified hydrogel solution was coated on the outer surface of the implanted xenogeneic decellularized artery, and the hydrogel was irradiated with 400-450 nm wavelength light (EFL light source) for 1 minute to form a gel.

[0214] Finally, the Bama pig's abdominal cavity was closed. Two months later, vascular dilatation was assessed by CT. Parameters were as follows: voltage = 120 kV, scan thickness = 5 mm, pitch = 0.992, reconstruction thickness = 1.25 mm, detector collimation width = 80 mm, rotation speed = 0.5.

[0215] As shown in Figure 19 (representative images), 8 weeks after xenograft transplantation, the transplanted artery in the saline group developed obvious aneurysm-like structures. IGF1C hydrogel significantly inhibited the expansion of the aneurysm.

[0216] Example 21: Ultrasound-guided hydrogel delivery to the abdominal aorta

[0217] Experimental Methods: A Bama pig weighing 25-35 kg was purchased from Tianjin Bainong Biological Co., Ltd. After isoflurane anesthesia, the back was disinfected and the skin was prepared. An ultrasound machine (Mindray, M9) was switched to abdominal high-penetration mode. The C5-1s probe was placed in the midline of the back and slowly moved to locate the abdominal aorta. The needle was inserted at a 60-degree angle in front of the probe.

[0218] Find the puncture needle in the ultrasound image and guide it to the site of the abdominal aortic aneurysm. During the needle insertion process, avoid organs such as the kidneys and do not penetrate the peritoneum. Add iodine haemoglobin to the hydrogel solution prepared in Example 15 (final concentration is 46%) and inject it into the adventitia of the abdominal aorta along with the puncture needle. Then, pass the 405nm laser optical fiber through the puncture needle to the gel site. After irradiation for 2 minutes, induce gel formation. Finally, remove the puncture needle and disinfect it locally with iodine tincture.

[0219] On the second day after surgery, a CT scan (GE Healthcare, Waukesha, WI, USA) was performed with the following parameters: voltage = 120 kV, slice thickness = 5 mm, pitch = 0.992, reconstruction thickness = 1.25 mm, detector collimation width = 80 mm, and rotation speed = 0.5.

[0220] The ultrasound-guided process is shown in Figure 20. As shown in the right figure of Figure 20 (representative images), the puncture needle can be inserted through the skin and reach the location of the abdominal aorta, delivering the hydrogel encapsulating IGF1C to the abdominal aorta.

[0221] The distribution of the hydrogel in the abdominal aorta is shown in FIG21 (representative images). The CT results show that the hydrogel can be distributed in the abdominal aortic aneurysm site.

[0222] Example 22: Ultrasound-guided delivery of IGF1C-loaded hydrogels to inhibit expansion of abdominal aortic aneurysms in Bama pigs

[0223] The present invention further develops an ultrasound-guided drug delivery method, which uses ultrasound technology to locally deliver IGF1C-loaded hydrogel to the abdominal aorta.

[0224] Experimental Method: Six Bama pigs weighing 25-35 kg were purchased from Tianjin Bainong Biological Co., Ltd. An abdominal aortic aneurysm model of Bama pigs was established according to the method in Example 23. No drug stimulation was applied to the transplanted xenograft blood vessels after surgery.

[0225] After 2 weeks, the model Bama pigs were divided into 2 groups. Normal saline group, ultrasound-guided hydrogel delivery treatment group. After isoflurane anesthesia, the back was disinfected and the skin was prepared. Turn on the ultrasound machine (Mindray, M9), adjust it to the abdomen-high penetration mode, place the C5-1s probe on the midline of the back, move slowly, and obtain the location of the abdominal aorta. Insert the puncture needle at 60° in front of the probe. Find the puncture needle in the ultrasound image and guide the puncture needle to the site of the abdominal aortic aneurysm. During the needle insertion process, avoid organs such as the kidneys and do not pass through the peritoneum. The normal saline group was injected with 2mL of normal saline, and the ultrasound-guided hydrogel delivery treatment group injected the IGF1C-loaded hydrogel solution prepared in Example 15 into the adventitia of the abdominal aorta. Subsequently, the optical fiber of the 405nm laser passed through the puncture needle to the glue site, and after irradiation for 2 minutes, it induced gelation. Finally, the puncture needle was pulled out and locally disinfected with iodine. Puncture treatment was performed once every two weeks.

[0226] As shown in Figure 22 (representative images), 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 medicament or preparation comprising the same.

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

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

4. The method of any one of claims 1-3, wherein the aneurysm is selected from abdominal aortic aneurysm or thoracic aortic aneurysm, or the arterial dissection is selected from abdominal aortic dissection or 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 the 3 heavy-chain CDRs and 3 light-chain CDRs of cetuximab; or the heavy-chain variable region and light-chain variable region of cetuximab; or the heavy chain and 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 medicament 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 (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) Gelation of the hydrogel is induced by light with a wavelength of approximately 400 - 450 nm (such as approximately 405 nm, such as an EFL light source).

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

14. The method of claim 13, wherein the local delivery is performed by puncture under ultrasound guidance into the lesion, preferably, the lesion is at an aneurysm or 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) The ultrasound device 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). (2) In front of the location of the probe, insert a puncture needle (such as at approximately 50 - 70°, such as at approximately 60°). (3) Locate the puncture needle in the ultrasound image. (4) The ultrasound image guides 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 induce gelation. (7) Withdraw the puncture needle and disinfect the local area (such as applying iodophor).

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