Use of antisense oligonucleotide in preparation of drug for treating disease caused by abnormal thyroid

Abstract

To provide, in the field of gene therapy for thyroid-associated ophthalmopathy, use of an antisense oligonucleotide in preparation of a drug for treating a disease caused by an abnormal thyroid.SOLUTION: According to experimental results of the present invention, an antisense oligonucleotide S-ASODN-1 having a specific sequence targeting an insulin-like growth factor 1 receptor (IGF-1R) can effectively reduce concentrations of IGF-1R and an anti-thyroid peroxidase antibody (TPO-Ab), improve a level of thyroid-stimulating hormone (TSH), and be used for treating TED.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to the field of gene therapy for thyroid-associated eye disease, and in particular to the use of fully thiolated antisense oligonucleotides or compositions thereof targeting the insulin-like growth factor-1 receptor (IGF-1R) gene in models of thyroid-associated eye disease and in the treatment of thyroid-associated eye disease. [Background technology]

[0002] Thyroid-associated eye disease (TAO), also known as thyroid eye disease (TED), Graves' eye disease or ophthalmopathy (GO), thyrotoxic exophthalmos, hypothyroid eye disease, and several other terms, is an eye disease associated with thyroid dysfunction. It is the most common orbital disease in adults and the most common external manifestation of toxic diffuse goiter (GD). It accounts for 25-40% of GD cases, 2% of GD cases, and a small number of hypothyroid and euthyroid patients. According to a statement from the China Center for Disease Control and Prevention, the overall human incidence of GD is approximately 1%, with a significantly higher incidence in women than in men. The incidence of TAO is estimated to be between 0.25-0.4%.

[0003] TAO is an organ-specific autoimmune orbital inflammatory disease associated with thyroid dysfunction. The body attacks orbital fibroblasts that overexpress IGF-1R, causing inflammation in the muscles and fat tissue behind the eye, pushing the eye forward and causing it to protrude outward. If this irreversible damage accumulates over a long period of time, it can lead to further blindness.

[0004] TAO is divided into two types. Active TAO, which usually lasts 1 to 3 years, is characterized by a progressive autoimmune / inflammatory reaction in the soft tissues of the orbit. Active TAO causes expansion and remodeling of the ocular soft tissues. The autoimmune / inflammatory reaction of active TAO resolves spontaneously, and the condition transitions to inactive TAO. Inactive TAO is a term used to describe the long-term / persistent sequelae of active TAO. The etiology of TAO is unknown. TAO is usually associated with Graves' hyperthyroidism, but it can also occur as part of other pathological autoimmune pathologies affecting the thyroid gland and producing pathological autoimmune pathologies in the orbit and periorbital tissues, and, rarely, in the pretibial skin (pretibial myxedema) or fingers (thyroid clubbed acromyopathy). TAO is an autoimmune ophthalmopathy in which the orbit and periocular soft tissues are primarily affected, with subsequent impact on the eye and vision. In TAO, inflammation and dilation of orbital soft tissues (primarily ocular muscles and fat) cause the eyeball to bulge forward (bulge) and away from its socket (a phenomenon called proptosis or exophthalmos). While TAO often does not result in blindness, the condition can cause sight-threatening lagophthalmos, troublesome diplopia (double vision), and compressive hypothyroidism optic neuropathy. TAO can precede, coincide with, or follow the systemic complications of thyroid dysfunction. Ocular clinical findings of TAO include upper eyelid retraction, eyelid lag, swelling, redness (erythema), conjunctivitis, and globe bulging (exophthalmos or proptosis), bulbar chemosis, periorbital edema, and altered ocular motility, with significant functional, social, and cosmetic impacts. Many signs and symptoms of TAO, including proteostasis and ocular hyperemia, result from expansion of orbital adipose tissue and periocular muscles. The increased volume of adipose tissue is due in part to new fat cell growth (adipogenesis) within the orbital fat. Accumulation of hydrophilic glycosaminoglycans (mainly hyaluronic acid) within the orbital adipose tissue and perimuscular connective tissue between extraocular muscle fibers further expands the adipose compartment and increases the size of the extraocular muscle body.Hyaluronic acid is produced by fibroblasts within the orbital fat and extraocular muscles, and its in vitro synthesis is stimulated by several cytokines and growth factors, including IL-1β, interferon-γ, platelet-derived growth factor, and thyroid-stimulating hormone (TSH).

[0005] For a long time, there has been no optimal treatment for moderately to severely active TED. The primary treatment is intravenous glucocorticoid infusion, which has problems such as poor improvement in exophthalmos and hormone-related systemic side effects, leaving significant unmet clinical needs. Second-line treatments include repeated hormone infusions or the combination of orbital radiation therapy or other immunomodulatory agents. Biologics such as teprotumumab, tocilizumab, and rituximab are also recommended as second-line treatments for moderately to severely active TED by the EUGOGO guidelines, the Chinese Guidelines for Clinical Diagnosis and Treatment of Thyroid Eye Disease (2022), and the American Thyroid Association and European Thyroid Association Thyroid Eye Disease Consensus. Teprotumumab is the first-line treatment, especially for TED with significant exophthalmos.

[0006] Infiltrating CD34+ fibrocytes derived from the myelomonocytic lineage enter the circulation and lay the foundation for disease. Their expressed and presented autoantigens may be similar to those found in the thyroid gland, including low levels of TSHR, thyroglobulin, and other thyroid antigens. Fibrocytes present antigens to antigen-specific T cells, which also support IgG1 production by B cells. These fibrocytes differentiate into CD34+ fibroblasts, which can further differentiate into myofibroblasts and adipocytes depending on the regulatory signals they receive. If resident orbital fibroblasts are CD34-, CD34+ fibroblasts encounter resident CD34- fibroblasts upon entering the orbit. Upon activation, these cells produce many pro- and anti-inflammatory factors and cytokines, including interleukin-1β, -6, -8, -10, -12, and -16, tumor necrosis factor-α, a chemokine called "regulated and activated normal T expressed and secreted" (or RANTES), CXCL12, and CD40 ligand (CD40LCD154).

[0007] These cytokines can act locally on almost all resident cells in the orbit. Almost all infiltrating and resident cell types express IGF-1R. Therefore, a therapeutic approach based on IGF-1R inhibition may cover all of these cells. Cytokine-activated fibroblasts synthesize hyaluronic acid (HA) and other glycosaminoglycans, which can expand the volume of orbital tissue and cause exophthalmos.

[0008] Additionally, thyroid-stimulating hormone receptor (TSHR) autoantibodies act as TSHR agonists, inducing excessive thyroid hormone secretion and disengaging the thyroid gland from pituitary control. TSHR autoantibodies are also the basis for Graves' ophthalmopathy and pretibial myxedema. The synergistic action of IGF-1R and TSHR autoantibodies causes expansion and inflammation of the retroorbital tissues.

[0009] Table 1 lists some of the drugs currently in clinical trials for treating thyroid-associated eye disease.

[0010] [Table 1]

[0011] Insulin-like growth factors (IGFs), also known as growth regulators, include IGF-1 and IGF-2. These growth factors can only exert their biological effects upon binding to their receptor, IGF-1R. The insulin-like growth factor-1 receptor, IGF-1R, belongs to the receptor tyrosine kinase family and is located on the cell membrane. After binding to IGFs, dimerization occurs, allowing the tyrosine domains to become accessible, leading to its own phosphorylation and subsequent activation of intracellular RAS-RAF-MAPK, PI3K-PKB / AKT, and other signaling pathways related to cell proliferation. Activation of IGF-1R is crucial for stimulating the growth and survival of tumor cells.

[0012] Table 2 shows the partial indication status of drugs currently under investigation that target the IGF-1R receptor, not including thyroid-associated eye disease.

[0013] [Table 2]

[0014] IGF1-1R inhibitors theoretically block the binding of IGF-1R to TSHR, blocking downstream signaling mediated by the TSHR / IGF-1R complex and improving TED. Recently, newly developed GIP-1R monoclonal antibodies have focused on TED. There is only one commercially available drug in the TED field: teprotumumab.

[0015] Antisense oligodeoxyribonucleotides (ASODNs) are artificially synthesized oligonucleotide fragments, typically 15–30 nucleotides in length. They primarily rely on complementary base pairing to interfere with the transcription and translation of target genes, enabling targeted gene therapy. ASODNs offer numerous potential targeting sites, rationally designed targeting, efficient in vivo and in vivo action, and the ability to be synthesized on a large scale. These advantages make them potentially valuable gene therapy agents. In recent years, breakthroughs in nucleotide chemical modification and delivery technologies have fueled a new wave of research and development into ASODN drugs. Several well-known pharmaceutical companies, both domestic and international, have identified antisense drugs as a key focus of their new drug development efforts. Currently, nine antisense oligonucleotide drugs are commercially available (Table 3), but none of them are targeted to thyroid-associated eye disease.

[0016] [Table 3]

[0017] Therefore, providing antisense oligonucleotides for treating thyroid-associated eye disease has important practical implications. Summary of the Invention [Problem to be solved by the invention]

[0018] In view of this, the present invention treats TED by designing an antisense oligonucleotide S-ASODN-1 with a unique sequence that targets the insulin-like growth factor-1 receptor IGF-1R.

[0019] This application is the first to discover that an antisense oligonucleotide, S-ASODN-1, with a specific sequence for the insulin-like growth factor-1 receptor (IGF-1R), effectively reduces the concentrations of IGF-1R and anti-thyroid peroxidase antibodies (TPO-Ab), increases thyroid-stimulating hormone (TSH) levels, and can be used to treat TED. Virtually no antisense oligonucleotides are currently approved or in clinical trials for the treatment of TED. Furthermore, not all antisense oligonucleotides for IGF-1R sequences can treat TED. For example, the other antisense oligonucleotide sequences described in this application, S-ASODN-2, S-ASODN-3, S-ASODN-4, and S-ASODN-5, cannot effectively regulate TPO-Ab and TSH levels and therefore cannot be used to treat TED. [Means for solving the problem]

[0020] The present disclosure relates to the use of a therapeutically effective amount of a thiolated antisense oligonucleotide or composition thereof targeting the IGF-1R gene in the preparation of a medicament for treating a disease caused by thyroid dysfunction, comprising: The thiolated antisense oligonucleotide targeting the IGF-1R gene is (I), having the nucleotide sequence set forth in SEQ ID No. 1, or (II) (I) is obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I), and has a nucleotide sequence that is the same or similar in function to the nucleotide sequence shown in (I); or (III), (I) or (II) has a nucleotide sequence having at least 80% nucleotide identity with the nucleotide sequence shown in (I), (II), Preferably, the thiolated antisense oligonucleotide targeting the IGF-1R gene has a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more nucleotide identity with the nucleotide sequence shown in (I) or (II). Provide use.

[0021] In some preferred embodiments, the sequence of the thiolated antisense oligonucleotide targeting the IGF-1R gene comprises a nucleotide sequence having 80% or more nucleotide identity to the sequence 5'-TCCTCCGGAGCCAGACTTCA-3' (SEQ ID NO: 1), preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more nucleotide identity, and more preferably the sequence of the thiolated antisense oligonucleotide targeting the IGF-1R gene is 5'-TCCTCCGGAGCCAGACTTCA-3' (SEQ ID NO: 1).

[0022] In some preferred embodiments, the disease caused by thyroid dysfunction comprises thyroid-associated eye disease.

[0023] In some preferred embodiments, the thiolated antisense oligonucleotides targeting the IGF-1R gene further comprise other chemical modifications.

[0024] In some preferred embodiments, the other chemical modification is one or more selected from a locked nucleic acid modification, a 2-position methoxyethyl group modification, and a 2-position oxymethyl modification.

[0025] In some preferred embodiments, the composition further comprises at least one other active agent.

[0026] In some preferred embodiments, the at least one other active agent is a therapeutic agent.

[0027] In some preferred embodiments, the therapeutic agent is one or more selected from a corticosteroid, a biologic, and a conventional immunosuppressant.

[0028] In some preferred embodiments, the corticosteroid is selected from glucocorticoids.

[0029] In some preferred embodiments, as described in Patent 202180081949.7, intravenous glucocorticoids (ivGCs) and oral glucocorticoids are used to treat patients with moderate to severe active TAO.

[0030] In some preferred embodiments, the biologic is one or more selected from a CD20+ B cell inhibitor, an IL-6R antibody, an IL-17A antagonist, an FcRn antagonist, an IL-11R blocking antibody, an anti-TNFα antibody, and a thyroid-stimulating hormone receptor TSHR inhibitor.

[0031] In some preferred embodiments, the conventional immunosuppressant is one or more selected from mycophenolate mofetil, cyclosporine, methotrexate, and azathioprine.

[0032] Chinese Guidelines for the Diagnosis and Treatment of Thyroid-Related Eye Diseases (2022) Chinese Journal of Ophthalmology, September 2022, Vol. 58, No. 9. Chin J Ophthalmol, September 2022, Vol. 58, No. 9.

[0033] In some preferred embodiments, the therapeutic agent is one or more selected from secukinumab, tocilizumab, satralizumab, bunakizumab, batoclimab, TOUR006, LASN01, infliximab, rituximab, and selenium.

[0034] In some preferred embodiments, as described in Patent 202180081949.7, the pharmaceutical composition for treating thyroid-associated eye disease further comprises a corticosteroid, rituximab or other anti-CD20 antibody, tocilizumab or other anti-IL-6 antibody, or selenium, infliximab or other anti-TNFα antibody, or a thyroid-stimulating hormone receptor (TSHR) inhibitor.

[0035] In some preferred embodiments, the thyroid-associated eye disease is manifested by one or more of the following symptoms: eyelid syndrome, exophthalmos, diplopia, ocular motility disorders, ocular chemosis, optic neuropathy, corneal degeneration, conjunctival corneal degeneration, and systemic symptoms in patients with hyperthyroidism.

[0036] In some preferred embodiments, the diplopia includes constant diplopia, non-constant diplopia, and intermittent diplopia.

[0037] 1) Blepharitis: This is an important sign of the onset of thyroid-related eye disease, and is mainly manifested as wide eyelid opening, with some sclera exposed, which is called eyelid retraction, and when the upper eyelid cannot drop along with the downward movement of the eyeball, it is called upper eyelid lag.

[0038] 2) Exophthalmos: Thyroid-associated eye disease often causes simultaneous exophthalmos in both eyes, but it can also occur anteroposteriorly. In the early stages, axial exophthalmos is common, while in the later stages, fibrosis and contracture of the extraocular muscles can cause the eyeballs to be fixed in an exophthalmos position, affecting aesthetics. In patients with hyperthyroidism, exophthalmos progresses rapidly. In some patients, exophthalmos becomes more pronounced after hyperthyroidism is controlled.

[0039] 3) Diplopia and ocular motility disorders: Thyroid-related eye disease inevitably affects the extraocular muscles, causing edema and inflammatory infiltration in the early stages and fibrosis in the later stages. When multiple extraocular muscles become fatigued, they can develop in succession, with the degree of fatigue varying from patient to patient. Diplopia can manifest as multipositional diplopia, and end-stage diplopia is constant. The frequency of fatigue in the extraocular muscles is, in order, the inferior rectus, medial rectus, superior rectus, and lateral rectus muscles. Rectus muscle fibrosis can cause restrictive extraocular muscle lesions.

[0040] 4) Bulbar chemosis: Conjunctival hyperemia and vascular dilation often occur at the horizontal muscle insertion point. Severe chemosis can protrude outside the palpebral fissure, appearing translucent or cloudy, causing incomplete closure of the palpebral fissure and subsequent corneal degeneration.

[0041] 5) Optic neuropathy: Overseas, it has been reported that optic neuropathy occurs in 5% to 10% of patients with Grave's ophthalmopathy, and that irreversible visual loss occurs in 30% of these patients. It is more common in elderly people, men, and smokers. This is due to increased intraorbital pressure caused by edema and thickened intraorbital tissue and muscles, which compresses the optic nerve at the apex of the orbit, resulting in decreased vision and visual field defects (especially in the inferior optic nerve). Some patients may also experience increased intraocular pressure.

[0042] 6) Corneal degeneration: Changes in the eyelids and severe exophthalmos cause incomplete closure of the palpebral fissure, resulting in lagophthalmos keratitis, which causes early loss of punctate epithelium beneath the cornea. In severe cases, corneal ulcers may develop, leading to further perforation and severe damage to visual function.

[0043] 7) Conjunctival corneal degeneration, intraorbital soft tissue edema, and increased intraorbital pressure can cause conjunctival hyperemia and edema, and in severe cases, the conjunctiva protrudes outside the palpebral fissure.

[0044] 8) Patients with hyperthyroidism also have other systemic symptoms such as irritability, increased basal metabolic rate, rapid pulse, weight loss, increased appetite, and shaking hands.

[0045] As used in this article, "thyroid-associated eye disease" (TAO), "thyroid eye disease" (TED), "Graves' ophthalmopathy," or "Graves' ophthalmopathy" (GO) refer to the same disease or condition and can be used interchangeably. All refer to inflammatory orbital pathology associated with several autoimmune thyroid diseases, most commonly Graves' disease (GD), but occasionally other conditions (e.g., Hashimoto's thyroiditis).

[0046] The terms "proptosis" and "exophthalmos" (also called exophthalmos, exophthalmia, and exorbitism) refer to the forward protrusion, shift, bulging, or protrusion of an organ. As used herein, the terms refer to the forward protrusion, shift, bulging, or protrusion of the eye away from the orbit. Some skilled in the art believe that proteostasis and exophthalmos have the same meaning and are generally used interchangeably, while others believe that there are subtle differences in meaning. Some refer to severe proteostasis with exophthalmos or to endocrine-related proteostasis. Others use the term exophthalmos to describe proteostasis associated with the eyes, for example, in subjects with TAO (TED or GO).

[0047] As used herein, the terms "proteophthalmos" and "exophthalmos" are interchangeable and refer to the forward projection, shift, bulging, or protrusion of the eye away from the orbit. Because the anterior opening contains only rigid bony structures for the enlarged orbit, any increase in orbital soft tissue contents originating from the lateral or posterior regions can cause the eye to shift forward. Proteophthalmos or exophthalmos can be the result of several disease processes, including infection, inflammation, tumors, wounds, cancer metastasis, endocrine disorders, vascular diseases, and extraorbital lesions. TAO (TED or GO) is currently recognized as the most common etiology of proteophthalmos in adults. Exophthalmos can be bilateral, as is common with TAO (TED or GO), or unilateral, as is common with orbital tumors.

[0048] In some preferred embodiments, the thiolated antisense oligonucleotide or composition thereof targeting the IGF-1R gene is prepared as a lyophilized agent or an injectable agent.

[0049] In some preferred embodiments, the thiolated antisense oligonucleotides or compositions thereof targeted to the IGF-1R gene are administered in combination with one or more thyroid eye disease treatments.

[0050] In some preferred embodiments, the thyroid eye disease treatment is radiation therapy or / and surgical treatment.

[0051] In some preferred embodiments, the dose of the thiolated antisense oligonucleotide targeting the IGF-1R gene is 2.5 mg / kg to 10 mg / kg.

[0052] The present invention has discovered that an antisense oligonucleotide, S-ASODN-1, with a specific sequence for the insulin-like growth factor-1 receptor (IGF-1R), effectively reduces the concentrations of IGF-1R and anti-thyroid peroxidase antibodies (TPO-Ab) and increases thyroid-stimulating hormone (TSH) levels, making it useful for treating TED. Virtually no antisense oligonucleotides are currently approved or in clinical trials for the treatment of TED. Furthermore, not all antisense oligonucleotides for IGF-1R sequences can treat TED. For example, the other antisense oligonucleotide sequences described herein, S-ASODN-2, S-ASODN-3, S-ASODN-4, and S-ASODN-5, cannot effectively regulate TPO-Ab and TSH levels and therefore cannot be used for the treatment of TED. [Brief explanation of the drawings]

[0053] In order to more clearly describe the technical solutions in the embodiments of the present invention or the prior art, the drawings that need to be used in the description of the embodiments or the prior art are briefly described below. [Figure 1] 1 shows a curve of changes in rat body weight during the administration period. [Figure 2] This shows the change in TPO-Ab concentration (IU / mL, Mean±SEM) after the action of S-ASODN-1 on a rat model of thyroid-associated eye disease. [Figure 3] This shows the change in TSH concentration (mU / L, Mean±SEM) after the action of S-ASODN-1 on a rat model of thyroid-associated eye disease. [Figure 4] This shows the change in IGF-1R concentration (ng / mL, Mean±SEM) after the action of S-ASODN-1 on a rat model of thyroid-associated eye disease. [Figure 5-1]These are pathological photographs (magnification 200x) of extraocular muscle lesions after treatment of a rat thyroid-associated eye disease model with the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group. [Figure 5-2] These are pathological photographs (magnification 200x) of extraocular muscle lesions after treatment of a rat thyroid-associated eye disease model with the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group. [Figure 5-3] These are pathological photographs (magnification 200x) of extraocular muscle lesions after treatment of a rat thyroid-associated eye disease model with the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group. [Figure 5-4] These are pathological photographs (magnification 200x) of extraocular muscle lesions after treatment of a rat thyroid-associated eye disease model with the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group. [Figure 5-5]These are pathological photographs (magnification 200x) of extraocular muscle lesions after treatment of a rat thyroid-associated eye disease model with the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group. [Figure 5-6] These are pathological photographs (magnification 200x) of extraocular muscle lesions after treatment of a rat thyroid-associated eye disease model with the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group. [Figure 5-7] These are pathological photographs (magnification 200x) of extraocular muscle lesions after treatment of a rat thyroid-associated eye disease model with the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group. [Figure 5-8] These are pathological photographs (magnification 200x) of extraocular muscle lesions after treatment of a rat thyroid-associated eye disease model with the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group. [Figure 5-9]These are pathological photographs (magnification 200x) of extraocular muscle lesions after treatment of a rat thyroid-associated eye disease model with the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group. [Figure 5-10] These are pathological photographs (magnification 200x) of extraocular muscle lesions after treatment of a rat thyroid-associated eye disease model with the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group. [Figure 6-1] 1 shows a curve of changes in rat body weight during the administration period in a comparative experiment. [Figure 6-2] This shows the change in TPOAB concentration (IU / mL, Mean±SEM) after the action of S-ASODN-1 on a rat model of thyroid-associated ophthalmopathy in a comparative experiment. [Figure 6-3] This shows the change in TSH concentration (mU / L, Mean±SEM) after the action of S-ASODN-1 on a rat model of thyroid-associated ophthalmopathy in a comparative experiment. [Figure 6-4] This shows the change in IGF-1R concentration (ng / mL, Mean±SEM) after the action of S-ASODN-1 on a rat model of thyroid-associated eye disease in a comparative experiment. DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention discloses the use of antisense oligonucleotides in the preparation of drugs for treating thyroid disorders, and those skilled in the art can realize this by referring to the contents of this document and appropriately modifying the process parameters. In particular, all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described in preferred embodiments, and it is clear that those skilled in the art can modify, appropriately change, and combine the methods and applications described in this document without departing from the content, spirit, and scope of the present invention to realize and apply the technology of the present invention.

[0055] The raw materials and reagents used in preparing the antisense oligonucleotides of the present invention to treat diseases caused by thyroid abnormalities are all commercially available.

[0056] The present invention will now be further described with reference to the following examples.

[0057] Example 1: Synthesis of fully thiolated antisense oligonucleotides

[0058] 1.S-ASODN-1(Sequence:5'-TCCTCCGGAGCCAGACTTCA-3'(SEQ ID NO:1))

[0059] The solid phase synthesis method was adopted for synthesis, and the equipment used was an OligoPilot 400 synthesis apparatus manufactured by GE, USA. The synthesis process was as follows:

[0060] 1) Deprotection A toluene solution of dichloroacetic acid was used as a deprotecting reagent to remove the 5'-DMT protecting group of the initiating nucleoside dA(bz) in the vector, liberating the 5'-hydroxyl.

[0061] 2) Coupling Using acetonitrile as a solvent and 5-ethylthiotetrazole as an activator, the dC(bz) phosphoramidite monomer was activated to form an activated intermediate, which was then condensed with the 5'-hydroxy of the nucleoside dA(bz) to perform coupling.

[0062] 3) Thiation Xanthogen hydride is a thiolating reagent that oxidizes the phosphite to a stable thiophosphate.

[0063] 4) Hydroxy protection Acetic anhydride was used as a protecting reagent to protect the 5'-hydroxyl of nucleosides that had not undergone coupling reaction. Based on the nucleotide sequence of S-ASODN-1, the above steps 1) to 4) were repeated to sequentially couple the corresponding nucleosides until the coupling of the S-ASODN sequence was completed.

[0064] 5) Deprotection The DMT protecting group of the last nucleoside dT was removed using dichloroacetic acid as a deprotecting reagent to obtain S-ASODN-1 bound to the vector.

[0065] 6) Aminolysis Concentrated aqueous ammonia was added to carry out an aminolysis reaction, hydrolyzing the ester bond between the vector and the nucleotide, and removing the protecting groups on phosphate, adenine, guanine, and cytosine. After filtration and rinsing with aqueous ethanol, the filtrate was collected.

[0066] 7) Purification The filtrate was subjected to reverse phase column chromatography and lyophilized to obtain the product, which had a purity of 93.2%. S-ASODN-2 sequence: 5'-TTCATTCCTTTTATTTGGGA-3' (SEQ ID NO: 2), S-ASODN-3 sequence: 5'-GGACCCTCCTCCGGAGCC-3' (SEQ ID NO: 3), S-ASODN-4 sequence: 5'-GAGAAACAGGAGCCCCCACA-3' (SEQ ID NO: 4), The synthesis method for S-ASODN-5 sequence: 5'-GCGCGGCTGGAAAGCGCGTT-3' (SEQ ID NO: 5) was the same as above, and the purities were 91.1%, 93.5%, 92.4% and 92.8%, respectively.

[0067] Example 2: Efficacy test of thyroid-associated ophthalmopathy in rat models

[0068] 1. Experimental Materials Samples: antisense oligonucleotides S-ASODN-1 (sequence: 5'-TCCTCCGGAGCCAGACTTCA-3' (SEQ ID NO: 1)), S-ASODN-2 (sequence: 5'-TTCATTCCTTTTATTTGGGA-3' (SEQ ID NO: 2)), S-ASODN-3 (sequence: 5'-GGACCCTCCTCCGGAGCC-3' (SEQ ID NO: 3)), S-ASODN-4 (sequence: 5'-GAGAAACAGGAGCCCCCACA-3' (SEQ ID NO: 4)), and S-ASODN-5 (sequence: 5'-GCGCGGCTGGAAAGCGCGTT-3' (SEQ ID NO: 5)), Positive drug: Teprotumumab (500 mg / bottle, Horizon Therapeutics USA, Inc.), Drug Solvent: Sodium Chloride Injection; Reagents used: Thyroglobulin (product number: T885815-100mg, Shanghai Macklin Biochemical Technology Co., Ltd.), Isoflurane (batch number: 20221201, 100mL, Jiangsu Hengfengqiang Biotechnology Co., Ltd.), Freund's complete adjuvant (5mL / bottle, Chondrex), TSH Elisa Reagent Kit (96T, CUSABIO), TPO-Ab Elisa Reagent Kit (96T, CUSABIO), IGF-1R Elisa Reagent Kit (48T, CUSABIO), Experimental animals: Sixty 7-week-old, female, SPF-level Wistar rats weighing 164.7g-179.9g were purchased from Beijing Charles River Laboratory Animal Technology Co., Ltd. The animals were housed in sterile, individually ventilated IVC cages, with six animals per cage. The bedding consisted of sterilized corncob bedding. The rats were fed a professionally prepared, sterilized diet and had free access to purified water. The temperature in the animal experiment room was maintained at approximately 25°C, the relative humidity was maintained at 40-70%, and light was on for 12 hours daily.

[0069] 2. Experimental Method 2.1 Animal grouping and model construction Sixty 7-week-old female SPF Wistar rats weighing 164.7g-179.9g were randomly divided into the control group (0.9% NaCl, 50μL / eye, qw×4), the model group (0.9% NaCl, 50μL / eye, qw×4), the S-ASODN-1 low-dose group (2.5mg / kg, 50μL / eye, qw×4), the S-ASODN-1 medium-dose group (5mg / kg, 50μL / eye, qw×4), and the S-ASODN-1 high-dose group (10mg / The mice were divided into four groups (6 mice per group): S-ASODN-2 high-dose group (10 mg / kg, 50 μL / eye, qw × 4), S-ASODN-3 high-dose group (10 mg / kg, 50 μL / eye, qw × 4), S-ASODN-4 high-dose group (10 mg / kg, 50 μL / eye, qw × 4), S-ASODN-5 high-dose group (10 mg / kg, 50 μL / eye, qw × 4), and a positive drug group (teprotumumab, 10 mg / kg, tail vein injection). See Table 4 for animal grouping information.

[0070] [Table 4]

[0071] remarks: [1] The first digit of the animal number indicates the group, the second letter indicates the sex (F for female, M for male), and the third, fourth, and fifth digits indicate the individual animal number. [2] qw×4 is administered once a week for a total of four doses.

[0072] 2.2 Experimental processing method Except for the control group, the remaining groups were given 0.6% NaI aqueous solution, and combined with intraperitoneal injection of thyroglobulin and Freund's incomplete / complete adjuvant emulsion to establish a Wistar rat thyroid eye disease model. Starting from the 27th day after the model was established, the solution was administered by retrobulbar injection or intravenous administration to both eyes (OU). This was designated as D1 (or M27). The administration was performed once a week for four consecutive weeks, designated as D2 (M28), D3 (M29), D4 (M30)... and the period before D1 administration was designated as D0.

[0073] 2.3 Evaluation Indicators Clinical observation, body weight, serum thyroid peroxidase antibody (TPO-AB) and thyroid-stimulating hormone (TSH) concentrations on D0 and D23, intact right eye (OD) tissue insulin-like growth factor 1 receptor (IGF-1R) concentrations, OU extraocular muscle histopathological examination.

[0074] Weight measurements: Animals were weighed on the day they were received, on the day quarantine ended, on the day they were separated into groups, and once a week after separation. Animals were weighed when they were found dead or moribund.

[0075] Protein detection: On D0 and D23, serum samples were taken from animals in all groups and ELISA: TPOAb and TSH were measured. On D23, OD eyeballs were taken from animals in each group and ELISA: IGF-1R was measured.

[0076] Pathological detection: On M49 (D23), extraocular muscle tissues (all animals) of surviving animals in all groups were collected and macroscopically observed for lesions. After fixation for at least 48 h, the extraocular muscle tissues and thyroid tissues of all planned and non-planned animals were collected, dehydrated, embedded, sectioned, stained with hematoxylin and eosin, and read.

[0077] 2.4 Data Processing Generally, descriptive analysis was used for histopathological data. For quantitative indices such as body weight, the mean ± standard error (Mean ± SEM) was calculated for each group. Comparisons between two groups were performed using a t-test, with statistical significance indicated by P < 0.05. For comparisons between multiple groups, each indicator data was analyzed using the following procedure: Equality of variance was tested using Levene's test. If variances were equal (P > 0.05), statistical analysis was performed using one-way analysis of variance (ANOVA). If ANOVA was statistically significant (P ≤ 0.05), comprehensive comparisons were performed using the LSD method. If variances were not equal (P ≤ 0.05), statistical analysis was performed using Dunnett's T3 test.

[0078] The above statistical operations were performed using SPSS 25.0.

[0079] 3. Experimental Results 3.1 Observation of weight and general condition During the test period, an animal in model group number 2F003 developed abdominal distension on M27 (D1) and died on M29 (D3), an animal in S-ASODN-1 medium dose group number 4F005 died on M46 (D20), and no abnormal signs were observed in any of the remaining groups during the test period.

[0080] On D23, the mean fasting body weights of Wistar rats in the control group, model group, S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and positive drug group were 281.78±5.85g, 270.48±6.05g, 273.35±4.73g, 258.58±3.28g, 259.38±6.24g, 267.52±2.07g, 267.47±1.76g, 271.97±2.74g, 271.85±3.05g, and 262.40±2.55g, respectively. During the study period, compared with the normal control group, Wistar rats in the model group showed slower weight gain, with significant weight loss on M19, D0, and D7 (P<0.05). Compared with the model group, the weight gain trends of Wistar rats in the S-ASODN-1 low, medium, and high dose groups, S-ASODN-2 high dose group, S-ASODN-3 high dose group, S-ASODN-4 high dose group, S-ASODN-5 high dose group, and positive drug group were consistent during the treatment period, with no statistically significant differences between groups (P>0.05). See Figure 1.

[0081] 3.2 Detection of TPO-Ab protein High levels of thyroid peroxidase antibodies (TPO-Abs) indicate that the body's immune system has already launched an attack on the thyroid gland, a condition commonly associated with autoimmune thyroid diseases such as thyroid-associated eye disease.

[0082] The serum TPO-Ab concentrations of Wistar rats in the control group, model group, S-ASODN-1 low dose group, S-ASODN-1 medium dose group, S-ASODN-1 high dose group, S-ASODN-2 high dose group, S-ASODN-3 high dose group, S-ASODN-4 high dose group, S-ASODN-5 high dose group, and positive drug group on D0 before administration and D23 after administration are shown in the table below and Figure 2.

[0083] [Table 5]

[0084] During the study period, the serum TPO-Ab concentrations in Wistar rats at D0 before administration in each of the remaining groups were significantly elevated compared with those in the control group (P<0.001), demonstrating that each group had successfully established a mouse thyroid-associated eye disease model.

[0085] Compared to the baseline values ​​on Day 0, the serum TPO-Ab levels in Wistar rats in the S-ASODN-1 low-dose, S-ASODN-1 medium-dose, S-ASODN-1 high-dose, and positive drug groups decreased by approximately 70-170 IU / mL after the final administration on Day 23, a significant decrease of approximately 10-30 times that of the model group (P<0.001), demonstrating favorable therapeutic efficacy. In the S-ASODN-1 high-dose group, the TPO-Ab level after the final administration on Day 23 was only 48.57 IU / mL, a decrease of approximately 27.97 times that of the model group, demonstrating optimal therapeutic efficacy.

[0086] Compared with D0 before administration in each group, there was no statistical difference (P>0.05) between the S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, S-ASODN-5 high-dose group, and model group after the final administration on D23. There was no significant change in TPO-Ab concentrations in the S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, and S-ASODN-5 high-dose group, indicating poor or no therapeutic effect.

[0087] The reduction in TPO-Ab concentration in the positive drug group was approximately 22.50 times that of the model group. According to the above data, the order of therapeutic effect in each group was as follows: S-ASODN-1 high dose group > positive drug group > S-ASODN-1 medium dose group > S-ASODN-1 low dose group > S-ASODN-2 high dose group, S-ASODN-3 high dose group, S-ASODN-4 high dose group, S-ASODN-5 high dose group.

[0088] 3.3 Detection of TSH protein Under normal conditions, thyroid-stimulating hormone (TSH), also known as thyroid-stimulating hormone, secreted by the pituitary gland regulates thyroxine secretion, while thyrotropin-releasing hormone (TRH) in the hypothalamus also regulates TSH secretion. A typical negative feedback loop exists between thyroid hormones and TSH; elevated thyroid hormone levels inhibit TSH secretion, whereas low thyroid hormone levels stimulate the pituitary gland to secrete more TSH. Decreased serum thyroxine (T2 or T6) levels increase serum FSH levels and enhance the TSH response to TRH stimulation. Thyroid dysfunction alters these regulation and responses, and because thyroid function is closely related to thyroid-related eye disease, understanding thyroid function status is crucial for diagnosing thyroid-related eye disease.

[0089] The serum TSH concentrations of Wistar rats in the control group, model group, S-ASODN-1 low dose group, S-ASODN-1 medium dose group, S-ASODN-1 high dose group, S-ASODN-2 high dose group, S-ASODN-3 high dose group, S-ASODN-4 high dose group, S-ASODN-5 high dose group, and positive drug group on D0 before administration and after the final administration on D23 are shown in the following table and Figure 3.

[0090] [Table 6]

[0091] During the study period, the serum TSH concentrations of Wistar rats in each of the remaining groups at D0 before administration were significantly lower than those in the control group (P<0.001), demonstrating that each group had successfully established a mouse thyroid-associated eye disease model.

[0092] Compared with the baseline values ​​on Day 0, serum TSH concentrations in Wistar rats treated with S-ASODN-1 at the low dose were significantly elevated on Day 23 (P<0.05). Serum TSH concentrations in Wistar rats treated with S-ASODN-1 at the medium dose, high dose, and positive drug groups were also significantly elevated (P<0.001). In these three groups, TSH concentrations increased to 0.64-1.70 IU / mL, approximately 3-15 times higher than in the control group, demonstrating favorable therapeutic efficacy. In the high-dose S-ASODN-1 group, TSH concentrations increased to 2.18 IU / mL on Day 23, approximately 15.45 times higher than in the control group, demonstrating optimal therapeutic efficacy.

[0093] There was no statistical difference between the S-ASODN-2 high-dose group, the S-ASODN-3 high-dose group, the S-ASODN-4 high-dose group, the S-ASODN-5 high-dose group and the model group (P>0.05).The changes in TSH levels before and after administration in the S-ASODN-2 high-dose group, the S-ASODN-3 high-dose group, the S-ASODN-4 high-dose group and the S-ASODN-5 high-dose group were not significant, indicating poor or no therapeutic effect.

[0094] The increase in TSH concentration in the positive drug group was approximately 11.45 times that of the model group. According to the above data, the order of therapeutic effect in each group was as follows: S-ASODN-1 high dose group > positive drug group > S-ASODN-1 medium dose group > S-ASODN-1 low dose group > S-ASODN-2 high dose group, S-ASODN-3 high dose group, S-ASODN-4 high dose group, S-ASODN-5 high dose group.

[0095] 3.4 Detection of IGF-1R The results of detecting the tissue insulin-like growth factor 1 receptor (IGF-1R) concentration of the complete OD (right eye) on D23 are shown in the table below and in Figure 4.

[0096] [Table 7]

[0097] During the study period, compared with the control group, the IGF-1R concentrations in the right eye tissue of Wistar rats in the model group, S-ASODN-1 low-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, and S-ASODN-5 high-dose group were significantly elevated (P<0.001), the IGF-1R concentrations in the right eye tissue of Wistar rats in the S-ASODN-1 medium-dose and positive drug groups were significantly elevated (P<0.05), and there was no statistically significant difference in the IGF-1R concentrations in the right eye tissue of Wistar rats in the S-ASODN-1 high-dose group (P>0.05).

[0098] Compared with the model group, the concentrations of IGF-1R in the right eye tissue of Wistar rats in the positive drug group and the S-ASODN-1 high-dose group were significantly decreased (P<0.001), the concentrations of IGF-1R in the right eye tissue of Wistar rats in the S-ASODN-1 low-dose group, S-ASODN-2 high-dose group, S-ASODN-3 high-dose group, S-ASODN-4 high-dose group, and S-ASODN-5 high-dose group were significantly decreased (P<0.05), and the concentrations of IGF-1R in the right eye tissue of Wistar rats in the S-ASODN-1 medium-dose group were significantly decreased (P<0.01).

[0099] 3.5 Pathological detection The following was revealed from the pathological photographs (magnification 200 times) of extraocular muscle lesions in a rat thyroid-associated ophthalmopathy model shown in Figures 5-1 to 5-10.

[0100] Control group: No obvious abnormalities were found in the extraocular muscles of the left and right eyes of 6 / 6 animals.

[0101] Model group: 1 / 5 animals had slight swelling of extraocular muscle fibers in the left extraocular muscles, 4 / 5 animals had slight muscle fiber degeneration and necrosis in the left extraocular muscles, and 5 / 5 animals had slight muscle fiber degeneration and necrosis in the right extraocular muscles.

[0102] S-ASODN-1 low dose group: 3 / 6 animals had slight swelling of extraocular muscle fibers in the left extraocular muscles, 3 / 6 animals had slight muscle fiber degeneration and necrosis in the left extraocular muscles, 4 / 6 animals had slight swelling of extraocular muscle fibers in the right extraocular muscles, and 2 / 6 animals had slight muscle fiber degeneration and necrosis in the right extraocular muscles.

[0103] S-ASODN-1 medium dose group: 4 / 5 animals had slight swelling of extraocular muscle fibers in the left extraocular muscles, 1 / 5 animals had slight muscle fiber degeneration and necrosis in the left extraocular muscles, 3 / 5 animals had slight swelling of extraocular muscle fibers in the right extraocular muscles, and 2 / 5 animals had slight muscle fiber degeneration and necrosis in the right extraocular muscles.

[0104] S-ASODN-1 high dose group: No obvious abnormalities were observed in the extraocular muscles of the left and right eyes of 6 / 6 animals.

[0105] S-ASODN-2 high dose group: 6 / 6 animals had slight muscle fiber degeneration and necrosis in the extraocular muscles of the left eye, 1 / 6 animals had slight swelling of the extraocular muscles of the right eye, and 5 / 6 animals had slight muscle fiber degeneration and necrosis in the extraocular muscles of the right eye.

[0106] S-ASODN-3 high dose group: 1 / 6 animals had slight swelling of extraocular muscle fibers in the left extraocular muscles, 5 / 6 animals had slight muscle fiber degeneration and necrosis in the left extraocular muscles, 3 / 6 animals had slight swelling of extraocular muscle fibers in the right extraocular muscles, and 3 / 6 animals had slight muscle fiber degeneration and necrosis in the right extraocular muscles.

[0107] S-ASODN-4 high dose group: 1 / 6 animals had slight swelling of extraocular muscle fibers in the left extraocular muscles, 5 / 6 animals had slight muscle fiber degeneration and necrosis in the left extraocular muscles, and 6 / 6 animals had slight muscle fiber degeneration and necrosis in the right extraocular muscles.

[0108] S-ASODN-5 high dose group: 1 / 6 animals had slight swelling of extraocular muscle fibers in the left extraocular muscles, 5 / 6 animals had slight muscle fiber degeneration and necrosis in the left extraocular muscles, 2 / 6 animals had slight swelling of extraocular muscle fibers in the right extraocular muscles, and 4 / 6 animals had slight muscle fiber degeneration and necrosis in the right extraocular muscles.

[0109] Positive drug group: No obvious abnormalities were found in the extraocular muscles of the left and right eyes of 6 / 6 animals.

[0110] See Table 8 for a summary of lesion numbers.

[0111] [Table 8]

[0112] Conclusion: In this experiment, repeated intraperitoneal injections of bovine thyroglobulin (Tg) induced orbital tissue lesions in Wistar rats. Serum TPO-Ab levels were significantly elevated, and the disease course was consistent in both eyes, demonstrating the successful establishment of a rat model. By retrobulbar injection of 50 μL / eye once weekly for 4 consecutive weeks, the high-dose S-ASODN-1 group demonstrated superior regulation of serum TPO-Ab protein expression, serum TSH protein expression, and IGF-1R expression in the right eye compared with the control group. Pathological sections of extraocular muscles also demonstrated a reduction in tissue lesions, demonstrating a favorable effect in treating thyroid eye disease.

[0113] 4. Comparative Experiment 4.1. Experimental materials Sample: thiolated antisense oligonucleotide S-ASODN-1 (sequence: 5'-TCCTCCGGAGCCAGACTTCA-3' (SEQ ID NO: 1)), Positive agent 2: miR-143 mimic, forward: 5′-CACAGAUAGAAGGGCCUCGU-3′ (SEQ ID NO: 6), reverse: 5′-CCAGGUGAAGCUACUGCAAG-3 (SEQ ID NO: 7); Drug Solvent: Sodium Chloride Injection; Reagents used: Thyroglobulin (product number: T885815-100mg, Shanghai Macklin Biochemical Technology Co., Ltd.), Isoflurane (batch number: 20221201, 100mL, Jiangsu Hengfengqiang Biotechnology Co., Ltd.), Freund's complete adjuvant (5mL / bottle, Chondrex), TSH Elisa Reagent Kit (96T, CUSABIO), TPO-Ab Elisa Reagent Kit (96T, CUSABIO), IGF-1R Elisa Reagent Kit (48T, CUSABIO), Experimental animals: 24 7-week-old, female SPF Wistar rats weighing 165.2-175.6g were purchased from Beijing Charles River Laboratory Animal Technology Co., Ltd. The animals were housed in sterile, individually ventilated IVC cages, with five animals per cage. The bedding consisted of sterilized corncob bedding. The rats were fed a professionally prepared, sterilized diet and had free access to purified water. The room temperature for the animal experiment was maintained at approximately 25°C, the relative humidity at 40-70%, and light was on for 12 hours daily.

[0114] 4.2.Experimental Method 4.2.1 Animal grouping and model construction Twenty-four 7-week-old, female, SPF-level Wistar rats weighing 165.2 g–175.6 g were randomly assigned by body weight to a control group (0.9% NaCl, 50 μL / eye, qw × 4), a model group (0.9% NaCl, 50 μL / eye, qw × 4), a high-dose S-ASODN-1 group (10 mg / kg, 50 μL / eye, qw × 4), and a positive drug group (miR-143 simulant, 10 mg / kg, tail vein injection), with six rats per group. See Table 1 for animal grouping information.

[0115] [Table 9]

[0116] remarks: [1] The first digit of the animal number indicates the group, the second letter indicates the sex (F for female, M for male), and the third, fourth, and fifth digits indicate the individual animal number. [2] qw×4 is administered once a week for a total of four doses.

[0117] 4.2.2 Experimental processing method Except for the control group, the remaining groups were given a 0.6% NaI solution, which was then combined with intraperitoneal injections of thyroglobulin and Freund's incomplete / complete adjuvant emulsion to establish a Wistar rat thyroid eye disease model. Starting from the 27th day after the model was established, the solution was administered by retrobulbar injection or intravenous administration to both eyes (OU), once a week for 4 consecutive weeks (D1).

[0118] 4.2.3 Evaluation indicators Clinical observations, body weight, serum thyroid peroxidase antibody (TPO-AB) and thyroid-stimulating hormone (TSH) concentrations before administration on D1 and on D23, and intact OD (right eye) tissue insulin-like growth factor 1 receptor (IGF-1R) concentrations.

[0119] Weight measurements: Animals were weighed on the day they were received, on the day quarantine ended, on the day they were separated into groups, and once a week after group separation. Animals were weighed when they were found dead or moribund.

[0120] Protein detection: On D1 and D23, serum samples were taken from animals in all groups and ELISA: TPOAb and TSH were measured. On D23, OD eyeballs were taken from animals in each group and ELISA: IGF-1R was measured.

[0121] 4.2.4 Data Processing Generally, descriptive analysis was used for histopathological data. For quantitative indices such as body weight, the mean ± standard error (Mean ± SEM) was calculated for each group. Comparisons between two groups were performed using a t-test, with statistical significance indicated by P < 0.05. For comparisons between multiple groups, each indicator data was analyzed using the following procedure: Equality of variance was tested using Levene's test. If variances were equal (P > 0.05), statistical analysis was performed using one-way analysis of variance (ANOVA). If ANOVA was statistically significant (P ≤ 0.05), comprehensive comparisons were performed using the LSD method. If variances were not equal (P ≤ 0.05), statistical analysis was performed using Dunnett's T3 test.

[0122] The above statistical operations were performed using SPSS 25.0.

[0123] Experimental Results 4.3.1 Observation of weight and general condition No abnormal signs were observed in any of the animals in each group during the study period.

[0124] On day 23, the mean fasting body weights of Wistar rats in the control group, model group, S-ASODN-1 high-dose group, and positive drug 2 group were 290.12±2.98g, 281.40±3.87g, 288.87±3.51g, and 289.35±2.41g, respectively. During the study period, Wistar rats in the model group showed slower weight gain than those in the normal control group, with significant weight loss on days 19, 0, and 7 (P<0.01). Compared with the model group, the weight gain trends of Wistar rats in the S-ASODN-1 high-dose group and positive drug 2 group during the treatment period were consistent, with no statistically significant differences between groups (P>0.05). See Figure 6-1.

[0125] 4.3.2 Detection of TPO-Ab protein High levels of thyroid peroxidase antibodies (TPO-Abs) indicate that the body's immune system has already launched an attack on the thyroid gland, a condition commonly associated with autoimmune thyroid diseases such as thyroid-associated eye disease.

[0126] The serum TPO-Ab concentrations of Wistar rats in the control group, model group, S-ASODN-1 high-dose group, and positive drug 2 group on D23 before and after administration on D1 are shown in the table below and in Figure 6-2.

[0127] [Table 10]

[0128] During the study period, the serum TPO-Ab concentrations in Wistar rats before D1 administration in each of the remaining groups were significantly elevated compared with those in the control group (P<0.001), demonstrating that each group had successfully established a mouse thyroid-associated eye disease model.

[0129] Compared to pre-dose levels on Day 1 in each group, serum TPO-Ab levels in Wistar rats in the high-dose S-ASODN-1 and anticoagulant groups decreased by 23-154 IU / mL after the final dose on Day 23, a significant decrease of 1-10 times that of the model group (P<0.001 and P<0.05), demonstrating favorable therapeutic effects. The TPO-Ab levels in the anticoagulant 2 group decreased by 1.80 times that of the model group, while the TPO-Ab levels in the high-dose S-ASODN-1 group after the final dose on Day 23 were only 51.90 IU / mL, a decrease of 11.93 times that of the model group and 6.66 times that of the anticoagulant 2 group, demonstrating optimal therapeutic effects.

[0130] According to the above data, the order of treatment effect in each group was S-ASODN-1 high dose group > positive drug 2 group.

[0131] 4.3.3 Detection of TSH protein Under normal conditions, thyroid-stimulating hormone (TSH), also known as thyroid-stimulating hormone, secreted by the pituitary gland regulates thyroxine secretion, while thyrotropin-releasing hormone (TRH) in the hypothalamus also regulates TSH secretion. A typical negative feedback loop exists between thyroid hormones and TSH; elevated thyroid hormone levels inhibit TSH secretion, whereas low thyroid hormone levels stimulate the pituitary gland to secrete more TSH. Decreased serum thyroxine (T2 or T6) levels increase serum FSH levels and enhance the TSH response to TRH stimulation. Thyroid dysfunction alters these regulation and responses, and because thyroid function is closely related to thyroid-related eye disease, understanding thyroid function status is crucial for diagnosing thyroid-related eye disease.

[0132] Before administration on D1 and after the final administration on D23, the serum TSH concentrations of Wistar rats in the control group, model group, S-ASODN-1 high-dose group, and positive drug group are shown in the following table and Figure 6-3.

[0133] [Table 11]

[0134] During the test period, the serum TSH concentrations of Wistar rats in each of the remaining groups before D1 administration were significantly lower than those in the control group (P<0.001), demonstrating that each group had successfully established a mouse thyroid-associated eye disease model.

[0135] Compared to pre-dose levels on Day 1 in each group, serum TSH levels in Wistar rats in the high-dose S-ASODN-1 and anti-drug groups were significantly elevated after the final administration on Day 23 (P<0.001 and P<0.05). TSH levels rose to 1.21-2.00 IU / mL, an increase of 7-16 times that of the model group, demonstrating favorable therapeutic effects. In the anti-drug 2 group, TSH levels rose 7.67 times that of the model group. In the high-dose S-ASODN-1 group, TSH levels rose to 2.00 IU / mL after the final administration on Day 23, an increase of 16.11 times that of the model group and 2.10 times that of the anti-drug 2 group, demonstrating optimal therapeutic effects.

[0136] According to the above data, the order of treatment effect in each group was S-ASODN-1 high dose group > positive drug 2 group.

[0137] 4.3.4 IGF-1R detection The results of detecting the concentration of insulin-like growth factor 1 receptor (IGF-1R) in intact OD (right eye) tissue on D23 are shown in the table below and in Figure 6-4.

[0138] [Table 12]

[0139] During the test period, compared with the control group, the concentrations of IGF-1R in the right eye tissue of Wistar rats in the model group and positive drug 2 groups were significantly increased (P<0.001 and P<0.01), while there was no statistical difference in the concentrations of IGF-1R in the right eye tissue of Wistar rats in the S-ASODN-1 high-dose group (P>0.05).

[0140] Compared with the model group, the concentration of IGF-1R in the right eye tissue of Wistar rats in the positive drug group and the high-dose S-ASODN-1 group was significantly decreased (P<0.001).

[0141] It should be pointed out that the above is only a preferred embodiment of the present invention, and those skilled in the art may make some improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered as part of the protection scope of the present invention.

Claims

1. A drug for treating a disease caused by thyroid dysfunction, The medicament comprises a therapeutically effective amount of a thiolated antisense oligonucleotide or composition thereof targeting the IGF-1R gene; The thiolated antisense oligonucleotide targeting the IGF-1R gene is (I) having the nucleotide sequence set forth in SEQ ID No. 1; or (II) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I), and having the same or similar function as the nucleotide sequence shown in (I); or (III), (I) or (II) has a nucleotide sequence having at least 80% nucleotide identity with the nucleotide sequence shown in (II), A drug characterized by:

2. The thiolated antisense oligonucleotide targeting the IGF-1R gene has a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more nucleotide identity with the nucleotide sequence shown in (I) or (II). The drug according to claim 1.

3. The disease caused by thyroid dysfunction includes thyroid-associated eye disease. The drug according to claim 1.

4. The thiolated antisense oligonucleotide targeting the IGF-1R gene further comprises other chemical modifications. The drug according to claim 1.

5. the other chemical modification is one or more selected from a locked nucleic acid modification, a 2-position methoxyethyl group modification, and a 2-position oxymethyl modification; The drug according to claim 4.

6. The composition further comprises at least one other active agent. The drug according to any one of claims 1 to 5.

7. said at least one other active agent is a therapeutic agent; The drug according to claim 6.

8. The therapeutic agent is one or more selected from the group consisting of corticosteroids, biologics, and conventional immunosuppressants. The drug according to claim 7.

9. The corticosteroid is selected from glucocorticoids i.v.GC; The drug according to claim 8.

10. the biologic is one or more selected from the group consisting of a CD20+ B cell inhibitor, an IL-6R antibody, an IL-17A antagonist, an FcRn antagonist, an IL-11R blocking antibody, an anti-TNF α antibody, and a thyroid stimulating hormone receptor (TSHR) inhibitor; The drug according to claim 8.

11. The conventional immunosuppressant is one or more selected from mycophenolate mofetil, cyclosporine, methotrexate, and azathioprine; The drug according to claim 8.

12. the therapeutic agent is one or more selected from secukinumab, tocilizumab, satralizumab, bunakizumab, batoclimab, TOUR006, LASN01, infliximab, rituximab, and selenium; The drug according to claim 7 or 8.

13. The thyroid-associated eye disease is manifested by one or more of the following symptoms: eyelid syndrome, exophthalmos, diplopia, ocular motility disorder, ocular chemosis, optic neuropathy, corneal degeneration, conjunctival corneal degeneration, systemic symptoms in patients with hyperthyroidism; The drug according to claim 3.

14. The diplopia includes constant diplopia, non-constant diplopia, and intermittent diplopia. The drug according to claim 13.

15. The thiolated antisense oligonucleotide or composition thereof targeting the IGF-1R gene is prepared as a lyophilized agent or an injection. The drug according to claim 1.

16. The thiolated antisense oligonucleotide or composition thereof targeting the IGF-1R gene is administered in combination with one or more thyroid eye disease treatments. The drug according to claim 1.

17. The thyroid eye disease treatment is radiation therapy or / and surgical treatment. The drug according to claim 16.

18. the dose of the thiolated antisense oligonucleotide targeting the IGF-1R gene is 2.5 mg / kg to 10 mg / kg; The drug according to claim 1 .

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